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Urocortin-1 (UCN1) is a member of the corticotropin releasing hormone (CRH) family of peptides that acts through CRH-receptor 1 (CRHR1) and CRH-receptor 2 (CRHR2). UCN1 can induce the adrenocorticotropin hormone and downstream glucocorticoids through CRHR1 and promote beneficial metabolic effects through CRHR2. UCN1 has a short half-life and has been shown to improve experimental autoimmune disease. A pegylated UCN1 peptide (PEG-hUCN1) was generated to extend half-life and was tested in multiple experimental autoimmune disease models and in healthy mice to determine effects on corticosterone induction, autoimmune disease, and glucocorticoid induced adverse effects. Cardiovascular effects were also assessed by telemetry. PEG-hUCN1 demonstrated a dose dependent 4-6-fold elevation of serum corticosterone and significantly improved autoimmune disease comparable to prednisolone in several experimental models. In healthy mice, PEG-hUCN1 showed less adverse effects compared with corticosterone treatment. PEG-hUCN1 peptide induced an initial 30% reduction in blood pressure that was followed by a gradual and sustained 30% increase in blood pressure at the highest dose. Additionally, an adeno-associated viral 8 (AAV8) UCN1 was used to assess adverse effects of chronic elevation of UCN1 in wild type and CRHR2 knockout mice. Chronic UCN1 expression by an AAV8 approach in wild type and CRHR2 knockout mice demonstrated an important role of CRHR2 in countering the adverse metabolic effects of elevated corticosterone from UCN1. Our findings demonstrate that PEG-hUCN1 shows profound effects in treating autoimmune disease with an improved safety profile relative to corticosterone and that CRHR2 activity is important in metabolic regulation. SIGNIFICANCE STATEMENT: This study reports the generation and characterization of a pegylated UCN1 peptide and the role of CRHR2 in UCN1-induced metabolic effects. The potency/selectivity, pharmacokinetic properties, pharmacodynamic effects, and efficacy in four autoimmune models and safety profiles are presented. This pegylated UCN1 shows potential for treating autoimmune diseases with reduced adverse effects compared to corticosterone treatment. Continuous exposure to UCN1 through an AAV8 approach demonstrates some glucocorticoid mediated adverse metabolic effects that are exacerbated in the absence of the CRHR2 receptor.

Introduction: There is evidence that supports a role for Vitamin D (Vit. D) in muscle. The exact mechanism by which Vit. D deficiency impairs muscle strength and function is not clear. Methods: Three-week-old mice were fed diets with varied combinations of Vit. D and Ca2+ deficiency. Behavioral testing, genomic and protein analysis, and muscle histology were performed with a focus on neuromuscular junction (NMJ)-related genes. Results: Vit. D and Ca2+ deficient mice performed more poorly on given behavioral tasks than animals with Vit. D deficiency alone. Genomic and protein analysis of the soleus and tibialis anterior muscles revealed changes in several Vit. D metabolic, NMJ-related, and protein chaperoning and refolding genes. Conclusions: These data suggest that detrimental effects of a Vit. D deficient or a Vit. D and Ca2+ deficient diet may be a result of differential alterations in the structure and function of the NMJ and a lack of a sustained stress response in muscles. This article is protected by copyright. All rights reserved.

article i nfo Cortical bone, the dominant component of the human skeleton by volume, plays a key role in protecting bones from fracture. We analyzed the cortical bone effects of teriparatide treatment in postmenopausal women with osteoporosis who had previously received long-term alendronate (ALN) therapy or were treatment naive (TN). Tetracycline-labeled paired iliac crest biopsies obtained from 29 ALN-pretreated and 16 TN women were evaluated for dynamic histomorphometric parameters of bone formation at the periosteal, endocortical and intracortical bone compartments, before and after 24 months of teriparatide treatment. At baseline, the frequen- cy of specimens without any endocortical and periosteal tetracycline labeling, and the percentage of quiescent osteons, was higher in the ALN than the TN group. Endocortical and periosteal mineralizing surface (MS/BS%), periosteal bone formation rate (BFR/BS), mineral apposition rate (MAR) and the number of intracortical forming osteons were significantly lower in the ALN-pretreated patients than in the TN group. Following teriparatide treatment, the frequency of endocortical and periosteal unlabeled biopsies decreased; in the ALN-pretreated group the percentage of quiescent osteons decreased and, in contrast, forming and resorbing osteons were in- creased. Teriparatide treatment resulted in significant increases of MAR in the endocortical, and MS/BS% in the periosteal compartment in the ALN-pretreated group. Most indices of bone formation remained lower in the ALN-pretreated group compared with the TN group at study end. Endocortical wall width was increased in both ALN-pretreated and TN groups. Cortical porosity and cortical thickness were significantly increased in the ALN-pretreated group after teriparatide treatment. Our results suggest that 24 months of teriparatide treatment increases cortical bone formation and cortical turnover in patients who were either TN or had previous ALN therapy.

Sclerostin antibodies increase bone mass by stimulating bone formation. However, human and animal studies show that bone formation increases transiently and returns to pre-treatment level despite ongoing antibody treatment. To understand its mechanism of action, we studied the time course of bone formation, correlating the rate and extent of accrual of bone mass and strength after sclerostin antibody treatment. Ovariectomized (OVX) rats were treated with a sclerostin-antibody (Scle-ab) at 20mg/kg sc once weekly and sacrificed at baseline and 2, 3, 4, 6, and 8weeks post-treatment. In Scle-ab treated rats, serum PINP and OCN rapidly increased at week 1, peaked around week 3, and returned to OVX control levels by week 6. Transcript analyses from the distal femur revealed an early increase in bone formation followed by a sustained decrease in bone resorption genes. Lumbar vertebral (LV) osteoblast surface increased 88% by week 2, and bone formation rate (BFR/BS) increased 138% by week 4. Both parameters were below OVX control by week 8. Bone formation was primarily a result of modeling based formation. Endocortical and periosteal BFR/BS peaked around week 4 at 313% and 585% of OVX control, respectively. BFR/BS then declined but remained higher than OVX control on both surfaces through week 8. Histomorphometric analyses showed LV-BV/TV did not further increase after week 4, while BMD continued to increase at LV, mid femur (MF), and femoral neck (FN) through week 8. Biomechanical tests showed a similar improvement in bone strength through 8weeks in MF and FN, but bone strength plateaued between weeks 6 and 8 for LV. Our data suggest that bone formation with Scle-ab treatment is rapid and modeling formation dominated in OVX rats. Although transient, the bone formation response persists longer in cortical than trabecular bone.

