Serum TRACP 5b Research Articles

Efficacy and Safety of Denosumab for the Treatment of Osteoporosis in Patients with Chronic Kidney Disease

Background: Chronic kidney disease (CKD) is an independent risk factor for osteoporosis and may lead to metabolic abnormalities that accelerate bone loss. Bisphosphonates, the most widely used treatment for osteoporosis, are contraindicated in patients with severe renal impairment. In the present study, we assessed whether denosumab is a safe and effective treatment for osteoporosis in patients with CKD. Methods and Findings: A total of 143 patients with osteoporosis who were treated with denosumab were analyzed retrospectively. Of these patients, 40 had been previously treated with bisphosphonates. All patients received supplemental vitamin D. Effectiveness was assessed by analyzing changes in bone mineral density (BMD) and serum tartrate-resistant acid phosphatase (TRACP)-5b as a marker for serum bone resorption. More than fifty percent of the patients treated with bisphosphonates showed low BMD at the time their therapy was changed to denosumab. Denosumab was associated with a larger increase in both lumbar and femur neck BMD than were bisphosphonates (+4.8% and +5.5%, respectively). Denosumab decreased serum TRACP-5b while increasing BMD (P<0.001), and was well tolerated. Serum calcium levels decreased shortly after the injection of denosumab, but recovered within 14 days. Supplemental vitamin D (0.5 to 1.0 μg/day) appeared to prevent hypocalcemia and to support efficacy of denosumab. Conclusions: Denosumab increases BMD in the lumbar vertebra and femur neck in patients with CKD. The effect of denosumab on BMD is greater than that of bisphosphonates in these patients.

Serum N-terminal telopeptide of type I collagen as an early marker of fracture nonunion in rabbits.

The aim of the present study was to establish an experimental animal model of fracture nonunion, and to investigate the changes in serum biomarker concentrations in fracture nonunion. A total of 20 purebred New Zealand rabbits were divided into two group: A bone defect group and a bone fracture group. In the bone defect group, a 15-mm section of bone (including the periosteum) was removed from the mid-radius, and the medullary cavities were closed with bone wax. In the bone fracture group, the mid-radius was fractured. X-rays were taken and blood samples were collected preoperatively and at 2, 3, 4, 5, 6, 7, 8, 10 and 12 weeks after the surgical procedure. The serum concentrations of osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP) served as markers of bone formation, and those of C-terminal telopeptide of type I collagen (CTX), N-terminal telopeptide of type I collagen (NTX) and tartrate-resistant acid phosphatase 5b (TRACP 5b) served as markers of bone resorption. The concentration levels of the markers were measured using a biotin double-antibody sandwich enzyme-linked immunosorbent assay. In the bone defect group, bone callus was observed on X-ray at 2 weeks in three rabbits and the bone calluses stabilized at 5 weeks; however, none of the bones had healed at 8 weeks. In the bone fracture group, the fracture line was distorted at 2 weeks and bone calluses formed at 6–8 weeks. In the bone defect group, the serum BSAP and TRACP 5b concentrations increased following the surgical procedure, peaked at 4 weeks, began to decrease at 5 weeks and stabilized after 6 weeks. The serum OC concentrations did not change significantly following the surgical procedure. The serum CTX concentrations fluctuated during the first 4 weeks, peaked at 5 weeks, then decreased and stabilized after 6 weeks. The serum NTX concentrations fluctuated during the first 4 weeks, were significantly lower at 5 weeks compared with the other time points and stabilized after 6 weeks. These results suggested that a bone nonunion model can be established in New Zealand rabbits by resecting a 15-mm section of bone from the mid-radius prior to bone wax blocking. Measurement of the serum BSAP, CTX, NTX, and TRACP 5b concentrations may be useful for the early detection of bone nonunion. The serum NTX concentrations changed significantly in rabbits with bone nonunion. Further studies are required in order to determine the feasibility of using serum NTX concentrations for the early diagnosis of bone nonunion.

Tartrate-Resistant Acid Phosphatase 5b in Young Patients With Sickle Cell Disease and Trait Siblings: Relation to Vasculopathy and Bone Mineral Density.

