Force-sensing treadmill gait analysis system can detect gait abnormalities in haemophilia patients without arthropathy.

BACKGROUND: Joint damage in patients with haemophilia (PwH) is commonly assessed by imaging, but few reports have described how structural changes in joints, for example, haemophilic arthropathy (HA)-affect gait ability. OBJECTIVES: We evaluated gait function among PwH with HA, PwH without HA, and people without haemophilia (non-PwH) using a Zebris FDM-T treadmill (FDM-T), an easy-to-use gait assessment instrument with a force sensor matrix. METHODS: The following gait parameters were collected: centre of pressure trajectory intersection (COPi) anterior/posterior variability, COPi lateral variability, COPi anterior/posterior symmetry, COPi lateral symmetry, single-limb support line (SLSL) length, and SLSL variability. Participants walked at their typical gait speed. The physical function of the PwH was assessed by the Hemophilia Joint Health Score (HJHS). Parameters were compared among the three groups. RESULTS: Twelve PwH with HA, 28 PwH without HA, and 12 non-PwH were enrolled. Gait speed significantly differed between groups (non-PwH, 3.1 ± 0.7; PwH without HA, 2.0 ± 0.7; PwH with HA; 1.5 ± 0.4). The COPi anterior/posterior variability, COPi lateral variability, SLSL length, and SLSL variability were greater in the PwH groups than in the non-PwH group. The COPi lateral symmetry differed between PwH with HA and the other groups. The HJHS was not correlated with gait parameters among PwH with HA. CONCLUSIONS: Gait parameters and speed were abnormal in both PwH with HA and PwH without HA. The FDM-T can be used to identify early stages of physical dysfunction that cannot be detected by conventional functional assessments such as the HJHS.

The effects of emicizumab on in vitro coagulation and fibrinolysis parameters in patients with disseminated intravascular coagulation with and without addition of anti-FVIII antibody.

BACKGROUND: Emicizumab (Emi) is used as haemostatic prophylaxis for patients with haemophilia A (PwHA). Disseminated intravascular coagulation (DIC) is a condition characterized by persistent systemic activation of coagulation, but there is yet no information on coagulation and fibrinolysis potentials in Emi-treated PwHA with DIC. AIM: To examine the effect of Emi on coagulation and fibrinolysis potentials in HA-model DIC plasmas. METHODS: Plasma from a patient with sepsis-DIC (seven patients) was treated with anti-factor (F)VIII monoclonal antibody (HA-model DIC plasma) and incubated with Emi (50 µg/mL). The plasma was then assessed using clot-fibrinolysis waveform analysis (CFWA). Coagulation and fibrinolysis parameters were expressed as ratios relative to normal plasma (|min1|-ratio and |FL-min1|-ratio, respectively). PATIENTS AND RESULTS: In case 1, coagulant potential was slightly high and fibrinolytic potential was extremely low, presenting a coagulant-dominant state (|min1|-ratio/|FL-min1|-ratio: 1.1/.38). In cases 2-5, fibrinolytic potential was not suppressed, but there were marked hypercoagulant potentials, indicating relative coagulant-dominant states. In case 6, coagulant and fibrinolytic potentials were increased but well balanced (|min1|-ratio/|FL-min1|-ratio: 1.38/1.28). In case 7, both potentials were severely deteriorated in not only CFWA but also the thrombin/plasmin generation assay. The addition of Emi into the HA-model DIC plasmas increased |min1|-ratio values in all cases, but the coagulant potentials did not exceed the initial ones (DIC plasma before treatment with anti-FVIII antibody). CONCLUSIONS: The presence of Emi in the HA-model DIC plasma improved coagulation potentials, but did not increase coagulation potentials beyond those of DIC plasma in non-HA states.

The in vitro effect of anticoagulant agents on coagulation and fibrinolysis in the presence of emicizumab in the plasmas from patients with haemophilia A.