There is evidence that supports a role for Vitamin D (Vit. D) in muscle. The exact mechanism by which Vit. D deficiency impairs muscle strength and function is not clear.Three-week-old mice were fed diets with varied combinations of Vit. D and CaVit. D and CaThese data suggest that detrimental effects of a Vit. D deficient or a Vit. D and Ca

We compared teriparatide (TPTD) and strontium ranelate (SR) efficacy on bone formation activity in a mature rat model of estrogen-deficiency bone loss. Rats were ovariectomized (OVX) at age 6 months and permitted to lose bone for 2 months to establish osteopenia before initiation of treatment with TPTD (5 or 15 μg/kg · d sc) or SR (150 or 450 mg/kg · d oral gavage). After 3 wk, RT-PCR analyses of bone formation genes in the distal femur metaphysis showed significant elevation of collagen 1α2, osteocalcin, bone sialoprotein, alkaline phosphatase, and Runx2 gene expression at both TPTD doses, relative to OVX controls. SR had no significant effect on expression of these genes. TPTD treatment for 12 wk dose dependently increased lumbar vertebral (LV) and femoral midshaft bone mineral content (BMC) and bone mineral density over pretreatment and age-matched OVX controls. SR 150 increased BMC, and SR 450 increased BMC and bone mineral density of femoral midshaft and LV over OVX controls. There were significant dose-dependent TPTD increases of LV and femoral neck strength, and TPTD 15 also increased midshaft strength compared with pretreatment and age-matched OVX controls. SR did not enhance bone strength relative to pretreatment or age-matched OVX controls. Histomorphometry of the proximal tibial metaphysis showed dose-dependent effects of TPTD on trabecular area, number, width, and osteoblast surface, bone mineralizing surface, and bone formation rate relative to pretreatment and age-matched OVX controls, whereas SR had no effect on these parameters. These findings confirmed the bone anabolic efficacy of teriparatide, but not SR in mature, osteopenic, OVX rats.

Type-I procollagen aminoterminal propeptide (PINP) is a useful biomarker for bone formation activity that is used to monitor treatment of bone disorders including osteoporosis. Studies with human patients under long-term therapy for osteoporosis by daily injection of parathyroid hormone (PTH) demonstrated that the circulating level of PINP at 3 months of treatment, measured by radioimmunoassay, was a good predictor for bone mineral density (BMD) at 18 months. It is important to have PINP assays for other species to elucidate processes of bone formation and for the development of new therapeutic options that can enhance bone formation activity. Currently, only a human PINP radioimmunoassay is commercially available for clinical use, which may not be cross reactive with PINP from other species. For example, rat PINP has little amino acid sequence homology to human PINP. Therefore, we developed a new, highly sensitive, high-throughput mass spectrometry-based assay for PINP from rat plasma or serum that does not rely on antibody reagents. Circulating levels of PINP showed age-dependent changes in rats. Prednisolone treatment, which is known to retard bone formation activity, led to a significant decrease in PINP, whereas PTH treatment dose-dependently increased PINP. The throughput of the assay parallels that of most antibody-based assays so that it can handle a large number of samples that are generated from preclinical animal studies. PINP in rats may serve as a biomarker for bone formation activity, and this assay could be instrumental in studying bone physiology in rat experimental models.

Overexpression of Wnt10b from the osteocalcin promoter in transgenic mice increases postnatal bone mass. Increases in osteoblast perimeter, mineralizing surface, and bone formation rate without detectable changes in pre-osteoblast proliferation, osteoblast apoptosis, or osteoclast number and activity suggest that, in this animal model, Wnt10b primarily increases bone mass by stimulating osteoblastogenesis. Introduction: Wnt signaling regulates many aspects of development including postnatal accrual of bone. Potential mechanisms for how Wnt signaling increases bone mass include regulation of osteoblast and/or osteoclast number and activity. To help differentiate between these possibilities, we studied mice in which Wnt10b is expressed specifically in osteoblast lineage cells or in mice devoid of Wnt10b. Materials and Methods: Transgenic mice, in which mouse Wnt10b is expressed from the human osteocalcin promoter (Oc-Wnt10b), were generated in C57BL/6 mice. Transgene expression was evaluated by RNase protection assay. Quantitative assessment of bone variables was done by radiography, μCT, and static and dynamic histomorphometry. Mechanisms of bone homeostasis were evaluated with assays for BrdU, TUNEL, and TRACP5b activity, as well as serum levels of C-terminal telopeptide of type I collagen (CTX). The endogenous role of Wnt10b in bone was assessed by dynamic histomorphometry in Wnt10b−/− mice. Results: Oc-Wnt10b mice have increased mandibular bone and impaired eruption of incisors during postnatal development. Analyses of femoral distal metaphyses show significantly higher BMD, bone volume fraction, and trabecular number. Increased bone formation is caused by increases in number of osteoblasts per bone surface, rate of mineral apposition, and percent mineralizing surface. Although number of osteoclasts per bone surface is not altered, Oc-Wnt10b mice have increased total osteoclast activity because of higher bone mass. In Wnt10b−/− mice, changes in mineralizing variables and osteoblast perimeter in femoral distal metaphyses were not observed; however, bone formation rate is reduced because of decreased total bone volume and trabecular number. Conclusions: High bone mass in Oc-Wnt10b mice is primarily caused by increased osteoblastogenesis, with a minor contribution from elevated mineralizing activity of osteoblasts.