Bone involvement is a frequent cause of acute morbidity in sickle cell disease (SCD). Tartrate-resistant acid phosphatase 5b (TRACP 5b), a bone resorption marker, is produced specifically by activated osteoclasts. We assessed bone mineral density (BMD) in 30 young patients with SCD and 17 asymptomatic patients with sickle cell trait (SCT) compared with 32 healthy controls and determined TRACP 5b levels in relation to vascular complications. Serum ferritin, alkaline phosphatase (ALP), and TRACP 5b were measured. Echocardiography was performed with assessment of BMD using dual energy X-ray absorptiometry (DXA). The BMD was decreased in patients with SCD compared with SCT and controls (P = .005), with no significant difference between the latter 2 groups. Patients with SCD had higher incidence of bone complications than SCT group and controls (P = .03). The SCD group with abnormal DXA scan had higher ferritin and ALP than normal BMD. Serum TRACP 5b was significantly higher in patients with SCD than SCT and controls (P = .003). The TRACP 5b levels were associated with severe vaso-occlusive crisis (P = .022). Patients treated with hydroxyurea and those on chelation therapy had lower TRACP 5b levels than untreated patients. The TRACP 5b level was positively correlated with lactate dehydrogenase, while there was no relation with ferritin, ALP, or BMD. We suggest that bone complications frequently occur in SCD as reflected by low BMD and high ALP and TRACP 5b. Hemolysis and iron overload may be involved in the occurrence of these complications. The lack of correlation between abnormal DXA scan and high TRACP 5b suggests that bone disease in SCD is multifactorial.

Effects of icariin total flavonoids capsule on bone mineral density and bone histomorphometry in growing rats

Objective: To investigate the effect of icariin total flavonoids capsules (ITFC) on bone mineral density (BMD) and bone histomorphometry in growing rats and its anti-osteoporosis mechanism. Methods: Thirty female SD rats were randomly divided into 3 groups:normal control group, ITFC-1 group and ITFC-2 group. Rats in ITFC-1 group and ITFC-2 group were fed with 50 mg·kg-1·d-1 or 100 mg·kg-1·d-1 ITFC, respectively, and those in normal control group were fed with equal volume of distilled water. The whole body BMD was measured after 4, 8 and 12 weeks, and BMDs of the right femur and lumbar vertebrae were measured after 12 weeks. The serum levels of tartaric acid phosphatase 5b (TRACP 5b) and bone alkaline phosphatase (BALP) were measured by ELISA. Bone morphometry was performed on the right tibia. Results: There were no significant differences in the body weight increase between normal control group and two ITFC groups (all P>0.05). There were also no significant differences in whole body BMDs after 4 and 8 weeks between normal control group and ITFC groups (all P>0.05). After 12 weeks, the whole body BMD, BMD of bone in vitro, serum BALP level and trabecular area in ITFC-1 group and ITFC-2 group were significantly higher, trabecular separation was significantly lower than that in normal control group (all P<0.05); and the trabecular width and the number in ITFC-2 group were also significantly higher, and serum TRACP 5b level was significantly lower than that in normal control group (all P<0.05). The BMD of bone in vitro, serum BALP level, trabecular number and area in ITFC-2 group were significantly higher, and serum TRACP 5b level was significantly lower than that in ITFC-1 group (all P<0.05). Conclusion: ITFC can prevent osteoporosis by increasing bone density and bone formation, decreasing bone resorption and improving microstructure of bone.

Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.

BackgroundChildren with longstanding use of antiepileptic drugs (AEDs) are susceptible to developing low bone mineral density and an increased fracture risk. However, the literature regarding the effects of AEDs on growth in epileptic children is limited. The aim of this study was to investigate the potential effects of valproate (VPA) and/or oxcarbazepine (OXC) therapy on growth velocity and bone metabolism.MethodsSeventy-three ambulatory children (40 boys and 33 girls) with epilepsy, aged between 1 and 18 years (mean age 9.8 ± 4.1 years), were evaluated for growth velocity before and for 1 year after VPA and/or OXC treatment. The bone resorption marker serum tartrate-resistant acid phosphatase 5b (TRAcP5b) and the bone formation marker serum bone-specific alkaline phosphatase (BAP) were measured post-AEDs therapy for 1 year.ResultsThe difference in growth velocity (ΔHt) and body weight change (ΔWt) between pre- and post-AEDs treatment were -1.0 ± 2.8 cm/year (P < 0.05) and 0.1 ± 3.9 kg/year (P = 0.84), respectively. The study population had serum TRAcP5b-SDS of -1.6 ± 1.2 and BAP-SDS of 1.7 ± 3.7 compared with sex- and age-matched healthy children. Significant correlation between serum TRAcP 5b and BAP activities was noted (r = 0.60, p < 0.001). There was a positive correlation between growth velocity and serum TRAcP 5b activity after AED treatment (r = 0.42, p < 0.01). No correlation was found between ΔHt, ΔWt, serum TRAcP 5b, BAP activity and types of AEDs.ConclusionGrowth velocity was significantly decreased in epileptic children after 1 year of VPA and/or OXC treatment. The effect of VPA and/or OXC therapy on dysregulation of bone metabolism might play a crucial role in physical growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12887-016-0597-7) contains supplementary material, which is available to authorized users.