INTRODUCTION: Emicizumab is used as hemostatic prophylaxis for patients with hemophilia A (PwHA), irrespective of the presence of inhibitors. Although bacterial infection can lead to a procoagulant state, there is limited information on coagulation and fibrinolysis potentials in emicizumab-treated PwHA and on the use of anticoagulants in such cases. AIM: We examined whether anticoagulants affect the coagulation and fibrinolysis potentials in plasma from PwHA spiked with emicizumab. METHODS: Plasma from PwHA was in vitro supplemented with emicizumab (50 µg/mL; emi-plasma) and anticoagulants (recombinant thrombomodulin (rTM), nafamostat mesylate (NM), unfractionated heparin (UFH), or low-molecular-weight heparin (LMH)). PwHA plasma spiked with rFVIII (1 IU/mL) was used as a reference (ref-plasma). The coagulation and fibrinolysis potentials in plasma was measured by thrombin and plasmin generation assay (T/P-GA) and clot-fibrinolysis waveform analysis (CFWA). RESULTS: In T/P-GA and CFWA, coagulation potentials (maximum coagulation velocity; |min1|, and peak thrombin; Th-Peak) in plasma rose with increasing concentrations of emicizumab and rFVIII, but fibrinolytic potentials (peak plasmin; Plm-Peak, and maximum fibrinolytic velocity; |FL-min1|) remained unchanged. Adding rTM, NM, and UFH to emi-plasma suppressed coagulation and fibrinolysis potentials, similar to ref-plasma. Regarding the heparin, UFH and LMH inhibited the improved coagulation in emi-plasma. UFH inhibited fibrinolysis as well, but LMH did not. CONCLUSIONS: Anticoagulants could exhibit the inhibitory effects on the coagulation and fibrinolysis potentials in plasma from PwHA spiked with emicizumab, similar to those in normal plasma.

Coagulant potentials of emicizumab in the plasmas from infant and toddler patients with hemophilia A.

BACKGROUND: Emicizumab significantly reduces bleedings in patients with hemophilia A (PwHA). A clinical study (HAVEN 7; NCT04431726) for PwHA aged less than or equal to 12 months is ongoing, but emicizumab-driven coagulation potential in PwHA in early childhood remains to be clarified. AIM: To investigate the in vitro or in vivo coagulation potential of emicizumab in plasmas obtained from infant and toddler PwHA. METHODS: Twenty-seven plasma samples from 14 infant/toddler PwHA (aged 0-42 months, median 19 months) who received emicizumab (n = 9), factor (F)VIII products (n = 8), or no treatment (n = 10) were obtained. FVIII activity in FVIII-treated plasmas was cancelled by the addition of anti-FVIII monoclonal antibody (mAb). Emicizumab-treated plasmas (in vivo) and emicizumab-spiked plasmas (in vitro) were analyzed. Emicizumab-untreated plasma or emicizumab-treated plasma supplemented with two anti-emicizumab mAbs were used as references. Adjusted maximum coagulation velocity (Ad|min1|) by clot waveform analysis and peak thrombin (Peak-Th) by thrombin generation assay was assessed. RESULTS: Ad|min1| values in 24 samples were improved by the presence of emicizumab. Values did not improve in the three remaining samples (aged 1, 23, and 31 months). Although the presence of emicizumab showed an age-dependent increase in Peak-Th in 20 samples, this increase was not observed in seven samples (aged 0, 1, 1, 2, 8, 19, and 36 months). Emicizumab-dependent increases in both Ad|min1| and Peak-Th were shown in 18 samples, and increases in either parameter were shown in eight samples. One sample (from patient aged 1 month) showed no increase in both, however. CONCLUSION: Emicizumab could improve coagulant potential in plasmas from infant/toddler patients with hemophilia A.

The use of infrared thermography for non-invasive detection of bleeding and musculoskeletal abnormalities in patients with hemophilia: an observational study.

BACKGROUND: In patients with hemophilia (PwH), bleeding often occurs in joints and muscles, and early detection of hemorrhage is important to prevent the onset and progression of mobility impairment. Complex-Image analysis such as ultrasonography, computed tomography, and magnetic resonance imaging are used to detect bleeding. On the other hand, no simple and rapid method to detect the active bleeding has been reported. Local inflammatory responses occur when blood leaks from damaged vessels, and the temperature at the site of active bleeding could be expected to increase in these circumstances, leading to an increase in surrounding skin temperature. Therefore, the purpose of this study was to investigate whether the measurement of skin temperature using infrared thermography (IRT) can be used as a diagnostic aid to detect active bleeding. METHODS: Fifteen PwH (from 6 to 82 years old) complaining of discomfort such as pain were examined. Thermal images were obtained simultaneously at the affected sides and comparable unaffected sides. The average skin temperature of the affected side and of the unaffected side were measured. The temperature differences were calculated by subtracting the average skin temperature at the unaffected side from the affected side. RESULTS: In eleven cases with active bleeding, the skin temperature at the affected side was more than 0.3 °C higher (0.3 °C to 1.4 °C) compared to the unaffected side. In two cases without active bleeding, there were no significant differences in skin temperature between the affected and unaffected sides. In two cases with previous rib or thumb bone fracture, the skin temperature at the affected side was 0.3 °C or 0.4 °C lower than that of the unaffected side, respectively. In two cases with active bleeding in which longitudinal evaluation was conducted, the difference in skin temperature decreased after hemostatic treatment. CONCLUSION: The analysis of skin temperature deference using IRT was a useful supportive tool to readily assess musculoskeletal abnormalities and bleeding in PwH as well as to determine the success of the hemostatic treatment.