Serum PINP has emerged as a reliable marker of bone turnover in humans and is routinely used to monitor bone formation. However, the effects of PTH (1-34) on bone turnover have not been evaluated following short-term treatment. We present data demonstrating that PINP is an early serum biomarker in the rat for assessing bone anabolic activity in response to treatment with PTH (1-38). Rat serum PINP levels were found to increase following as few as 6 days of treatment with PTH (1-38) and these increases paralleled expression of genes associated with bone formation, as well as, later increases in BMD. Additionally, PINP levels were unaffected by treatment with an antiresorptive bisphosphonate. PINP may be used to detect PTH-induced early bone formation in the rat and may be more generally applicable for preclinical testing of potential bone anabolic drugs.

Bone microarchitecture measured at the iliac crest at 6 mo was confirmed to be a reasonable surrogate for, and a predictor of, architecture and strength of the femoral neck and lumbar vertebra after 18 mo of teriparatide treatment. However, the data taken together showed the importance of cortical bone volume for vertebra to assess pharmacological effects on bone quality. Introduction: Improvements in bone architecture with teriparatide treatment are suggested to contribute to fracture risk reduction in osteoporotic patients. Teriparatide significantly improves microarchitecture in the iliac crest of humans by stimulating bone modeling and remodeling processes that differ dramatically from those induced by antiresorptives. The relationship between improvements of bone microarchitecture and improvements of bone strength with teriparatide treatment has not yet been fully studied. Materials and Methods: Ovariectomized monkeys were administered vehicle (n = 20); teriparatide 1.0 μg/kg/d (n = 19); or teriparatide 5.0 μg/kg/d (n = 21) for 18 mo. Iliac crest biopsies were obtained at 6 and 15 mo after initiation of treatment. Animals were killed after 18 mo of treatment, and adjacent vertebrae or contralateral proximal femora were processed for biomechanical or histomorphometric analyses. Pearson correlation analyses were performed to assess the relationship between biomechanical and static histomorphometric parameters of lumbar vertebra, femoral neck, and iliac crest biopsies. Results: Static histomorphometric parameters of the 6- and 15-mo biopsies were significantly correlated with the vertebral and femoral neck parameters obtained at 18 mo of teriparatide treatment. Iliac crest biopsy parameters at 6 and 15 mo also correlated with vertebral and femoral neck strength at 18 mo. Static histomorphometry of the lumbar vertebra and femoral neck at 18 mo also significantly correlated with strength at these sites. However, cortical bone volume of the lumbar vertebrae had the strongest correlation with vertebral and femoral neck strength (r = 0.74 and 0.71, respectively). Conclusions: Teriparatide dose dependently improved cortical and trabecular microarchitecture of vertebra and femoral neck, as well as trabecular microarchitecture of the iliac crest. Bone microarchitecture at all sites was significantly correlated with lumbar vertebra and femoral neck strength. Cortical bone volume of vertebra had the strongest correlation with vertebral and femoral neck strength. Therefore, structural improvement seemed to be part of the mechanism for improved strength observed with teriparatide treatment. Trabecular bone architecture of the iliac crest at 6 mo also correlated with vertebral and femoral neck strength, as did femoral neck (cortical and trabecular) histomorphometry and trabecular histomorphometry of vertebra after 18 mo of treatment. Because clinical assessment of cortical bone volume is not readily possible for vertebra noninvasively, these findings confirm the importance of iliac crest biopsies to monitor skeletal health and show that biopsies are a reasonable surrogate to assess spine and femoral neck structure and function.

Parathyroid hormone (PTH) and glycogen synthase kinase-3 (GSK-3) inhibitor 603281-31-8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281-31-8 in rat osteopenia model. Female 6-month-old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1-38) at 10 microg/kg/day s.c. or 603281-31-8 at 3 mg/kg/day p.o. for 60 days. Twenty-four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P

Transiliac bone biopsies were obtained from 55 women treated with teriparatide or placebo for 12–24 months. We report direct evidence that modeling bone formation at quiescent surfaces was present only in teriparatide-treated patients and bone formation at remodeling sites was higher with teriparatide than placebo. Introduction: Recombinant teriparatide [human PTH(1-34)], a bone formation agent for the treatment of osteoporosis when given once daily subcutaneously, increases biochemical markers of bone turnover and activation frequency in histomorphometry studies. Materials and Methods: We studied the mechanisms underlying this bone-forming action of teriparatide at the basic multicellular unit by the appearance of cement lines, a method used to directly classify surfaces as modeling or remodeling osteons, and by the immunolocalization of IGF-I and IGF-II. Transiliac bone biopsies were obtained from 55 postmenopausal women treated with teriparatide 20 or 40 μg or placebo for 12–24 months (median, 19.8 months) in the Fracture Prevention Trial. Results: A dose-dependent relationship was observed in modeling and mixed remodeling/modeling trabecular hemiosteons. Trabecular and endosteal hemiosteon mean wall thicknesses were significantly higher in both teriparatide groups than in placebo. There was a dose-dependent relationship in IGF-II immunoreactive staining at all bone envelopes studied. The greater local IGF-II presence after treatment with teriparatide may play a key role in stimulating bone formation. Conclusions: Direct evidence is presented that 12–24 months of teriparatide treatment induced modeling bone formation at quiescent surfaces and resulted in greater bone formation at remodeling sites, relative to placebo.

Teriparatide, human PTH (1-34), a new therapy for osteoporosis, elicits markedly different skeletal responses depending on the treatment regimen. In order to understand potential mechanisms for this dichotomy, the present investigation utilized microarrays to delineate the genes and pathways that are regulated by intermittent (subcutaneous injection of 80 μg/kg/day) and continuous (subcutaneous infusion of 40 μg/kg/day by osmotic mini pump) PTH (1–34) for 1 week in 6-month-old female rats. The effect of each PTH regimen was confirmed by histomorphometric analysis of the proximal tibial metaphysis, and mRNA from the distal femoral metaphysis was analyzed using an Affymetrix microarray. Both PTH paradigms co-regulated 22 genes including known bone formation genes (i.e., collagens, osteocalcin, decorin, and osteonectin) and also uniquely modulated additional genes. Intermittent PTH regulated 19 additional genes while continuous treatment regulated 173 additional genes. This investigation details for the first time the broad profiling of the gene and pathway changes that occur in vivo following treatment of intermittent versus continuous PTH (1–34). These results extend previous observations of gene expression changes and reveal the in vivo regulation of BMP3 and multiple neuronal genes by PTH treatment. © 2005 Wiley-Liss, Inc.