Abstract P6-16-01: Differences in patterns of change of bone turnover markers during treatment with bone-modifying agents of breast cancer patients with bone metastases

Abstract Background: Bone-modifying agents have demonstrated their efficacy for treatment by suppressing osteoclast function. The activity of bone-modifying agents can be monitored by means of bone resorption markers such as c-terminal crosslinking telopeptide of type I collagen (1CTP) and N-telopeptide of type I collagen (NTX) as well as bone forming marker bone-specific alkaline phosphatase (BAP). In contrast to these markers which indirectly indicate bone turnover, tartrate-resistant acid phosphatase-5b (Tracp-5b) has been established as a direct marker showing osteoclast number and activity. The aim of this study was to identify the relative significance of these bone turnover markers as indicators of treatment efficacy induced by bone-modifying agents for breast cancer patients with bone metastases. Patients and Methods: For this study, 52 breast cancer patients with bone metastases treated with bone-modifying agents were recruited. Zoledronic acid and denosumab were administered as bone-modifying agents to 36 and 22 patients, respectively (for 6 patients, denosumab was used after zoledronic acid). Serum Tracp-5b, 1CTP, NTX and BAP were measured with, respectively, the EIA (enzyme immunoassay), RIA (two-antibody radioimmunoassay), ELISA (enzyme-linked immunosorbent assay) and CLEIA (chemiluminescent enzyme immunoassay) method. Blood samples were obtained pretreatment and 1, 3 and 6 months after treatment. Changes in these bone turnover markers were statistically analyzed with Friedman's test, and correlation between serum markers and clinicopathological factors was calculated with Mann-Whitney's test. Results: Serum tracp-5b decreased significantly after treatment (p&amp;lt;0.0001). The baseline median value of Tracp-5b (457.5mU/dl, range: 173-1630mU/dl) had been reduced to 137mU/dl (91-795mU/dl) 1 month after treatment, but no further reduction was observed after that. For 13 out of 15 patients to whom Tracp-5b was administered, abnormally high levels (above 420mU/dl) decreased to normal range with one month treatment. Serum NTX was also significantly reduced after treatment (p=0.0007). The median baseline value (16.5nmolBCE/L, 6.1-52.2nmolBCE/L) was diminished after 1 month (to 10.9nmolBCE/L, 7.0-49.5nmolBCE/L), and further reduction of NTX was observed after 3 months (9.55nmolBCE/L, 6.4-56.0nmolBCE/L). Similarly, baseline BAP (15.1μg/L, 6.4-81.3μg/L) decreased significantly (p=0.0032), a reduction which was obtained after 3 months (10.15μg/L, 6.1-51.7μg/L), but not after 1 month (13.0μg/L, 7.7-137.0μg/L). On the other hand, reduction in 1CTP was not significant (p=0.83). Conclusion and discussion: Although baseline values of the bone turnover markers Tracp-5b, NTX and BAP decreased significantly after treatment with bone-modifying agents, the pattern of reduction for these three markers varied. Tracp-5b appears to reflect efficacy of bone-modifying agents most quickly and sensitively, possibly due to its direct link to the number and activity of osteoclasts. These findings may prove usefulness of Tracp-5b when considering the efficacy of various bone-modifying agents in clinical practice. Citation Format: Higuchi T, Nishimukai A, Yanai A, Miyagawa Y, Murase K, Imamura M, Ozawa H, Takatsuka Y, Miyoshi Y. Differences in patterns of change of bone turnover markers during treatment with bone-modifying agents of breast cancer patients with bone metastases. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-16-01.

High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay.

BackgroundBone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment.MethodsA total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals.ResultsThe average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24).ConclusionsHigh UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.

Whole-body vibration can attenuate the deterioration of bone mass and trabecular bone microstructure in rats with spinal cord injury

Experimental, controlled study. To examine the effects of whole-body vibration (WBV) on bone mass and trabecular bone microstructure (TBMS) during the early stage in juvenile rats with spinal cord injury (SCI). Studied at the Kio University in Japan. Thirty-four 8-week-old male Wistar rats were divided into 3 groups: the SCI group, the sham-operation group (SHAM) and the SCI+WBV group. WBV started on the 8th day after SCI. After 1 or 2 weeks of WBV treatment, measurements of tissue mineral density, trabecular bone mineral content (BMC) and parameters of TBMS were obtained by scanning the proximal tibias with x-ray micro-computed tomography. Serum levels of osteocalcin (OC) and of tartrate-resistant acid phosphatase 5b (TRACP 5b) were measured with ELISA. BMC, volume bone mineral density, bone volume (BV), BV fraction (BV/tissue volume) and connectivity density (Conn.D) of TBMS parameters were significantly higher in SCI+WBV rats than in SCI rats after 2-week WBV. The BMC and BV/TV of bone mass index correlated well with Conn.D, suggesting the preservation of Conn.D. induced by WBV. SCI+WBV rats showed a decrease in serum OC after 1-week WBV, but a quick recovery from that after 2-week WBV. There was no difference in serum TRACP 5b among the 3 groups throughout the experimental period. WBV treatment could attenuate the bone deterioration that occurs during the early stage in juvenile rats with SCI. In a clinic, this early WBV intervention may be an effective rehabilitation modality for preventing bone fragility in SCI patients.

