Frequency of Change in Assessment from Bony Union to Nonunion after Lumbar Interbody Fusion: A Multicenter Study.

INTRODUCTION: Assessments of early postoperative bony union after posterior lumbar interbody fusion via computed tomography (CT) have revealed cases in which interbody fixation by bony union resulted in nonfusion due to bone absorption. The apparent bone union state reverted to a nonunion state several months later, exhibiting a so-called "fake union" phenomenon. Additionally, few reports have evaluated the effect of teriparatide on bony union. The present study aimed to evaluate the frequency of change in assessment from fusion to nonfusion in the postoperative follow-up of lumbar interbody fusion, compare the late postoperative bony union rates in groups with or without early postoperative fusion, and examine the effect of postoperative teriparatide in those groups. METHODS: Sixty-nine subjects enrolled from multiple hospitals were prospectively evaluated following single-level lumbar interbody fusion. The patients were randomly allocated into treatment with or without weekly postoperative teriparatide. The subjects were then classified as having bony union or nonfusion at 2 months postoperatively, and fusion rates at 6 months were compared. For the evaluation of bony union, blinded radiological examinations were performed via CT. Additional comparisons were made according to teriparatide use. RESULTS: The rate of nonunion at 6 months postoperatively in patients with fusion at 2 months postoperatively was 27.8%. Among subjects with bony union at 2 months postoperatively, the fusion rate at 6 months in those who received teriparatide was 93.3% (p=0.027) versus 57.1% in those who did not. CONCLUSIONS: The rate of nonunion at 6 months postoperatively in patients exhibiting union at 2 months after surgery was 27.8%. Postoperative weekly teriparatide treatment significantly reduced the rate of fake union.

Teriparatide Improves Bone and Lipid Metabolism in a Male Rat Model of Type 2 Diabetes Mellitus.

Osteoporosis is a complication of diabetes mellitus (DM). The pathology of diabetic osteoporosis is distinct from postmenopausal osteoporosis, and there are no specific treatment guidelines for diabetic osteoporosis. In the current study, this issue was addressed by evaluating the effect of osteoporosis medications, such as the anabolic agent PTH [teriparatide (TPTD)] and the antiresorptive agents calcitonin [elcatonin (ECT)] and bisphosphonate [risedronate (RIS)], on bone metabolism as well as on glucose and lipid metabolism in spontaneously diabetic Torii (SDT) fatty rats, which are a model of type 2 DM (T2DM). The medicines were injected subcutaneously into 8-week-old male SDT fatty rats three times weekly for 8 weeks. TPTD treatment in SDT fatty rats increased the osteoblast number and function on trabecular bone in vertebrae, and increased the trabecular bone mass, bone mineral density (BMD), and mechanical strength of vertebrae. Additionally, TPTD improved cortical bone structure and increased BMD. RIS decreased the osteoclast number and function, which led to an increase in vertebral bone mineral content and BMD in the femoral diaphysis, and mechanical strength was increased in the vertebrae. ECT showed no clear effects on bone mass or metabolism. Similar to diabetic lesions, all of the drugs had no effects on hyperglycemia, pancreas morphology, or serum insulin and glucagon levels. However, triglyceride levels and lipid droplets in fatty liver were decreased in the TPTD group. These results suggest that TPTD may be useful for treating fatty liver in addition to osteoporosis in T2DM.

Parathyroid Hormone Shifts Cell Fate of a Leptin Receptor-Marked Stromal Population from Adipogenic to Osteoblastic Lineage.