Osteoprotegerin (OPG), a secreted member of the tumor necrosis factor receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Because OPG functions physiologically as a locally generated (paracrine) factor, we used high-throughput screening to identify small molecules that enhance the activity of the promoter of the human OPG gene. We found three structurally unrelated compounds that selectively increased OPG gene transcription, OPG mRNA levels, and OPG protein production and release by osteoblastic cells. Structural analysis of one compound, a benzamide derivative, led to the identification of four related molecules, which are also OPG inducers. The most potent of these compounds, Cmpd 5 inhibited osteoclast formation and parathyroid hormone-induced calvarial bone resorption. In vivo, Cmpd 5 completely blocked resorptive activity (serum calcium, osteoclast number) in parathyroid hormone-treated rats. Furthermore, Cmpd 5 reduced the ability of a rat breast cancer to metastasize to bone. Finally, the compound also prevented bone loss in a rat adjuvant arthritis model. These results provide proof of the concept that low molecular weight compounds can enhance OPG production in ways that can result in effective therapies.

The Cbfa1/Runx2 (referred to herein as Cbfa1) transcription factor has been shown to be essential for osteoblast differentiation and bone formation during embryogenesis. PTH given intermittently is a proven bone anabolic agent. Here, we investigated whether PTH regulates the expression and/or activity of Cbfa1 in osteoblastic cells and in a rat metatarsal organ culture assay. PTH was found to regulate Cbfa1 mRNA in the rat osteosarcoma cell line UMR106 in a concentration-dependent manner. The effect of PTH was mimicked by forskolin, an activator of adenylate cyclase leading to the protein kinase A pathway. PTH administered intermittently for 5 d in vivo was found to stimulate Cbfa1 protein in the rat proximal tibiae metaphysis. To demonstrate PTH regulation of Cbfa1 activity, a construct containing six tandem Cbfa1 binding elements fused to luciferase was shown to be rapidly stimulated in response to PTH. This stimulation preceded the effects on mRNA regulation and resulted from a protein kinase A-mediated increase in Cbfa1 activity. Finally, using a neonate rat metatarsal organ culture system, we demonstrated dose-dependent anabolic responsiveness to PTH and to Cbfa1 overexpression from an adenoviral construct. We further showed that Cbfa1 antisense oligonucleotides that blocked adenoviral Cbfa1-induced anabolic effects in this organ culture model also abolished the PTH-mediated anabolic increase. These findings suggest a requirement for Cbfa1 in mediating the anabolic effects of PTH. Thus, regulation of Cbfa1 expression or activity is an important mechanism by which PTH controls osteoblast function.

In the context of drug development programs, studies of the nonhuman primate (NHP) often play a prominent role in skeletal assessment. Many therapeutic agents with a mechanism of action involving the skeleton use the monkey for bone efficacy and safety assessments because of the similarities between the skeletons of NHPs and humans. This chapter provides scientists engaged in drug development programs an understanding of the unique role that the NHP can play in characterizing skeletal effects. Basic principles of skeletal structure and function are reviewed, with an emphasis on comparisons of the NHP skeleton to that of humans and rodents. A comprehensive set of methodologies to assess the NHP skeleton are available and are summarized in this chapter. These methods include bone densitometry, biomechanical testing, biomarkers of bone formation and resorption, histology, and histomorphometry. Practical aspects for implementing these measures in routine toxicity studies are briefly described in this chapter.

Long-term effects of a new selective estrogen receptor modulator (SERM) arzoxifene were examined in ovariectomized (OVX) rats. Arzoxifene was administered postoperatively (po) at 0.1 mg/kg per day or 0.5 mg/kg per day to 4-month-old rats, starting 1 week after OVX for 12 months. At study termination, body weights for arzoxifene groups were 16–17% lower than OVX control, which was caused by mainly reduced gain of fat mass. Longitudinal analysis of the proximal tibial metaphysis (PTM) by computed tomography (CT) at 0, 2, 4, 6, 9, and 12 months showed that OVX induced a 22% reduction in bone mineral density (BMD) at 2 months, which narrowed to a 12% difference between sham-operated (sham) and OVX rats by 12 months. Both doses of arzoxifene prevented the OVX-induced decline in BMD. Histomorphometry of the PTM showed that arzoxifene prevented bone loss by reducing osteoclast number in OVX rats. Arzoxifene maintained bone formation indices at sham levels and preserved trabecular number above OVX controls. Micro-CT analysis of lumbar vertebrae showed similar preservation of BMD compared with OVX, which were not different from sham. Compression testing of the vertebra and three-point bending testing of femoral shaft showed that strength and toughness were higher for arzoxifene-treated animals compared with OVX animals. Arzoxifene reduced serum cholesterol by 44–59% compared with OVX. Uteri wet weight from arzoxifene animals was 38–40% of sham compared with OVX rats, which were 29% of sham. Histology of the uterine endometrium showed that cell heights from both doses of arzoxifene were not significantly different from OVX controls. In summary, treatment of OVX rats with arzoxifene for nearly one-half of a lifetime maintained beneficial effects on cholesterol and the skeleton. These data suggest that arzoxifene may be a useful therapeutic agent for osteoporosis in postmenopausal women.