Evaluation of bone involvement in patients with Gaucher disease: a semi-quantitative magnetic resonance imaging method (using ROI estimation of bone lesion) as an alternative method to semi-quantitative methods used so far.

The aim of this study was to evaluate bone involvement in patients with Gaucher disease (GD) and to propose a novel semi-quantitative magnetic resonance imaging (MRI) staging. MRI of the lumbar spine, femur, and tibia was performed in 24 patients with GD and 24 healthy controls. We also measured circulating levels of C-C motif ligand-3 (CCL-3) chemokine, C-telopeptide of collagen type-1 (CTX), and tartrate-resistant acid phosphatase isoform type-b (TRACP-5b). We used the following staging based on MRI data: stage I: region of interest (ROI) 1/2 of normal values and bone infiltration up to 30%; stage II: ROI 1/3 of normal values and bone infiltration from 30 to 60%; stage III: ROI 1/4 of normal values and bone infiltration from 60% to 80%; and stage IV: detection of epiphyseal infiltration, osteonecrosis and deformity regardless of the ROI's values. All but two patients had abnormal MRI findings: 9 (37.5%), 6 (25%), 3 (12.5%), and 4 (16.7%) had stages I-IV, respectively. Patients with GD had elevated chitotriosidase, serum TRACP-5b, and CCL-3 levels (P<0.001). We propose an easily reproducible semi-quantitative scoring system and confirm that patients with GD have abnormal MRI bone findings and enhanced osteoclast activity possibly due to elevated CCL-3.

Biomarkers for Bisphosphonate-Related Osteonecrosis of the Jaw.

The aim of this study was to investigate a possible biomarker for bisphosphonate-related osteonecrosis of the jaw (BRONJ) in an animal model. Forty-eight Sprague-Dawley rats were randomly divided into the bisphosphonate group (n = 36), who were injected once a week with zoledronic acid, and the control group (n = 12), who were injected once a week with saline. After 6 weeks, surgical intervention was performed, and injections were continued up to 8 weeks. Rats in the bisphosphonate group were then further classified to the ONJ group, and the non-ONJ group, and biomarkers, including CTx, Glu-OC, TRACP 5b, RANKL, and OPG, were assessed at baseline (T0), at surgical intervention (T1), and at sacrifice (T2). Histomorphometric analysis for quantification of osteoclasts was performed. Repeated measures analysis of variance revealed that TRACP 5b levels and the RANKL/OPG ratio were significantly decreased over time in the ONJ group compared with the non-ONJ group (p < .05). At T2, the area under the curve was 0.807 for TRACP 5b (sensitivity: 88.9%, specificity 66.7% at cutoff) and 0.765 for the RANKL/OPG ratio (sensitivity: 77.8%, specificity 62.9% at cutoff). TRACP 5b showed a lower least significant change (29.6%) with lower intra-assay coefficient of variability (CV; 6.32%) and interassay CV (11.20%) compared with those of the RANKL/OPG ratio (39.27%) and showed a higher signal-to-noise ratio (2.76) than that of the RANKL/OPG ratio (1.62). N.Oc/T.Ar and N.Oc/B.Ar demonstrated significantly decreased number of osteoclasts in ONJ group versus non-ONJ group. These results show that serum TRACP 5b and the RANKL/OPG ratio were possible biomarkers for BRONJ. These data may provide useful additional information for future ONJ research. Further studies are needed to validate these results in humans with ONJ.

Topical Treatment with Xiaozheng Zhitong Paste (XZP) Alleviates Bone Destruction and Bone Cancer Pain in a Rat Model of Prostate Cancer-Induced Bone Pain by Modulating the RANKL/RANK/OPG Signaling.

To explore the effects and mechanisms of Xiaozheng Zhitong Paste (XZP) on bone cancer pain, Wistar rats were inoculated with vehicle or prostate cancer PC-3 into the tibia bone and treated topically with inert paste, XZP at 15.75, 31.5, or 63 g/kg twice per day for 21 days. Their bone structural damage, nociceptive behaviors, bone osteoclast and osteoblast activity, and the levels of OPG, RANL, RNAK, PTHrP, IGF-1, M-CSF, IL-8, and TNF-α were examined. In comparison with that in the placebo group, significantly reduced numbers of invaded cancer cells, decreased levels of bone damage and mechanical threshold and paw withdrawal latency, lower levels of serum TRACP5b, ICTP, PINP, and BAP, and less levels of bone osteoblast and osteoclast activity were detected in the XZP-treated rats (P<0.05). Moreover, significantly increased levels of bone OPG but significantly decreased levels of RANL, RNAK, PTHrP, IGF-1, M-CSF, IL-8, and TNF-α were detected in the XZP-treated rats (P<0.05 for all). Together, XZP treatment significantly mitigated the cancer-induced bone damage and bone osteoclast and osteoblast activity and alleviated prostate cancer-induced bone pain by modulating the RANKL/RANK/OPG pathway and bone cancer-related inflammation in rats.