Cisplatin Augments Antitumor T-Cell Responses Leading to a Potent Therapeutic Effect in Combination With PD-L1 Blockade.

BACKGROUND: Immune checkpoint inhibitors have marked antitumor effect. However, monotherapy benefits only a limited population of patients, and further improvement of their effects is required. Here the therapeutic effect and immune response during anti-PD-L1 plus cisplatin combination therapy were investigated in a mouse model. MATERIALS AND METHODS: E.G7-OVA, expressing ovalbumin (OVA) gene as a model tumor antigen, was subcutaneously inoculated into syngeneic mice and treated with anti-PD-L1 with/without cisplatin. The tumor growth and activation status of immune cells were evaluated. RESULTS: The anti-PD-L1 plus cisplatin combination resulted in a potent antitumor effect leading to tumor shrinkage compared to anti-PD-L1 or cisplatin alone, even though each alone, significantly inhibited tumor growth compared to the control group. During treatment, all groups, including that treated with cisplatin alone, had increased CD8+ T-cell infiltration into tumor tissues compared with the control group, and the therapeutic effect was diminished by CD8+ cell depletion. Aside from its direct cytotoxic effect, cisplatin alone increased chemokine levels and expression of immune checkpoint molecules on CD8+ T-cells in the tumor site. The combination effectively activated OVA-specific CD8+ T-cells. Furthermore, anti-PD-L1 alone and in combination with cisplatin, but not cisplatin alone, induced interferon-gamma-producing CD4+ T-cells. CONCLUSION: These findings provide a rationale for anti-PD-L1 plus cisplatin becoming a promising combination therapy for patients with cancer.

Continuous administration of bevacizumab plus capecitabine, even after acquired resistance to bevacizumab, restored anti-angiogenic and antitumor effect in a human colorectal cancer xenograft model.

Vascular endothelial growth factor (VEGF)-neutralizing therapy with bevacizumab has become increasingly important for treating colorectal cancer. It was demonstrated that second-line chemotherapy together with bevacizumab after disease progression (PD) on first-line therapy including bevacizumab showed clinical benefits in metastatic colorectal and breast cancers (ML18147 trial, TANIA trial). One of the rationales for these trials was that the refractoriness to first-line therapy is caused by resistance to not so much bevacizumab as to the chemotherapeutic agents. Nevertheless, resistance to bevacizumab cannot be ruled out because VEGF-independent angiogenesis has been reported to be a mechanism of resistance to anti-VEGF therapy. In this study, we used a xenograft model with the human colon cancer HT-29 cells to investigate the mechanisms underlying the effect of continued administration of bevacizumab plus capecitabine even after resistance to bevacizumab was acquired. The combination of capecitabine plus bevacizumab exhibited significantly stronger antitumor and anti-angiogenic activities than did monotherapy with either agent. Capecitabine treatment significantly increased the intratumoral VEGF level compared with the control group; however, the combination with bevacizumab neutralized the VEGF. Among angiogenic factors other than VEGF, intratumoral galectin-3, which reportedly promotes angiogenesis both dependent on, and independently of VEGF, was significantly decreased in the capecitabine group and the combination group compared with the control group. In an in vitro experiment, 5-fluorouracil (5-FU), an active metabolite of capecitabine, inhibited galectin-3 production by HT-29 cells. These results suggested that capecitabine has a dual mode of action: namely, inhibition of tumor cell growth and inhibition of galectin-3 production by tumor cells. Thus, capecitabine and bevacizumab may work in a mutually complementary manner in tumor angiogenesis inhibition to overcome the resistance caused by angiogenic factors other than VEGF. These results suggest the clinical relevance and the mechanism of action of treatment with bevacizumab in combination therapy beyond PD.