Continuous infusion of PTH in vivo results in active bone resorption. To investigate the molecular basis of the catabolic effect of PTH in vivo, we evaluated the role of OPG and RANKL, which are known to influence osteoclast formation and function. Weanling rats fed a calcium-free diet were parathyroidectomized and infused with PTH via an Alzet pump to examine: 1) the changes of serum-ionized calcium and osteoclast number, 2) the expression of OPG/RANKL mRNA and protein, and 3) the expression of osteoblast phenotype bone formation-associated genes such as osteoblast specific transcription factor, osteocalcin, bone sialoprotein, and type I collagen. PTH (1–38) (0.01–20 μg/100 g) continuous infusion for 1–24 h resulted in a dose-dependent increase in serum-ionized calcium in parathyroidectomized rats and a corresponding dose-dependent increase in osteoclast number, indicating an increased bone resorption. At 20 μg/100 g PTH dose level, serum-ionized calcium was 2.1-fold of the vehicle control and not different from the Sham-parathyroidectomized rats, and osteoclast number was 3-fold of the vehicle control and 1.7-fold of the Sham-parathyroidectomized rats. In the distal femur, RANKL mRNA expression was increased (27-fold) and OPG mRNA expression was decreased (4.6-fold). The changes in RANKL and OPG mRNA levels were rapid (as early as 1 h), dose dependent, and sustained over a 24-h period that was examined. Immunohistochemical evaluation of bone sections confirmed that OPG level was reduced in proximal tibial metaphysis upon PTH infusion. Circulating OPG protein level was also decreased by 32% when compared with the parathyroidectomized control. The expression of genes that mark the osteoblast phenotype was significantly decreased [osteoblast specific transcription factor (2.3-fold), osteocalcin (3-fold), bone sialoprotein (2.8-fold), and type I collagen (5-fold)]. These results suggest that the catabolic effect of PTH infusion in vivo in this well-established resorption model is associated with a reciprocal expression of OPG/RANKL and a co-ordinate decrease in the expression of bone formation-related genes. We propose that the rapid and sustained increase in RANKL and decrease in OPG initiate maintain and favor the cascade of events in the differentiation/recruitment and activation of osteoclasts.

A neonatal rat metatarsal organ culture model has been employed to study the anabolic effects of Sonic/Indian hedgehog and BMP-4. In this culture system, a significant increase in endochondral ossification is measured by an increase in length of mineralized bone, after daily treatment for 7 days with Sonic hedgehog protein (Shh-N). Previous evidence indicated that PTH related protein (PTHrP) is a critical target of hedgehog function in endochondral ossification. Using a PTHrP blocking antibody, it is shown that hedgehog mediates this activity via pathways other than through PTHrP. A dose-related increase in endochondral ossification is observed when metatarsals are treated with 25 ng/ml recombinant human bone morphogenetic protein 4 (BMP-4). However, this activity is not evident at higher doses of BMP-4 (200 ng/ml). High doses of BMP-4 resulted in increased expression of noggin messenger RNA and cotreatment of noggin and Shh-N resulted in reversal of the anabolic action of Shh-N. This observation suggests that BMP-4 signaling can influence the Shh-N mediated increase in endochondral ossification. Finally, we show that the Shh-N and BMP-4 mediated increase in endochondral ossification is reversed by treatment with antisense oligonucleotides targeted against Cbfa1. Thus, this report identifies Shh-N as an inducer of endochondral ossification that mediates its effect via BMP-4 and Cbfa1-dependent pathways.

STUDY DESIGN Controlled animal experiments. OBJECTIVE To test the dose and efficacy of teriparatide in a rat spinal fusion model. SUMMARY OF BACKGROUND DATA Teriparatide was shown to enhance spinal fusion in rats and rabbits previously, but the dose-dependent effect of teriparatide in spinal fusion in rats was not well characterized. METHODS A 0.5 × 0.5 cm trabecular bone graft was taken and implanted onto the L5 and L6 transverse processes of the same rat. Rats were randomly assigned into 3 groups: saline vehicle control (Vehicle), teriparatide 4 μg/kg per day (PTH4), and teriparatide 23 μg/kg per day (PTH23) subcutaneous injections for 4 weeks (5 d per wk). The L5-L6 spinal segments were harvested at week 4, and assessments included radiography, micro-computed tomography, manual palpation, and histomorphometry. L3 vertebra, femurs, and serum bone markers were examined. RESULTS The average radiographical score of L5-L6 fusion in Vehicle, PTH4, and PTH23 groups was 1.53, 2.87, and 4.11, respectively, with the PTH23 being significantly higher (P = 0.001 vs. Vehicle). The average micro-computed tomographic score of L5-L6 fusion in Vehicle, PTH4, and PTH23 groups was 1.53, 2.40, and 3.74, respectively (P = 0.001, PTH23 vs. Vehicle and PTH4). Manual palpation showed that fusion rate was 20%, 50%, and 67.7% in Vehicle, PTH4, and PTH23 groups, respectively. The bone mineralization apposition rate at the fusion site was significantly increased in a dose-dependent manner among the groups. Teriparatide significantly increased vertebral and femoral bone mineral density, bone mineral content, and trabecular area in a dose-dependent manner relative to Vehicle. No difference was found between the circulating Procollagen type I N-terminal propeptide and intact osteocalcin levels in the serum at 4 weeks after treatments. CONCLUSION Teriparatide at 23 μg/kg per day for 4 weeks showed anabolic skeletal effects and significantly enhanced spinal fusion rate in rats, whereas teriparatide at 4 μg/kg per day had also anabolic effects but did not significantly enhance spinal fusion rate. Higher doses of teriparatide may be needed to promote spinal fusion in short-term application.

Publisher Summary This chapter provides an understanding of the identification, synthesis, biological, and pharmacological activities of non-secosteroidal ligands and modulators that are capable of binding in the traditional ligand-binding pocket of vitamin D receptor (VDR) to manipulate its physiologic and homeostatic functions in the various target tissues where it is expressed. The two main reasons for pursuing the design and development of novel non-secosteroidal ligands include the need for new and effective pharmacological agents for human disease treatment as well as the identification of research tools to further elucidate the biological function of vitamin D and VDR. The unique structural features of nonsecosteroidal ligands may afford advantageous properties that are inherently different from the liabilities surrounding 1α,25(OH)2D3 or its secosteroidal analogs. These properties include altered DBP binding, reduced 24-hydroxylase metabolism, distinct VDR modulating behavior, or enhanced pharmacokinetic and pharmacodynamic profiles. Five classes of compounds that can be chemically classified as non-secosteroids include molecules based on a diarylmethane chemical scaffold (LG190119, VDRM-2), C/D ring-modified scaffolds, bis- and tris-aromatic triols (CD4420), podocarpic acid derivative (VDRL-1), and steroidal compounds such as the secondary bile acid lithocholic acid and its derivatives. The structure and chemistry of these molecules are described in the chapter.