The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer.

In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

Effects of Cabozantinib Alone and in Combination with Bortezomib in the 5TGM1 Murine Multiple Myeloma Model

Introduction: Cabozantinib (cabo) is an inhibitor of tyrosine kinases including MET, VEGFR2, RET, and the TAM family kinases TYRO3, AXL, and MER. Cabo has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is the second most common hematologic malignancy, and represents ~2% of all cancer deaths. MM is a monoclonal B-cell (plasma cell) neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. Thus, the primary objectives of this research were to A): determine the activity of cabo on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model (study 1), and B): investigate the impact of cabo on overall survival of these mice when dosed alone or in combination with bortezomib (btz) (study 2).Methods: Female C57BL/KaLwRij mice were allocated to treatment groups (n=15-16 per group) with equivalent average body weights. Four experimental groups were utilized in each of 2 studies: a vehicle control group, and groups receiving single agent btz (0.5 mg/kg ip twice a week) or cabo (10 mg/kg, PO QD). Study 1 also included a higher dose cabo group (30 mg/kg, PO QD), and study 2 included a combination group: btz (0.5 mg/kg ip twice a week) plus cabo (10 mg/kg, PO QD). In study 2, each single agent group also received the vehicle from the alternate single-agent group via the appropriate route and schedule.On day 0, animals were inoculated with 5TGM1 mouse myeloma cells by IV administration. Dosing began on day 1 and continued daily until euthanasia at day 35 (study 1) or day 70 (study 2). Body weights were determined twice a week and blood samples were collected on days -1, 15, 22, and 34 for analysis of paraprotein (IgG2b) and TRACP 5b. In study 1 the development of osteolytic lesions was detected by radiography at the end of the study. Some animals were euthanized before the end of the experiment due to paraplegia. Animals euthanized within four days of the end of the experiment in study 1 were included in the analyses.Results: In study 1, btz reduced serum IgG2b levels, and decreased the frequency of soft tissue lesions, but did not show bone protective properties. Cabo exhibited bone protective effects: mean and total area of osteolytic lesions were reduced at the 30 mg/kg dose, and serum TRACP 5b values and osteoclast counts at the tumor-bone interface were reduced at both the 10 and 30 mg/kg doses. Relative bone area did not differ from control according to histomorphometry. The rise in serum IgG2b started earlier than vehicle control in both cabo-treated groups, but a significant difference was not observed in relative IgG2b at sacrifice. Cabo dose dependently increased the necrotic tumor area in bone, indicating the possibility that the rise in IgG2b may have been due to lysis of plasma cells. Both doses of cabo decreased the frequency of soft tissue lesions.In study 2, the median survival times were 36 d (vehicle), 43 d (btz), 48 d (cabo), and 55 d (cabo+btz). The prolongation of OS compared to vehicle was statistically significant for the cabo group but not for the btz group. Prolongation of OS in the combination group was significant compared to btz alone, but not when compared to cabo alone.Conclusions: Cabo showed both bone-protective and anti-tumor effects in this murine model of MM. In addition, statistically-significant prolongation of overall survival was observed with the combination of cabo+btz compared to btz alone, and with single-agent cabo compared to vehicle. Based on these results, further investigation of cabozantinib alone or in combination with other agents in multiple myeloma is warranted. DisclosuresAftab:Exelixis: Employment, Equity Ownership. Suominen:Pharmatest: Employment; Exelixis: Research Funding. Clary:Exelixis: Employment, Equity Ownership. Käkönen:Pharmatest: Employment; Exelixis: Research Funding. Fagerlund:Pharmatest: Employment; Exelixis: Research Funding. Alhoniemi:Pharmatest: Employment; Exelixis: Research Funding. Rissanen:Pharmatest: Employment; Exelixis: Research Funding. Halleen:Pharmatest: Employment; Exelixis: Research Funding.