Intermittent parathyroid hormone (iPTH) treatment induces bone anabolic effects that result in the recovery of osteoporotic bone loss. Human PTH is usually given to osteoporotic patients because it induces osteoblastogenesis. However, the mechanism by which PTH stimulates the expansion of stromal cell populations and their maturation toward the osteoblastic cell lineage has not be elucidated. Mouse genetic lineage tracing revealed that iPTH treatment induced osteoblastic differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs), which carried the leptin receptor (LepR)-Cre. Although these findings suggested that part of the PTH-induced bone anabolic action is exerted because of osteoblastic commitment of MSPCs, little is known about the in vivo mechanistic details of these processes. Here, we showed that LepR+ MSPCs differentiated into type I collagen (Col1)+ mature osteoblasts in response to iPTH treatment. Along with osteoblastogenesis, the number of Col1+ mature osteoblasts increased around the bone surface, although most of them were characterized as quiescent cells. However, the number of LepR-Cre-marked lineage cells in a proliferative state also increased in the vicinity of bone tissue after iPTH treatment. The expression levels of SP7/osterix (Osx) and Col1, which are markers for osteoblasts, were also increased in the LepR+ MSPCs population in response to iPTH treatment. In contrast, the expression levels of Cebpb, Pparg, and Zfp467, which are adipocyte markers, decreased in this population. Consistent with these results, iPTH treatment inhibited 5-fluorouracil- or ovariectomy (OVX)-induced LepR+ MSPC-derived adipogenesis in BM and increased LepR+ MSPC-derived osteoblasts, even under the adipocyte-induced conditions. Treatment of OVX rats with iPTH significantly affected the osteoporotic bone tissue and expansion of the BM adipose tissue. These results indicated that iPTH treatment induced transient proliferation of the LepR+ MSPCs and skewed their lineage differentiation from adipocytes toward osteoblasts, resulting in an expanded, quiescent, and mature osteoblast population. © 2019 American Society for Bone and Mineral Research.

Weekly Teriparatide Administration and Preoperative Anterior Slippage of the Cranial Vertebra Next to Fusion Segment < 2 mm Promote Osseous Union After Posterior Lumbar Interbody Fusion.

STUDY DESIGN: A multicenter case-control study. OBJECTIVE: The aim of this study was to investigate the independent predictors of osseous union after posterior lumbar interbody fusion (PLIF). SUMMARY OF BACKGROUND DATA: PLIF is usually performed to treat lumbar degenerative diseases in elderly patients. Some patients exhibit intervertebral pseudoarthrosis. METHODS: We analyzed 66 elderly patients with osteoporosis who underwent PLIF from 2011 to 2014 (all women, mean age 71 years, follow-up period ≥6 months). Patients were randomly allocated to receive either treatment with weekly teriparatide, starting at 1 week postoperatively, or no teriparatide. Preoperative lumbar spine radiographs were obtained, and the amount of anterior slippage was measured. Osseous union was assessed by computed tomography at 6 months postoperatively. RESULTS: Thirty-three patients (50%) showed complete osseous union, while 33 did not. Teriparatide was administered in 20 (61%) patients of the union group and in 9 (27%) patients of the nonunion group (P < 0.01). The preoperative anterior slippage of the cranial vertebra next to fusion segment < 2 mm was observed in 16 (49%) and 4 (12%) patients in the union and nonunion groups, respectively (P < 0.01). Multivariate regression analysis showed that teriparatide administration (odds ratio, 4.75; 95% confidence interval: 1.51-14.90; P < 0.01) and preoperative anterior slippage of the cranial vertebra next to fusion segment < 2 mm (odds ratio, 5.90; 95% confidence interval: 1.53-22.70; P < 0.01) were independently associated with osseous union within 6 months after PLIF. At 6 months postoperatively, the mean femoral neck bone mineral density significantly increased by 1.1% in the union group and decreased by 1.3% in the nonunion group (P < 0.05). CONCLUSION: Weekly teriparatide administration and preoperative anterior slippage of the cranial vertebra next to fusion segment < 2 mm were independent predictors of osseous union within 6 months after PLIF. Our findings suggest that biological and mechanical factors may influence the improvement of spinal fusion. LEVEL OF EVIDENCE: 4.

Enhancement of ghrelin-signaling system by Rikkunshi-To attenuates teriparatide-induced pica in rats.

Teriparatide is clinically used for the treatment of osteoporosis; however, nausea is often observed in patients. Its insufficient control affects the ability to continue teriparatide therapy. Rikkunshi-To (RKT), a traditional Japanese herbal medicine, improves the gastrointestinal function via activation of the ghrelin-signaling system. We investigated the therapeutic effects of RKT on teriparatide-induced nausea in rats and the involvement of ghrelin in these effects. We previously reported that ovariectomized rats showed pica (kaolin ingestion), a behavior that can be used to assess nausea in rats, after the subcutaneous administration of teriparatide; thus, the behavior was used as an index of nausea. Ovariectomized rats were fed diets with or without RKT (1%) for 2 weeks, and then they received the subcutaneous injection of teriparatide (400 µg/kg). Teriparatide significantly increased the incidence of pica, while suppressing intestinal motility and plasma ghrelin levels in rats fed normal diets; however, rats fed diets with RKT showed improvements in all of the teriparatide-induced adverse reactions. These therapeutic effects were antagonized by a ghrelin receptor antagonist ([D-Lys3]-GHRP-6; 200 nmol/rat). These findings suggest that the enhancement of ghrelin-signaling is involved in RKT's therapeutic effect, and that RKT is a potentially useful treatment for teriparatide-induced nausea.