Vitamin D(3) analogues were shown to be beneficial for osteoporosis and other indications, but their narrow therapeutic window between efficacy and hypercalcemia has limited their clinical utility. A nonsecosteroidal, tissue-selective, orally bioavailable, vitamin D receptor (VDR) ligand was ascertained to be efficacious in bone while having modest calcemic effects in vivo. This compound (VDRM2) potently induced Retinoid X Receptor alpha (RXR)-VDR heterodimerization (EC(50) = 7.1 +/- 1.6 nM) and induced osteocalcin promoter activity (EC(50) = 1.9 +/- 1.6 nM). VDRM2 was less potent in inducing Ca(2+) channel transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression (EC(50) = 37 +/- 12 nM). VDRM2 then was evaluated in osteopenic ovariectomized (OVX) rats and shown to dose-dependently restore vertebral bone mineral density (BMD) from OVX to sham levels at 0.08 microg/kg per day. Hypercalcemia was observed at a dose of 4.6 microg/kg per day of VDRM2, suggesting a safety margin of 57 [90% confidence interval (CI) 35-91]. 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D], ED71, and alfacalcidol restored BMD at 0.030, 0.0055, and 0.046 microg/kg per day, respectively, whereas hypercalcemia was observed at 0.22, 0.027, and 0.23 microg/kg per day, indicating a safety margin of 7.3, 4.9, and 5.0, respectively (90% CIs 4.1-13, 3.2-7.7, and 3.5-6.7, respectively). Histomorphometry showed that VDRM2 increased cortical bone area and stimulated the periosteal bone-formation rate relative to OVX at doses below the hypercalcemic dose. By contrast, ED71 increased the periosteal bone-formation rate only above the hypercalcemic dose. VDRM2 suppressed eroded surface on trabecular bone surfaces at normal serum calcium dosage levels, suggesting dual anabolic and antiresorptive activity. In summary, vitamin D analogues were more potent than VDRM2, but VDRM2 had a greater safety margin, suggesting possible therapeutic potential.

The level of increased bone formation after 24 months of treatment with teriparatide (rhPTH (1–34), TPTD) is similar in patients who were either treatment-naive (TN) or had lower bone turnover initially due to previous alendronate (ALN) therapy. Bone anabolic effects of TPTD in postmenopausal women with osteoporosis may be blunted during the initial phase after switching from ALN to TPTD. To explore the long-term implications, we examined histomorphometric and biochemical markers of bone turnover of patients on TPTD therapy after long-term ALN treatment. Paired biopsies were obtained after tetracycline double labeling at baseline and after 24 months of TPTD treatment from 29 ALN-pretreated (64.5 ± 16.4 months) and 16 TN patients. Biochemical markers were measured at baseline, during the treatment, or at study end. Compared with the baseline, after 24-month TPTD, activation frequency (Ac.F.) and osteoid surface (OS) increased in both groups: 0.11–0.34 cycles per year, 3.96–9.8% in the ALN-pretreated group and 0.19–0.33 cycles per year, 6.2–11.3% (p

Objective The purpose of this study was to use microarray technology to: (1) understand the early molecular events underlying the damage of articular cartilage initiated by this surgical procedure, and (2) determine whether these changes mimic those that are occurring in human osteoarthritic (OA) cartilage. Design Cartilage was harvested from both medial and lateral sides of the tibial plateaus and femoral condyles of both meniscal tear (MT) and sham surgery groups on days 3, 7 and 21 post-surgery. mRNA prepared from these rat cartilage samples was used for microarray analysis. Results Statistical analysis identified 475 genes that were differentially expressed between the sham and MT groups, at one or more of the time points that were analyzed. By integrating these genes with OA-related genes reported previously in a rat OA model and in human OA array studies, we identified 20 commonly changed genes. Six out of these 20 genes (Col5A1, Col6A2, INHBA, LTBP2, NBL1 and SERPINA1) were differentially expressed in two animal models and in human OA. Pathway analysis identified some key features of OA pathology, namely cartilage extracellular matrix remodeling, angiogenesis, and chondrocyte cell death that were recapitulated in the animal models. The rat models suggested increased inflammation and cholesterol metabolic pathways may play important role in early cartilage degeneration. Conclusion We identified a large number of differentially expressed genes in the articular cartilage of the MT model. While there was lack of overall identity in cartilage gene expression between the rat models and human OA, several key biological processes were recapitulated in the rat MT OA model.

PTH has been shown to enhance fracture repair; however, exactly when and where PTH acts in this process remains to be elucidated. Therefore, we conducted a longitudinal, region-specific analysis of bone regeneration in mature, osteopenic rats using a cortical defect model. Six-month-old rats were ovariectomized, and allowed to lose bone for 2 months, before being subjected to bilateral 2-mm circular defects in their femoral diaphyses. They were then treated for 5 wk with hPTH1–38 at doses of 0, 3, 10, or 30 μg/kg · d and scanned weekly by in vivo quantitative computed tomography. Quantitative computed tomography analyses showed temporal, dose-dependent increases in mineralization in the defects, intramedullary (IM) spaces, and whole diaphyses at the defect sites. Histomorphometry confirmed PTH stimulation of primarily woven bone in the defects and IM spaces, but not the periosteum. After necropsy, biomechanical testing identified an increase in strength at the highest PTH dose. Serum procollagen type I N-terminal propeptide concentration showed a transient increase due to drilling, but procollagen type I N-terminal propeptide also increased with PTH treatment, whereas tartrate-resistant acid phosphatase unexpectedly decreased. Analyses of lumber vertebra confirmed systemic efficacy of PTH at a nonfracture site. In summary, PTH dose dependently induced new bone formation within defects, at endocortical surfaces, and in IM spaces, resulting in faster and greater bone healing, as well as efficacy at other skeletal sites. The effects of PTH were kinetic, region specific, and most apparent at high doses that may not be entirely clinically relevant; therefore, clinical studies are necessary to clarify the therapeutic utility of PTH in bone healing.