Abstract 734: Effects of Cabozantinib in the 5TGM1 murine multiple myeloma model

Abstract Cabozantinib is an inhibitor of tyrosine kinases including MET, VEGFR2, and RET, and has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is the second most common hematologic malignancy, and represents ∼2% of all cancer deaths. MM is a monoclonal B-cell (plasma cell) neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. Thus, our aim was to determine the activity of cabozantinib on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model. Four experimental groups were included: negative control group receiving vehicle, positive control group receiving bortezomib (0.5 mg/kg ip twice a week), low dose cabozantinib group (10 mg/kg, PO QD) and high dose cabozantinib group (30 mg/kg, PO QD). Female C57BL/KaLwRij mice were allocated to treatment groups (n=15 per group) with equivalent average body weights. On day 0, animals were inoculated with 5TGM1 mouse myeloma cells by IV administration. Dosing began on day 1 and continued daily until euthanasia at day 35. Body weights were determined twice a week and blood samples were collected on days -1, 15, 22, and 34 for analysis of paraprotein (IgG2b) and TRACP 5b. The development of osteolytic lesions was detected by radiography at the end of the study. Some animals (4/15) were euthanized before the end of the experiment due to paraplegia in control and bortezomib groups, but none in cabozantinib groups. Animals euthanized within four days of the end of the experiment were included in the analysis. Bortezomib reduced serum IgG2b levels and decreased the frequency of soft tissue lesions, but did not show bone protective properties. Cabozantinib exhibited bone protective effects: mean and total area of osteolytic lesions were reduced at the 30 mg/kg dose, and serum TRACP 5b values and osteoclast counts at the tumor-bone interface were reduced at both the 10 and 30 mg/kg doses. Relative bone area did not differ from control according to histomorphometry. The rise in serum IgG2b started earlier than vehicle control in both cabo-treated groups, but a significant difference was not observed in relative IgG2b at sacrifice. Cabozantinib dose dependently increased the necrotic tumor area in bone, indicating the possibility that the rise in IgG2b may have been due to lysis of plasma cells. Both cabozantinib doses decreased the frequency of soft tissue lesions. In summary, cabozantinib showed both bone-protective and anti-tumor effects in this murine model of MM. Based on these promising results, further investigation of cabozantinib in multiple myeloma is warranted. Citation Format: Mari I. Suominen, Douglas O. Clary, Rami Käkönen, Katja M. Fagerlund, Esa Alhoniemi, Jukka P. Rissanen, Jussi M. Halleen, Dana T. Aftab. Effects of Cabozantinib in the 5TGM1 murine multiple myeloma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 734. doi:10.1158/1538-7445.AM2014-734

Abstract 1803: A new spectrum-selective cathepsin inhibitor, VBY-825, inhibits bone destruction in a syngeneic 5TGM1 multiple myeloma mouse model

Abstract Multiple myeloma (MM) is the second most common blood cancer after non Hodgkin lymphoma. It is a monoclonal B-cell neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, fractures and hypercalcemia. Chemo- or radiotherapy may induce remissions, but MM is generally thought to be incurable. Our aim was to observe the effects of a cathepsin inhibitor VBY-825 on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model using immunocompetent C57BL/KaLwRij mice. VBY-825 is a potent inhibitor of cathepsins K, L, B, V, and S. 5TGM1 cells were inoculated via tail vein in 7 weeks old female C57BL/KaLwRij mice, which were divided to 4 groups: Control group received vehicle of VBY-825 (5% dextrose 10 ml/kg daily), Control group received bortezomib vehicle (3 ml/kg twice a week), Reference group received bortezomib (0.5 mg/kg twice a week) and Study group received VBY-825 (100 mg/kg daily). Administration of all compounds began one day before tumor cell inoculation and continued until day 34. Disease progression was followed by measuring the serum levels of paraprotein (IgG2b) and TRACP 5b, radiography, and body weight. The animals were sacrificed 5 weeks after inoculation, examined macroscopically, and their bones were collected for histomorphometric analysis. The reference compound bortezomib had no effects on body weight but it delayed the disease progression based on IgG2b measurements. It also decreased the number and total area of osteolytic lesions, but not mean osteolytic lesion area (MOLA). VBY-825 had no effect on body weight or IgG2b level, frequency of soft tissue tumors or intraosseous tumor area. VBY-825 decreased total and MOLA, consistent with inhibited resorption. There was also a trend of increased relative trabecular bone area. Serum TRACP 5b activity in the VBY-825 treated group did not differ from the respective vehicle group, whereas the number of osteoclasts at tumor-bone interface was increased in VBY-825 treated animals. These findings suggest that VBY-825 decreased osteoclast function and resorption activity without decreasing the number of osteoclasts In conclusion, VBY-825 had no effects on tumor growth but it inhibited bone destruction in this mouse model of MM, which is consistent with its potency on cathepsin S and K, which are known to be important in osteoclast-mediated bone resorption. VBY-825 is a promising candidate for the treatment of tumor-associated bone disease. Citation Format: Mari I. Suominen, Johanna Tuomela, Esa Alhoniemi, Katja M. Fagerlund, Jukka P. Rissanen, Jussi M. Halleen, Leslie J. Holsinger. A new spectrum-selective cathepsin inhibitor, VBY-825, inhibits bone destruction in a syngeneic 5TGM1 multiple myeloma mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2014-1803

Temporal changes in systemic and local expression of bone turnover markers during six months of sclerostin antibody administration to ovariectomized rats