Superior antitumor activity of trastuzumab combined with capecitabine plus oxaliplatin in a human epidermal growth factor receptor 2-positive human gastric cancer xenograft model.

In the treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced gastric or gastroesophageal junction cancer, it has been reported that the combination of trastuzumab with capecitabine plus cisplatin, or with 5-fluorouracil (5-FU) plus cisplatin, significantly increased overall survival compared with chemotherapy alone (ToGA trial). In addition, adjuvant therapy with capecitabine plus oxaliplatin (XELOX) improved the survival of patients who received curative D2 gastrectomy (CLASSIC trial). However, the efficacy of the combination of trastuzumab with XELOX for patients with HER2-positive gastric cancer remains unknown. The aim of this study, was to investigate the efficacy of the combination of trastuzumab with XELOX in a HER2-positive human gastric cancer xenograft model. Combination treatment with these three agents (trastuzumab 20 mg/kg, capecitabine 359 mg/kg and oxaliplatin 10 mg/kg), was found to exhibit a significantly stronger antitumor activity in NCI-N87 xenografts compared with either trastuzumab or XELOX alone. In this model, treatment with trastuzumab alone or trastuzumab plus oxaliplatin enhanced the expression of thymidine phosphorylase (TP), a key enzyme in the generation of 5-FU from capecitabine in tumor tissues. In in vitro experiments, trastuzumab induced TP mRNA expression in NCI-N87 cells. In addition, NCI-N87 cells co-cultured with the natural killer (NK) cell line CD16(158V)/NK-92 exhibited increased expression of TP mRNA. When NCI-N87 cells were cultured with CD16(158V)/NK-92 cells in the presence of trastuzumab, the mRNA expression of cytokines reported to have the ability to induce TP was upregulated in tumor cells. Furthermore, a medium conditioned by CD16(158V)/NK-92 cells also upregulated the expression of TP mRNA in NCI-N87 cells. These results suggest that trastuzumab promotes TP expression, either by acting directly on NCI-N87 cells, or indirectly via a mechanism that includes trastuzumab-mediated interactions between NK and NCI-N87 cells. Therefore, the combination of trastuzumab with XELOX may be a potent therapy for HER2-positive gastric cancer.

Eldecalcitol replaces endogenous calcitriol but does not fully compensate for its action in vivo.

Calcitriol (1α,25-dihydroxyvitamin D3, 1α,25(OH)2D3) is an essential hormone that works in cooperation with parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23) to regulate calcium and phosphorus homeostasis. Previous in vivo studies in rats have shown that eldecalcitol, a vitamin D analog, is more active than calcitriol in stimulating calcium and phosphorus absorption in the intestine and in increasing serum FGF-23, but is not as active in suppressing blood PTH. However, those results are problematic because administration of exogenous eldecalcitol or calcitriol affects the synthesis and degradation of endogenous calcitriol, and competes for binding to vitamin D receptor (VDR) in target tissues. Therefore, we tried to evaluate the 'true biological activity in vivo' of each compound by comparing their biological activities with respect to their blood concentrations. In VDR gene knockout mice, calcitriol and eldecalcitol did not affect either serum or urinary calcium levels, and also did not induce the expression of target genes. These results indicate that the actions of eldecalcitol are mediated by VDR. In normal rats, concentrations of both calcitriol and eldecalcitol in the blood increased dose-dependently and had a linear correlation with administered dosage. The concentration of calcitriol in the blood was reduced by eldecalcitol treatment, falling to below the limit of detection at 0.1µg/kg eldecalcitol. Based on the concentration of each compound in the blood, eldecalcitol had approximately 1/4 to 1/7 the activity of calcitriol to increase serum calcium, FGF-23, and urinary calcium excretion, and to suppress blood PTH. Eldecalcitol dose-dependently increased urinary phosphorus excretion and reduced serum phosphorus. However, calcitriol did not change serum phosphorus. In accordance with serum chemistry and hormones, a concentration of eldecalcitol in the blood of 3-8 times that of calcitriol was required to stimulate target gene expressions in the kidneys (VDR, TRPV5, and calbindin-D28k) and bone (VDR, FGF-23, and RANKL). On the other hand, the blood concentrations of eldecalcitol needed to stimulate target genes in the intestine (TRPV6, calbindin-D9k, and VDR) were comparable to those of calcitriol. These results indicate that oral administration of eldecalcitol stimulates target gene expression in the intestine similarly to calcitriol, but to a much lesser extent than calcitriol in the kidneys and bones. The major finding of the present study is that eldecalcitol suppresses endogenous calcitriol and replaces it. However, it may not fully compensate for the action of calcitriol in vivo. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

A series of nonsecosteroidal vitamin D receptor agonists for osteoporosis therapy.