UNLABELLED: GSK-3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK-3 inhibitor was evaluated in pre-osteoblasts and in osteopenic rats. GSK-3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats. INTRODUCTION: Inactivation of glycogen synthase kinase -3 (GSK-3) leads to stabilization, accumulation, and translocation of beta-catenin into the nucleus to activate downstream Wnt target genes. To examine whether GSK-3 directly regulates bone formation and mass we evaluated the effect of 603281-31-8, a small molecule GSK-3 alpha/beta dual inhibitor in preosteoblastic cells and in osteopenic rats. MATERIALS AND METHODS: Murine mesenchymal C3H10T1/2 cells were treated with GSK-3 inhibitor (603281-31-8) and assayed for beta-catenin levels, activity of Wnt-responsive promoter, expression of mRNA for bone formation, and adipogenic markers and alkaline phosphatase activity. In vivo, 6-month-old rats were ovariectomized (OVX), allowed to lose bone for 1 month, and treated with GSK-3 inhibitor at 3 mg/kg/day orally for 60 days. At the end of treatment, BMD was measured by DXA, bone formation rate by histomorphometry, vertebral strength (failure in compression), and the expression levels of osteoblast-related genes by real-time PCR. RESULTS: Treatment of C3H10T1/2 cells with the GSK-3 inhibitor increased the levels of beta-catenin accompanied by activation of Wnt-responsive TBE6-luciferase reporter gene. This was associated with an increased expression of mRNA for bone sialoprotein (1.4-fold), collagen alpha1 (I) (approximately 2-fold), osteocalcin (1.2-fold), collagen alpha1(V) (1.5-fold), alkaline phosphatase (approximately 160-fold), and runx2 (1.6-fold), markers of the osteoblast phenotype and bone formation activity. Alkaline phosphatase mRNA expression paralleled alkaline phosphatase activity. The mRNA levels of collagens alpha1 (I), alpha1 (V), biglycan, osteonectin, and runx-2 increased on treatment with the GSK-3 inhibitor in rat femur compared with the OVX control. DXA analyses revealed significant increases in BMC and BMD in cancellous and cortical bone of OVX rats treated with GSK-3 inhibitor. This was associated with increased strength (peak load, energy, and stiffness) assessed by lumbar vertebra load to failure in compression. Histomorphometric analyses showed that 603281-31-8 robustly increased bone formation but did not exclude a small effect on osteoclasts (resorption). CONCLUSIONS: An orally active, small molecule GSK-3 inhibitor induced osteoblast differentiation and increased markers of bone formation in vitro, and increased markers of bone formation, bone mass, and strength in vivo, consistent with a role for the canonical Wnt pathway in osteogenesis.

The pharmacological preservation of bone in the ovariectomized rat by estrogen, selective estrogen receptor modulators (SERMs), and bisphosphonates has been well described. However, comprehensive molecular analysis of the effects of these pharmacologically diverse antiresorptive agents on gene expression in bone has not been performed. This study used DNA microarrays to analyze RNA from the proximal femur metaphysis of sham and ovariectomized vehicle-treated rats, and ovariectomized rats treated for 35 days with maximally efficacious doses of 17-α ethinyl estradiol, the benzothiophene SERM, raloxifene, the benzopyran SERM, ( S )-3-(4-hydroxyphenyl)-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2 H -1-benzopyran-7-ol (EM652), and the aminobisphosphonate, alendronate. Ovariectomy resulted in 644 significant probe set changes relative to sham control rats ( p < 0.05), whereas E2, raloxifene, EM652, and alendronate regulated 613, 765, 652, and 737 probe sets, respectively, relative to ovariectomized control rats. An intersection of these data sets yielded 334 unique genes that were altered after ovariectomy and additionally changed by one or more antiresorptive treatment. Clustering analysis showed that the transcript profile was distinctly different for each pharmaceutical agent and that raloxifene maintained more genes at sham levels than any other treatment. In addition, E2 and alendronate suppressed a cluster of genes associated with bone formation activity below that of sham, whereas raloxifene had little effect on these genes. These data indicate stronger suppressive effects of E2 and alendronate on bone formation activity and that ovariectomy plus raloxifene resembles sham more closely than ovariectomized animals treated with E2, EM652, or alendronate.

The skeletal efficacy of raloxifene (Ral) plus weekly teriparatide [recombinant human parathyroid hormone (1-34), TPTD] combinations relative to each treatment alone or sequentially were evaluated in osteopenic, ovariectomized rats. In the first study, 6-month-old Sprague-Dawley rats were ovariectomized (Ovx) and permitted to lose bone for 1 month before treatment for the following 3 months. Raloxifene (Ral, 1 mg/kg/day orally) was evaluated alone and in combination with TPTD (10 or 30 microg/kg/week) administered weekly by subcutaneous injection. QCT, biomechanical testing, and histomorphometry were used to quantitate skeletal effects. Weekly TPTD alone at either dose had no skeletal effect relative to Ovx. Daily Ral prevented further loss of vertebral bone mineral density (BMD), resulting in BMD that was significantly greater than Ovx, but significantly less than age-matched, sham-Ovx, vehicle controls (sham). The raloxifene plus 30 microg/kg/week TPTD group had vertebral BMD that was significantly greater than Ovx, Ral alone, and both TPTD dose-alone groups. Therefore, the Ral plus TPTD group completely restored bone mass to sham levels. Compression testing of lumbar vertebra L5 confirmed increased strength for both Ral plus TPTD combinations relative to Ovx, with strength not different from sham. Histomorphometry of the proximal tibial metaphysis showed that Ovx significantly increased eroded surface and bone formation compared to sham. Raloxifene treatment restored eroded surface and bone formation rate back to sham levels. Raloxifene plus TPTD at 30 microg/kg/week resulted in a significantly higher mineral appositional rate compared to Ral and sham, which was not different from Ovx and TPTD alone. Raloxifene plus TPTD at both doses had eroded surfaces that were significantly less than Ovx but not different from sham or Ral alone. In a sequential study, 6-month-old Ovx rats were permitted to develop osteopenia for 2 months before a daily TPTD 80 microg/kg/day subcutaneous injection was initiated. Following 2 months of TPTD treatment, animals were either (1) continued on TPTD, (2) discontinued from TPTD, (3) switched to Ral 3 mg/kg/day, oral, or 17 alpha-ethynyl estradiol (EE2) 0.1 mg/kg/day, oral, for another 2 months. Raloxifene and EE2 maintained most of TPTD-induced new bone in Ovx rats by preventing the increase in bone turnover rate after withdrawal of TPTD. Raloxifene also restored the elevated bone formation activity induced by TPTD to the level of sham. These data suggest that Ral and TPTD have complementary interactions in osteopenic, Ovx rats. Raloxifene inhibited bone resorption, and reduced high bone turnover without significantly retarding TPTD stimulation of bone formation activity.