Direct cell-cell contact between mature osteoblasts and osteoclasts dynamically controls their functions in vivo.

Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial-temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell-cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell-cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell-cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.

Administration frequency as well as dosage of PTH are associated with development of cortical porosity in ovariectomized rats.

To investigate whether the administration frequency of parathyroid hormone (PTH) is associated with the development of cortical porosity, this study established 15 dosage regimens of teriparatide [human PTH(1-34), TPTD] with four distinct concentrations and four distinct administration frequencies of TPTD to 16-week-old ovariectomized rats. Our analyses demonstrated that the bone mineral density, mechanical properties, and bone turnover were associated with the total amount of TPTD administered. Our observations further revealed that the cortical porosity was markedly developed as a result of an increased administration frequency with a lower concentration of total TPTD administration in our setting, although the highest concentration also induced cortical porosity. Deconvolution fluorescence tiling imaging on calcein-labeled undecalcified bone sections also demonstrated the development of cortical porosity to be closely associated with the bone site where periosteal bone formation took place. This site-specific cortical porosity involved intracortical bone resorption and an increased number and proximity of osteocytic lacunae, occasionally causing fused lacunae. Taken together, these findings suggested the involvement of local distinctions in the rate of bone growth that may be related to the site-specific mechanical properties in the development of cortical porosity induced by frequent and/or high doses of TPTD.

Acute development of cortical porosity and endosteal naïve bone formation from the daily but not weekly short-term administration of PTH in rabbit.

Teriparatide [human parathyroid hormone (1-34)], which exerts an anabolic effect on bone, is used for the treatment of osteoporosis in patients who are at a high risk for fracture. That the once-daily administration of teriparatide causes an increase in cortical porosity in animal models and clinical studies has been a matter of concern. However, it is not well documented that the frequency of administration and/or the total dose of teriparatide affect the cortical porosity. The present study developed 4 teriparatide regimens [20 µg/kg/day (D20), 40 µg/kg/day (D40), 140 µg/kg/week (W140) and 280 µg/kg/week (W280)] in the rabbit as a model animal with a well-developed Haversian system and osteons. The total weekly doses were equivalent in the low-dose groups (D20 and W140) and in the high-dose groups (D40 and W280). After the short-term (1 month) administration of TPDT, micro-CT, histomorphometry and three-dimensional second harmonic generation (3D-SHG) imaging to visualize the bone collagen demonstrated that daily regimens but not weekly regimens were associated with the significant development of cortical porosity and endosteal naïve bone formation by marrow fibrosis. We concomitantly monitored the pharmacokinetics of the plasma teriparatide levels as well as the temporal changes in markers of bone formation and resorption. The analyses in the present study suggested that the daily repeated administration of teriparatide causes more deleterious changes in the cortical microarchitecture than the less frequent administration of higher doses. The findings of the present study may have some implications for use of teriparatide in clinical treatment.

Role of Weekly Teriparatide Administration in Osseous Union Enhancement within Six Months After Posterior or Transforaminal Lumbar Interbody Fusion for Osteoporosis-Associated Lumbar Degenerative Disorders: A Multicenter, Prospective Randomized Study.