Sclerostin (Scl) is an osteocyte protein that decreases bone formation, and its inhibition by neutralizing antibodies (Scl-Ab) increases bone formation, mass and strength. We investigated the effects of Scl-Ab in mature ovariectomized (OVX) rats with a mechanistic focus on longer-term responses of osteoclasts, osteoblasts and osteocytes. Four-month-old Sprague–Dawley rats had OVX or sham surgery. Two months later, sham controls received sc vehicle while OVX rats received vehicle (OVX-Veh) or Scl-Ab (25mg/kg) once weekly for 6 or 26weeks followed by necropsy (n=12/group). Terminal blood was collected for biochemistry, non-adherent marrow cells were harvested from femurs for ex vivo osteoclast formation assays, and vertebrae and tibiae were collected for dynamic histomorphometry and mRNA analyses. Scl-Ab treatment led to progressively thicker but fewer trabeculae in the vertebra, leading to increased trabecular bone volume and reduced trabecular surfaces. Scl-Ab also increased cortical bone volume in the tibia, via early periosteal expansion and progressive endocortical contraction. Scl-Ab significantly reduced parameters of bone resorption at week 6 relative to OVX-Veh controls, including reduced serum TRACP-5b, reduced capacity of marrow cells to form osteoclasts ex vivo, and >80% reductions in vertebral trabecular and tibial endocortical eroded surfaces. At week 26, serum TRACP-5b and ex vivo osteoclast formation were no longer reduced in the Scl-Ab group, but eroded surfaces remained >80% lower than in OVX-Veh controls without evidence for altered skeletal mRNA expression of opg or rankl. Scl-Ab significantly increased parameters of bone formation at week 6 relative to OVX-Veh controls, including increases in serum P1NP and osteocalcin, and increased trabecular, endocortical and periosteal bone formation rates (BFRs). At week 26, surface-referent trabecular BFR remained significantly increased in the Scl-Ab group versus OVX-Veh controls, but after adjusting for a reduced extent of trabecular surfaces, overall (referent-independent) trabecular BFR was no longer significantly elevated. Similarly, serum P1NP and osteocalcin were no longer significantly increased in the Scl-Ab group at week 26. Tibial endocortical and periosteal BFR were increased at week 6 in the Scl-Ab group versus OVX-Veh controls, while at week 26 only endocortical BFR remained increased. The Scl-Ab group exhibited significant increments in skeletal mRNA expression of several osteocyte genes, with sost showing the greatest induction in both the tibia and vertebra. We propose that Scl-Ab administration, and/or the gains in bone volume that result, may have increased osteocytic expression of Scl as a possible means of regulating gains in bone mass.

Clinical efficacy and treatment persistence of monthly minodronate for osteoporotic patients unsatisfied with, and shifted from, daily or weekly bisphosphonates: the BP-MUSASHI study.

SummaryThis multi-center, prospective, open-label, observational study evaluated the effects of once-monthly minodronate (50 mg) on treatment persistence, bone turnover markers, bone mineral density, low back pain, and upper gastrointestinal symptoms in outpatients with osteoporosis previously treated with daily or weekly bisphosphonate products.IntroductionThe purposes of this study were to investigate the effects of once-monthly oral minodronate (MIN 50 mg) on bone turnover markers and bone mineral density, low back pain, and upper gastrointestinal symptoms, as well as preference for and treatment persistence of MIN 50 mg among Japanese osteoporosis patients currently treated with daily or weekly bisphosphonates.MethodsStudy patients were allocated based on their preference to either the Switch group (patients willing to switch over to MIN 50 mg) or the Continue group (patients wanting to continue their current therapies). Patients’ treatment persistence and satisfaction levels with the therapies were assessed using a self-administered questionnaire. The study endpoints were serum TRACP-5b, serum P1NP, bone mineral density, upper gastrointestinal symptoms, and low back pain.ResultsIn total, 264 and 133 patients were allocated into the Switch and Continue groups, respectively. Approximately, 65 % of patients were willing to switch to MIN 50 mg, with the predominant reason being “less frequent dosing more convenient.” Treatment persistence was significantly higher in the Switch group (MIN 50 mg) than the Continue group. Almost all patients with abnormal bone metabolism markers demonstrated normalization after switchover. MIN 50 mg alleviated low back pain and upper gastrointestinal symptoms induced by prior bisphosphonate use.ConclusionsMIN 50 mg alleviates low back pain, reduces bone turnover markers and increases bone density, and induces fewer upper gastrointestinal symptoms after switchover from prior bisphosphonate products, and therefore, it may provide patients with a more convenient treatment option and enhance long-term treatment persistence.