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)2D3 had been replaced by aryl acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)2D3. An X-ray analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat model using immature rats, we identified a potent active vitamin D3 analog, compound 7e. In mature rats of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249) could be a possible new drug candidate for treating osteoporosis in human.

Fos plays an essential role in the upregulation of RANK expression in osteoclast precursors within the bone microenvironment.

Fos plays essential roles in the osteoclastic differentiation of precursor cells generated by colony-stimulating factor 1 (CSF-1) and receptor activator of NF-κB ligand (RANKL; also known as tumor necrosis factor ligand superfamily member 11, Tnsf11). RANKL-deficient (RANKL(-/-)) mice and Fos(-/-) mice exhibit osteopetrosis due to an osteoclast deficiency. We previously reported that RANK-positive osteoclast precursors are present in bone of RANKL(-/-) mice but not Fos(-/-) mice. Here we report the role of Fos in RANK expression in osteoclast precursors. Medullary thymic epithelial cells and intestinal antigen-sampling microfold cells have been shown to express RANK. High expression of RANK was observed in some epithelial cells in the thymic medulla and intestine but not in osteoclast precursors in Fos(-/-) mice. RANK mRNA and protein levels in bone were lower in Fos(-/-) mice than RANKL(-/-) mice, suggesting that Fos-regulated RANK expression is tissue specific. When wild-type bone marrow cells were inoculated into Fos(-/-) mice, RANK-positive cells appeared along bones. RANK expression in wild-type macrophages was upregulated by coculturing with RANKL(-/-) osteoblasts as well as wild-type osteoblasts, suggesting that cytokines other than RANKL expressed by osteoblasts upregulate RANK expression in osteoclast precursors. CSF-1 receptor-positive cells were detected near CSF-1-expressing osteoblastic cells in bone in Fos(-/-) mice. CSF-1 upregulated RANK expression in wild-type macrophages but not Fos(-/-) macrophages. Overexpression of Fos in Fos(-/-) macrophages resulted in the upregulation of RANK expression. Overexpression of RANK in Fos(-/-) macrophages caused RANKL-induced signals, but failed to recover the RANKL-induced osteoclastogenesis. These results suggest that Fos plays essential roles in the upregulation of RANK expression in osteoclast precursors within the bone environment.

Daily administration of eldecalcitol (ED-71), an active vitamin D analog, increases bone mineral density by suppressing RANKL expression in mouse trabecular bone.

Eldecalcitol (ED-71) is a new vitamin D3 derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED-71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED-71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED-71 (50 ng/kg body weight) to 8-week-old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED-71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle-arrested receptor activator of NF-κB (RANK)-positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED-71 affected neither the number of RANK-positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED-71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL-positive cell surface around the trabecular bone was significantly reduced in ED-71-treated mice than in the control mice. ED-71 administration also increased BMD in 12-week-old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED-71 increases BMD by suppressing RANKL expression in trabecular bone in vivo.

[Frontiers in vitamin D; basic research and clinical application. Inhibitory effects of eldecalcitol, an active vitamin D derivative, on bone resorption in vivo].

Eldecalcitol is a new vitamin D(3) derivative recently approved for the treatment of osteoporosis in Japan. Previous studies showed that daily administration of eldecalcitol increased bone mineral density (BMD) by suppressing bone resorption in animals and in patients with osteoporosis. We examined how eldecalcitol suppresses bone resorption in vivo . Daily administration of eldecalcitol into mice did not affect properties of osteoclast precursors, but suppressed RANKL expression in bone. These results suggest that daily administration of eldecalcitol increase BMD by suppressing RANKL expression in bone.

Lineage-committed osteoclast precursors circulate in blood and settle down into bone.

Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. We previously showed that cell cycle-arrested quiescent osteoclast precursors (QOPs) were detected along bone surfaces as direct osteoclast precursors. Here we show that receptor activator of NF-κB (RANK)-positive cells isolated from bone marrow and peripheral blood possess characteristics of QOPs in mice. RANK-positive cells expressed c-Fms (receptors of macrophage colony-stimulating factor) at various levels, but scarcely expressed other monocyte/granulocyte markers. RANK-positive cells failed to exert phagocytic and proliferating activities, and differentiated into osteoclasts but not into dendritic cells. To identify circulating QOPs, collagen disks containing bone morphogenetic protein-2 (BMP disks) were implanted into mice, which were administered bromodeoxyuridine daily. Most nuclei of osteoclasts detected in BMP-2-induced ectopic bone were bromodeoxyuridine-negative. RANK-positive cells in peripheral blood proliferated more slowly and had a much longer lifespan than F4/80 (a macrophage marker)-positive macrophages. When BMP disks and control disks were implanted in RANK ligand-deficient mice, RANK-positive cells were observed in the BMP disks but not in the controls. F4/80-positive cells were distributed in both disks. Administration of FYT720, a sphingosine 1-phosphate agonist, promoted the egress of RANK-positive cells from hematopoietic tissues into bloodstream. These results suggest that lineage-determined QOPs circulate in the blood and settle in the bone.

[Control of bone resorption by RANKL-RANK system].

Differentiation and function of osteoclasts is regulated by RANKL and OPG, both of which are produced by osteoblasts. Osteoclast precursors express RANK, the receptor of RANKL. The activation of the RANKL-RANK system stimulates osteoclast differentiation and function. OPG is a decoy receptor of RANKL, which inhibits the RANKL-RANK interaction. Several genetic disorders in RANKL, RANK and OPG genes cause osteoclast abnormalities. Therefore, the development of anti-osteoporosis drugs that suppress the RANKL-RANK system is advanced now. Recently, anti-RANKL antibody, denosumab, is developed and being used as a new treatment for osteoporosis.

Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis.

Novel vitamin D(3) analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog, LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor (VDR), the carboxyl groups had very unique hydrogen bonding interactions in VDR and mimicked 1α-hydroxy group and/or 3ß-hydroxy group of 1α,25-dihydroxyvitamin D(3). A highly potent analog, 6a, with good in vitro activity and pharmacokinetic profiles was identified from an SAR study. Compound 6a showed significant prevention of bone loss in a rat osteoporosis model by oral administration.

Nobiletin, a polymethoxy flavonoid, suppresses bone resorption by inhibiting NFκB-dependent prostaglandin E synthesis in osteoblasts and prevents bone loss due to estrogen deficiency.

Nobiletin, a polymethoxy flavonoid, prevents cancer and inflammation, but the roles of nobiletin in bone are unclear. We examined the effects of nobiletin on bone resorption in vitro and on bone mass in ovariectomized (OVX) mice in vivo. In vitro, nobiletin suppressed osteoclast formation and bone resorption induced by interleukin (IL)-1. Nobiletin suppressed the expression of cyclooxygenase-2, NFκB-dependent transcription, and prostaglandin E (PGE) production induced by IL-1 in osteoblasts. OVX mice showed severe bone loss in the femur by increased bone resorption due to estrogen deficiency, and nobiletin significantly restored the bone mass. Nobiletin could be beneficial to bone health in postmenopausal women.

Eldecalcitol is less effective in suppressing parathyroid hormone compared to calcitriol in vivo.

Eldecalcitol is a vitamin D3 analog under clinical development for the treatment of osteoporosis. Previous studies have shown that the binding activities of eldecalcitol to the serum vitamin D binding protein (DBP) and the vitamin D receptor (VDR) are 421.9% and 44.6% of those of calcitriol, respectively, and also, suppressed parathyroid hormone (PTH) production by only 3.5% of calcitriol in vitro using bovine parathyroid cell primary culture. Here, we compared in vivo activities of eldecalcitol on serum calcium, BMD and PTH with those of calcitriol. Six-week old male rats were given either vehicle (medium chain triglyceride; n=6), eldecalcitol (0.025, 0.05, 0.1, 0.25, 0.5 microg/kg; n=6) or calcitriol (0.25, 0.5, 1.0, 2.5, 5 microg/kg; n=6) daily for 14 days by oral gavages. Eldecalcitol was approximately five-times more potent than calcitriol in increasing serum calcium. Eldecalcitol significantly increased lumbar spine BMD, however, calcitriol had no effect on BMD at any given doses. On the contrary, eldecalcitol did not affect PTH mRNA synthesis at the normocalcemic doses, despite the BMD was higher than normal. These observations indicate that, as previous in vitro study suggested, eldecalcitol is less effective in suppressing PTH compared to calcitriol.