BACKGROUND: For elderly patients, posterior lumbar interbody fusion (PLIF) or transforaminal lumbar interbody fusion (TLIF) is usually performed to treat lumbar degenerative diseases. However, some patients exhibit pseudarthrosis following such procedures. The anabolic agent teriparatide is an approved treatment for promoting bone formation in osteoporotic patients. Our multicenter, prospective randomized study assessed the role of once-weekly teriparatide administration on patient outcomes following interbody fusion. METHODS: Patients were females who were ≥50 years of age, had a bone mineral density (BMD) of <80% of the sex-matched young adult mean and/or previous spinal compression or femoral fractures, and had lumbar degenerative disease. Patients were randomly allocated to receive either weekly teriparatide, administered subcutaneously starting at week 1, for 6 months postoperatively (the teriparatide arm), or no teriparatide (the control arm). Blinded radiographic evaluations were performed using dynamic radiography and computed tomography (CT) and assessed by modified intention-to-treat analysis and per-protocol analysis. Clinical and neurological symptoms were evaluated using the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOA-BPEQ) and the Oswestry Disability Index (ODI). RESULTS: Seventy-five patients were randomized to treatment, and 66 patients completed treatment. At 4 months postoperatively, bone fusion in the 2 center CT slices was significantly higher in the teriparatide arm compared with the control arm in the age-adjusted modified intention-to-treat analysis and was significantly higher at 6 months in the per-protocol analysis. Radiographic examinations showed no disc-space narrowing and no intervertebral disc instability. JOA-BPEQ and ODI results were improved postoperatively in both treatment arms. CONCLUSIONS: Weekly administration of teriparatide promoted bone formation at the surgical fusion site and decreased bone resorption, as indicated by bone metabolic marker results, within the early postoperative period. Our findings suggest that combining lumbar interbody fusion and teriparatide treatment may be an effective option for managing lumbar degenerative disease in elderly patients. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Increased cortical porosity is associated with daily, not weekly, administration of equivalent doses of teriparatide.

INTRODUCTION: The pharmacokinetic profile of parathyroid hormone (PTH) determines its effects on bone resorption and formation. When administered intermittently, anabolic effects are favored in comparison with the continuous treatment. Among the intermittent treatment regimens, lower frequency of administration may have a lower effect on bone remodeling. We therefore hypothesized that weekly administration of teriparatide will produce less increase in intracortical remodeling and porosity than reported using daily treatment. METHODS: We treated 17 female New Zealand white rabbits aged 6months for 1month with teriparatide [human PTH(1-34)] as follows. (i) Vehicle-treated Control (n=4); (ii) 20µg/kg daily (n=3); (iii) 40µg/kg daily (n=3); (iv) 140µg/kg weekly (n=3); (v) 280µg/kg weekly (n=4). Proximal femurs were imaged ex vivo using micro-CT (Scanco Viva CT-40) at 15µmvoxel size. Areas, pore size, and porosity were analyzed on the total, compact cortex (CC), and transitional zones in a 10mm length region of interest (ROI) starting at the midshaft using StrAx1.0. RESULTS: Compared to controls, the 20µg/kg daily was associated with 3.0% higher porosity in the transitional zone (p=0.09) while the 40µg/kg daily was associated with a higher porosity in the cortex (8.7%; p=0.04) and in the transitional zone (5.7%; p=0.007). The daily regimens were also associated with a greater proportion of porosity due to pores >15µm2; particularly in the transitional zone where 20 and 40µg/kg daily increased porosity 2 fold (p=0.06) and 5 fold (p=0.04) relative controls respectively. The 140 and 280µg/kg weekly were not associated with an increase in porosity. There was no difference in total, compact or transitional zone cross sectional areas between the groups. CONCLUSION: Effects of intermittent teriparatide depend on the dose and frequency of administration. Daily dosing, particularly the higher dose, but not weekly dosing, increased cortical porosity. Work is needed to investigate the effects of the regimens on bone formation.

Frequency of Teriparatide Administration Affects the Histological Pattern of Bone Formation in Young Adult Male Mice.