Clinical significance of serum BAP, TRACP 5b and ICTP as bone metabolic markers for bone metastasis screening in lung cancer patients

BackgroundWe investigated the clinical significance of serum bone-specific alkaline phosphatase (BAP), tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and type I collagen carboxyterminal telopeptide (ICTP) as bone metabolic markers for bone metastasis (BM) screening in lung cancer patients. MethodsNewly diagnosed advanced lung cancer patients with (N=130) and without (N=135) BM were enrolled in the study. Serum BAP, TRACP 5b and ICTP were measured before the treatment. ResultsBAP, TRACP 5b and ICTP values were higher in patients with BM compared with patients without BM (all P<0.0001). Area under ROC curve (AUC) of BAP, TRACP 5b and ICTP was 0.760, 0.753 and 0.835 (all P<0.0001), respectively. The cut-off values for BAP, TRACP 5b and ICTP were 21.8μg/l, 7.8 U/l and 8.8μg/l, respectively. When TRACP 5b and ICTP were combined, AUC was elevated to 0.895 (P<0.0001), and the cut-off values were TRACP 5b 7.6 U/l and ICTP 8.4μg/l. ConclusionsWe conclude that serum BAP, TRACP 5b and ICTP may serve as useful tools for BM screening in lung cancer patients.

Induction of Apoptosis in the Bone Marrow Promotes Regenerative Actions of Parathyroid Hormone (PTH) in Bone.

PTH has bone regenerative properties via mechanisms that remain elusive. This study aimed to determine the impact of marrow apoptosis on PTH anabolic actions. Studies were in conformance with FASEB animal use principles. Adult mice were treated with pro‐apoptotic etoposide (ETOP) or vehicle (VEH), daily 5d then 3d/wk for 6wks. PTH or VEH was administered daily for: VEH, PTH, ETOP and PTH+ETOP and skeletal phenotyping, flow cytometry, and serum bone markers analyzed. MicroCT and histomorphometry of tibiae revealed ETOP decreased and PTH increased bone volume and area, the greatest increase with PTH + ETOP. ETOP (5d) increased early marrow apoptotic cells (AnnexinV+PI‐) as well as MER+CD11b+F4/80+ and MER+CD68+F4/80+ myeloid cells. At 6wks, CD68+, Gr1+CD11b+F4/80+, CD45+and Lin−CD29+sca1+ cells were increased in ETOP w/ or w/o PTH. Serum TRAcP5b was increased in ETOP and to a greater extent with PTH. P1NP levels were increased in PTH and PTH+ETOP. PTH+ETOP mice had reduced osteoclasts per tibial bone vs. VEH or ETOP. In another model, luciferase+bone marrow stromal cells were implanted to form ectopic ossicles and bone growth tracked via bioluminescence. ETOP mice had increased luciferase vs. VEH, and ETOP augmented the PTH increase. These data suggest that induction of apoptosis provides a conducive environment for PTH pro‐regenerative actions, effects which may be due to increased macrophage efferocytosis.

A pivotal role of bone remodeling in granulocyte colony stimulating factor induced hematopoietic stem/progenitor cells mobilization

The majority of hematopoietic stem/progenitor cells (HSPCs) reside in bone marrow (BM) surrounded by a specialized environment, which governs HSPC function. Here we investigated the potential role of bone remodeling cells (osteoblasts and osteoclasts) in homeostasis and stress-induced HSPC mobilization. Peripheral blood (PB) and BM in steady/mobilized state were collected from healthy donors undergoing allogeneic transplantation and from mice treated with granulocyte colony stimulating factor (G-CSF), parathyroid hormone (PTH), or receptor activator of nuclear factor kappa-B ligand (RANKL). The number and the functional markers of osteoblasts and osteoclasts were checked by a series of experiments. Our data showed that the number of CD45(-) Ter119(-) osteopontin (OPN)(+) osteoblasts was significantly reduced from 4,085 ± 135 cells/femur on Day 0 to 1,032 ± 55 cells/femur on Day 5 in mice (P = 0.02) and from 21.38 ± 0.66 on Day 0 to 14.78 ± 0.65 on Day 5 in healthy donors (P < 0.01). Decrease of osteoblast number leads to reduced level of HSPC mobilization regulators stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF), and OPN. The osteoclast number at bone surface (OC.N/B.s) was significantly increased from 1.53 ± 0.12 on Day 0 to 4.42 ± 0.46 on Day 5 (P < 0.01) in G-CSF-treated mice and from 0.88 ± 0.20 on Day 0 to 3.24 ± 0.31 on Day 5 (P < 0.01) in human. Serum TRACP-5b level showed a biphasic trend during G-CSF treatment. The ratio of osteoblasts number per bone surface (OB.N/B.s) to OC.N/B.s was changed after adding PTH plus RANKL during G-CSF treatment. In conclusion, short term G-CSF treatment leads to reduction of osteoblasts and stimulation of osteoclasts, and interrupting bone remodeling balance may contribute to HSPC mobilization.