OVX monkeys treated for 18 months with 1 or 5 g/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 g/kg/day teriparatide (parathyroid hormone (1-34); PTH1), or 5 g/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 CT, 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. J Bone Miner Res 2004;19:623- 629. Published online on January 12, 2004; doi: 10.1359/JBMR.040112

With the ready availability of several osteoporosis therapies, teriparatide [human PTH-(1-34)] is likely to be prescribed to postmenopausal women with prior exposure to agents that prevent bone loss, such as bisphosphonates, estrogen, or selective estrogen receptor modulators. Therefore, we evaluated the ability of once daily teriparatide to induce bone formation in ovariectomized (Ovx) rats with extended prior exposure to various antiresorptive agents, such as alendronate (ABP), 17 alpha-ethinyl estradiol (EE), or raloxifene (Ral). Sprague Dawley rats were Ovx and treated with ABP (28 microg/kg, twice weekly), EE (0.1 mg/kg per d), or Ral (1 mg/kg per d) for 10 months before switching to teriparatide 30 microg/kg per d for another 2 months. Analysis of the proximal tibial metaphysis showed that all three antiresorptive agents prevented ovariectomy-induced bone loss after 10 months, but were mechanistically distinct, as shown by histomorphometry. Before teriparatide treatment, ABP strongly suppressed activation frequency and bone formation rate to below levels in other treatment groups, whereas these parameters were not different from sham values for EE or Ral. Trabecular area for ABP, EE, and Ral were greater than that in Ovx controls. However, the trabecular bone effects of ABP were attributed not only to effects on the secondary spongiosa, but also to the preservation of primary spongiosa, which was prevented from remodeling. After 2 months of teriparatide treatment, lumbar vertebra showed relative bone mineral density increases of 18%, 7%, 11%, and 10% for vehicle/teriparatide, ABP/teriparatide, EE/teriparatide, and Ral/teriparatide, respectively, compared with 10 month levels. Histomorphometry showed that trabecular area was increased by 105%, 113%, 36%, and 48% for vehicle/teriparatide, ABP/teriparatide, EE/teriparatide, and Ral/teriparatide, respectively, compared with 10 month levels. Teriparatide enhanced mineralizing surface, mineral apposition rate, and bone formation rate in all groups. Compression testing of vertebra showed that teriparatide improved strength (peak load) and toughness in all groups to a proportionately similar extent compared with 10 month levels. These data showed a surprising ability of the rat skeleton to respond to teriparatide despite extensive pretreatment with ABP, EE, or Ral. Therefore, the mature skeleton of Ovx rats remains highly responsive to the appositional effects of teriparatide regardless of pretreatment status in terms of cancellous bone area or rate of bone turnover.

We report the consequences of prolonged treatment with recombinant human parathyroid hormone (1-34) (PTH) in male and ovariectomized female rats with mature skeletons. Intact male and osteopenic, ovariectomized, female F-344 rats were evaluated after 1 year of treatment with 0, 8, or 40 microg/kg/day s.c. PTH. Males and females were about 6 months of age at study initiation; females were ovariectomized (Ovx) for 5 weeks before initiation of PTH treatment. PTH did not affect the survival of either intact males or ovariectomized females. Qualitative histopathology showed expected changes associated with aging in kidneys and proximal tibiae, with no treatment-related anomalies after 1 year of PTH administration. PTH slightly increased the femoral length of ovariectomized females but not that of males. No significant differences in femoral length were observed between sham and Ovx controls. Proximal femora of the males and ovariectomized females given the high dose of 40 microg/kg showed 211 and 186% greater trabecular bone area, 118 and 94% greater cortical thickness, 170 and 189% greater trabecular number, and 321 and 404% greater connectivity (node-to-node struts) compared with respective vehicle controls. Increased trabecular and endocortical surface apposition coincided with a 78 and 70% loss of marrow space for males and females treated with PTH, respectively. Biomechanical strength (ultimate load) of the femoral neck increased by 73 and 76%, respectively, in males and ovariectomized females. Cortical bone analyses of the femoral midshaft showed 105 and 72% increases in bone mineral content, 67 and 55% increases in bone mineral density, and 22 and 10% increases in cross-sectional area for males and ovariectomized females, respectively, with altered shape of femora. Biomechanical analyses of the midshaft showed substantial increases in strength and stiffness but a reduction in ultimate strain, which was likely due to the altered geometry of the midshaft for PTH groups. Aging effects on strength of vertebra and femoral midshaft were reversed by PTH treatment. In summary, the 1-year treatment duration, which represents about 50% of lifetime, did not affect survival and was not associated with any treatment-related anomalies in the kidney or skeleton. PTH reversed the aging process in bones but not kidneys and substantially increased bone mass and strength to well beyond normally attained levels. However, compared with short-term studies reported previously, there seemed to be no advantages to extending PTH treatment to 12 months in rat bones.