Evidence supports that daily and once-weekly administration of teriparatide, human (h)PTH(1-34), enhance bone mass in osteoporotic patients. However, it is uncertain whether different frequencies of hPTH(1-34) administration would induce bone formation similarly in terms of quantity and quality. To investigate that issue, mice were subjected to different frequencies of PTH administration, and their bones were histologically examined. Frequencies of administration were 1 time/2 days, 1 time a day, and 2 and 4 times a day. Mice were allocated to either to control or to 3 different dosing regimens: 80 µg/kg of hPTH(1-34) per injection (80 µg/kg per dose), 80 µg/kg of hPTH(1-34) per day (80 µg/kg · d), or 20 µg/kg of hPTH(1-34) per day (20 µg/kg · d). With the regimens of 80 µg/kg per dose and 80 µg/kg · d, high-frequency hPTH(1-34) administration increased metaphyseal trabecular number. However, 4 doses per day induced the formation of thin trabeculae, whereas the daily PTH regimen resulted in thicker trabeculae. A similar pattern was observed with the lower daily hPTH(1-34) dose (20 µg/kg · d): more frequent PTH administration led to the formation of thin trabeculae, showing a thick preosteoblastic cell layer, several osteoclasts, and scalloped cement lines that indicated accelerated bone remodeling. On the other hand, low-frequency PTH administration induced new bone with mature osteoblasts lying on mildly convex surfaces representative of arrest lines, which suggests minimodeling-based bone formation. Thus, high-frequency PTH administration seems to increase bone mass rapidly by forming thin trabeculae through accelerated bone remodeling. Alternatively, low-frequency PTH administration leads to the formation of thicker trabeculae through bone remodeling and minimodeling.

Differences in vertebral, tibial, and iliac cancellous bone metabolism in ovariectomized rats.

Bone histomorphometry is usually performed on the iliac bone in humans and the tibia or vertebrae in rats. Bone metabolism differences among skeletal sites may be problematic when translating experimental results from rats to humans, but data on such differences in rats are lacking. Therefore, we examined the differences in bone structure and metabolism among skeletal sites using the lumbar vertebra (LV), tibia, and iliac bone obtained from ovariectomized or sham-operated rats preoperatively and at various times from 3 days to 26 weeks postoperatively. The trabeculae were thicker in the LV, where bone metabolism was less active than at other sites, and numerous fine trabeculae were observed in the tibia, where bone metabolism was more active. The iliac bone structure and metabolism were intermediate between those of the tibia and LV. Ovariectomy induced lower bone volume and higher bone metabolism in all skeletal sites, but the changes were greatest and occurred earliest in the tibia, followed by the iliac bone and then LV. Ovariectomy caused changes in bone metabolic markers, which occurred earlier than those in bone tissue. Activation frequency (Ac.f) increased after ovariectomy. At week 26 in ovariectomized rats, Ac.f was highest in the tibia (3.13 N/year) but similar between iliac bone (0.87 N/year) and LV (1.39 N/year). Ac.f is reportedly 0.3-0.4 N/year in the iliac bone of postmenopausal women, suggesting that bone turnover in rats is several times higher than in humans. The reference values reported here are useful for translating experimental results from rats to humans.

Comparison of treatment effects of teriparatide and the bisphosphonate risedronate in an aged, osteopenic, ovariectomized rat model under various clinical conditions.

Teriparatide and bisphosphonates are osteoporosis medications that increase bone mineral density (BMD) and prevent fracture, but each has a different mechanism of action. Teriparatide promotes bone formation, while bisphosphonates suppress bone resorption. In the clinical setting, however, drug selection is not always tailored to the particular clinical condition of the patient or mechanism of action of the drug. We compared the effects of teriparatide and the bisphosphonate risedronate on bone metabolism using two ovariectomized rat models to elucidate the optimal use of these two drugs in the clinical setting. We first performed dose-finding experiments to determine the equivalent effective doses of each drug (5.6 and 3.0 µg/kg for teriparatide and risedronate, respectively). We then compared the effects of these doses on bone metabolism after subcutaneous administration three times weekly for 4 months starting either the day after ovariectomy (preventive study) or 12 months after ovariectomy (therapeutic study). The increase in proximal tibial BMD under the physical conditions that increased bone turnover at 1 to 2 months after ovariectomy was greater in the risedronate group than in the teriparatide group. In contrast, the increases in lumbar vertebral BMD and bone strength under the physical conditions that significantly decreased BMD and bone strength at 12 months after ovariectomy were greater in the teriparatide group than in the risedronate group. The present study provides important information on the selection of antiosteoporotic drugs, including teriparatide and risedronate, in treatment protocols tailored to the clinical conditions of patients and drug mechanisms.

Early Effects of Single and Low-Frequency Repeated Administration of Teriparatide, hPTH(1-34), on Bone Formation and Resorption in Ovariectomized Rats.

Intermittent repeated administration of teriparatide (TPTD) has potent anabolic effects on bones in vivo. However, TPTD has both anabolic and catabolic effects on osteoblasts in vitro, and the mechanisms underlying its promotion of bone formation are unclear. This study aimed to elucidate the time-dependent changes in bone formation and resorption by examining changes in bone turnover markers and bone tissue over time after TPTD administration with low frequency in ovariectomized rats. The amount of serum osteocalcin, a bone formation marker, was transiently reduced after single TPTD administration, but increased thereafter, remaining increased for several days. In contrast, the amount of excreted urinary C-telopeptide, a bone resorption marker, increased transiently after single TPTD administration, and subsequently returned to control levels on the day after administration. Tissue histomorphometric analyses conducted 8 h after administration showed no changes in bone formation or bone resorption parameters. However, at 48 h, the bone formation parameters OS/BS and Ob.S/BS were increased, while the bone resorption parameter ES/BS was decreased. After repeated TPTD administration for 4 weeks, OS/BS, Ob.S/BS, and MS/BS increased, while Oc.S/BS decreased. Serum osteocalcin at 4 weeks after repeated administration was significantly correlated with OS/BS and Ob.S/BS. These present findings indicate that TPTD has dual, time-dependent effects on bone resorption and bone formation. Immediately after single administration, there was transient promotion of bone resorption and suppression of bone formation. However, sustained stimulation of bone formation occurred thereafter. Furthermore, these data suggest that this sustained bone formation led to anabolic effects with repeated TPTD administration.

Induction and antagonism of pica induced by teriparatide in rats.

Intermittent subcutaneous injection of teriparatide, an active fragment of human parathyroid hormone, is clinically used for the treatment of osteoporosis. Patients suffer from nausea, which is one of the side effects teriparatide induces; however, the etiology of teriparatide-induced nausea remains unknown. We have reported pica, kaolin ingestion behavior, can be used as an assessment of nausea-related response in rats. In this study, we investigated the characteristics of teriparatide-induced pica and the abilities of anti-emetic drugs to inhibit teriparatide-induced pica. Male and female adolescent (4-week-old), young (8-week-old), and adult (30-week-old) naive rats, and ovariectomized (OVX: 17-week-old) and sham-operated (17-week-old) rats subcutaneously received teriparatide (0.4 mg/kg, n=4), and their kaolin and food intakes were monitored for 24 h after the injection. Among the tested rats, we found that OVX rats, rather than male, female, and sham-operated rats, showed marked teriparatide-induced pica (0 mg/kg: 0.17±0.07 g, 0.4 mg/kg: 6.18±0.91 g). Teriparatide-induced pica in OVX rats was inhibited by intraperitoneal pretreatment with serotonin 5-HT3 (granisetron 0.5 mg/kg), dopamine D2 (prochlorperazine 0.5 mg/kg), neurokinin NK1 (fosaprepitant 1 mg/kg), and histamine H1 (diphenhydramine 10 mg/kg) receptor antagonists to 70%, 11%, 19%, and 59% of that in vehicle-treated control, respectively. These results suggest that teriparatide-induced pica in OVX rats has the potential to reflect teriparatide-induced nausea; 5-HT3, D2, NK1, and H1 receptor activation is involved in the development of this behavior; antagonists of these receptors have the potential to be medical candidates used as treatments for teriparatide-induced nausea in human patients.

Three-times-weekly administration of teriparatide improves vertebral and peripheral bone density, microarchitecture, and mechanical properties without accelerating bone resorption in ovariectomized rats.

Daily and weekly administration of teriparatide (PTH1-34) reduces the risk of osteoporotic bone fractures. However, their effects on markers of bone formation and bone resorption differ. These results indicate that the dosing frequency of teriparatide may affect bone metabolism and bone structure, with different effects on bone strength. In the present study, to evaluate the dose-related effects of a low administration frequency of teriparatide on bone status, we investigated the effects of three-times-weekly administration of teriparatide (1.1, 5.6, or 28.2 µg/kg) for 12 months on bone parameters, including bone metabolism markers, bone mineral density (BMD), micro-computed tomography, and bone strength, using 6-month-old ovariectomized (OVX) rats. Three-times-weekly administration of teriparatide dose-dependently increased the BMD of the lumbar vertebra and femur in OVX rats, and increased serum osteocalcin (a marker of bone formation), but not type I collagen C-telopeptide (a marker of bone resorption). The trabecular number and thickness increased in the vertebrae and femur, as in prior reports of daily teriparatide administration in OVX rats. Cortical thickness increased only toward the endocortical side of the femur, unlike with daily administration. Bone strength of the vertebrae and proximal and shaft of the femur was correlated with the changes in BMD and bone structure. These results demonstrate the effects of low frequency, intermittent administration of teriparatide on the biomechanical, and microstructural properties of bone in OVX rats.

Novel sandwich ELISAs for rat DMP1: age-related decrease of circulatory DMP1 levels in male rats.

Dentin matrix protein 1 (DMP1), a noncollagenous bone matrix protein produced by osteocytes, regulates matrix mineralization and phosphate homeostasis. The lack of a precise assay for circulating DMP1 levels impairs further investigation of the protein's biological significance. Because full-length precursor DMP1 is cleaved into NH2- and COOH-terminal fragments during the secretory process, we developed two new sandwich ELISAs for the NH2- and COOH-terminal fragments of rat DMP1. One of these ELISAs, ELISA 1-2, is based on two affinity-purified polyclonal antibodies against the DMP1-1 and DMP1-2 peptides of the NH2-terminal fragment, whereas the other, ELISA 4-3, is based on two affinity-purified polyclonal antibodies against the DMP1-3 and DMP1-4 peptides of the COOH-terminal fragment. The polyclonal antibodies were characterized in immunohistochemical and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) studies. Immunohistochemical analyses of rat bone using these polyclonal antibodies revealed DMP1 immunoreactivity in osteocytes and pericanalicular matrix, consistent with the previously reported osteocyte-specific expression of DMP1. LC-MS/MS analyses of rat plasma-derived immunoreactive products affinity-extracted with these antibodies revealed the presence of DMP1 in circulating blood. The ELISAs established with these antibodies met accepted standards for reproducibility, repeatability, precision, and accuracy. Circulating DMP1 and levels of other biochemical markers (osteocalcin, Trap5b, Dkk-1, and SOST) were measured in 2-, 4-, 8-, 12-, 18-, 24-, 72-, and 96-week-old Wistar male rats. Circulating DMP1 levels determined by ELISAs 1-2 and 4-3 significantly decreased with age. During rapid skeletal growth (2-12weeks), DMP1 levels measured by ELISA 4-3 were over three times higher than those measured by ELISA 1-2; however, DMP1 levels in old animals (72 and 96weeks) were almost the same when measured by either ELISA. DMP1 levels determined by both ELISAs were most highly positively correlated with the level of Dkk-1, second most highly correlated with the level of osteocalcin, and less highly correlated with the levels of Trap5b and SOST. These novel sandwich ELISAs for rat DMP1 are highly specific and allow precise measurements of circulating DMP1, which may be a new biochemical marker for osteocyte-mediated bone turnover.

Osteosarcoma in Sprague-Dawley rats after long-term treatment with teriparatide (human parathyroid hormone (1-34)).

Teriparatide, a therapeutic agent for osteoporosis, has been reported to increase the incidences of bone neoplasms such as osteosarcoma when administered subcutaneously to Fischer 344 (F344) rats for a long term, but its non-carcinogenic dose level following 2-year daily administration has not been established. Here we report detailed studies on the carcinogenicity of teriparatide following long-term administration. When teriparatide was administered subcutaneously to male and female Sprague-Dawley (SD) rats daily for 2 years, the incidence of osteosarcoma was increased at 13.6 µg/kg/day. The non-carcinogenic dose level was 4.5 µg/kg/day for both males and females. The development of osteosarcoma in SD rats depends on the dose level of, and treatment duration with, teriparatide. Responses of the bones to teriparatide were similar between F344 and SD rats in many aspects. These results suggested that the carcinogenic potential of teriparatide in SD rats is essentially the same as in F344 rats.

Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures.

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.

[Human parathyroid hormone (1-34) as a new therapeutic agent for osteoporosis].

Human parathyroid hormone (PTH) is used clinically for severe osteoporosis in North America and Europe. The clinical trial is now in progress in Japan. In this review, I introduce the additive effects of PTH with bone resorption inhibitors, the different effects on bone structure and quality between PTH and bone resorption inhibitor, and the bone-increasing effects of PTH involved in IGF/IRS, AP-1 and Wnt pathways. I focus on new topics including the presentations in ASBMR (American Society for Bone and Mineral Research) 2004.