Characterization, pharmacokinetics, and pharmacodynamics of anti-Siglec-15 antibody and its potency for treating osteoporosis and as follow-up treatment after parathyroid hormone use.

Recent studies have established the idea that Siglec-15 is involved in osteoclast differentiation and/or function, and it is anticipated that therapies suppressing Siglec-15 function can be used to treat bone diseases such as osteoporosis. We have produced rat monoclonal anti-Siglec-15 antibody (32A1) and successively generated humanized monoclonal anti-Siglec-15 antibody (DS-1501a) from 32A1. Studies on the biological properties of DS-1501a showed its specific binding affinity to Siglec-15 and strong activity to inhibit osteoclastogenesis. 32A1 inhibited multinucleation of osteoclasts and bone resorption (pit formation) in cultured mouse bone marrow cells. 32A1 also inhibited pit formation in cultured human osteoclast precursor cells. Maximum serum concentration and serum exposure of DS-1501a in rats were increased in a dose-dependent manner after single subcutaneous or intravenous administration. Furthermore, single administration of DS-1501a significantly suppressed bone resorption markers with minimal effects on bone formation markers and suppressed the decrease in bone mineral density (BMD) of the lumbar vertebrae in ovariectomized (OVX) rats. In histological analysis, the osteoclasts distant from the chondro-osseous junction of the tibia tended to be flattened, shrunken, and functionally impaired in 32A1-treated rats, while alkaline phosphatase-positive osteoblasts were observed throughout the metaphyseal trabeculae. In addition, we compared the efficacy of 32A1 with that of alendronate (ALN) as follow-up medicine after treatment with parathyroid hormone (PTH) using mature established osteoporosis rats. The beneficial effect of PTH on bone turnover disappeared 8 weeks after discontinuing the treatment. The administration of 32A1 once every 4 weeks for 8 weeks suppressed bone resorption and bone formation when the treatment was switched from PTH to 32A1, leading to the maintenance of BMD and bone strength. Unlike with ALN, the onset of suppression of bone resorption with 32A1 was rapid, while the suppression of bone formation was mild. The improvement of bone mass, beneficial bone turnover balance, and suppression of osteoclast differentiation/multinucleation achieved by 32A1 were supported by histomorphometry. Notably, the effects of 32A1 on bone strength, not only structural (extrinsic) but also material (intrinsic) properties, were significantly greater than those of ALN. Since the effect of 32A1 on BMD was moderate, its effect on bone strength could not be fully explained by the increase in BMD. The beneficial balance of bone turnover caused by 32A1 might, at least in part, be responsible for the improvement in bone quality. This is the first report describing the effects of anti-Siglec-15 antibody in OVX rats; the findings suggest that this antibody could be an excellent candidate for treating osteoporosis, especially in continuation therapy after PTH treatment, due to its rapid action and unprecedented beneficial effects on bone quality.

Anti-Siglec-15 antibody suppresses bone resorption by inhibiting osteoclast multinucleation without attenuating bone formation.

Anti-resorptive drugs are widely used for the treatment of osteoporosis, but excessive inhibition of osteoclastogenesis can suppress bone turnover and cause the deterioration of bone quality. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a transmembrane protein expressed on osteoclast precursor cells and mature osteoclasts. Siglec-15 regulates proteins containing immunoreceptor tyrosine-based activation motif (ITAM) domains, which then induce nuclear factor of activated T-cells 1 (NFATc1), a master transcription factor of osteoclast differentiation. Anti-Siglec-15 antibody modulates ITAM signaling in osteoclast precursors and inhibits the maturation of osteoclasts in vitro. However, in situ pharmacological effects, particularly during postmenopausal osteoporosis, remain unclear. Here, we demonstrated that anti-Siglec-15 antibody treatment protected against ovariectomy-induced bone loss by specifically inhibiting the generation of multinucleated osteoclasts in vivo. Moreover, treatment with anti-Siglec-15 antibody maintained bone formation to a greater extent than with risedronate, the first-line treatment for osteoporosis. Intravital imaging revealed that anti-Siglec-15 antibody treatment did not cause a reduction in osteoclast motility, whereas osteoclast motility declined following risedronate treatment. We evaluated osteoclast activity using a pH-sensing probe and found that the bone resorptive ability of osteoclasts was lower following anti-Siglec-15 antibody treatment compared to after risedronate treatment. Our findings suggest that anti-Siglec-15 treatment may have potential as an anti-resorptive therapy for osteoporosis, which substantially inhibits the activity of osteoclasts while maintaining physiological bone coupling.

Osteoclast differentiation by RANKL and OPG signaling pathways.

INTRODUCTION: In bone tissue, bone resorption by osteoclasts and bone formation by osteoblasts are repeated continuously. Osteoclasts are multinucleated cells that derive from monocyte-/macrophage-lineage cells and resorb bone. In contrast, osteoblasts mediate osteoclastogenesis by expressing receptor activator of nuclear factor-kappa B ligand (RANKL), which is expressed as a membrane-associated cytokine. Osteoprotegerin (OPG) is a soluble RANKL decoy receptor that is predominantly produced by osteoblasts and which prevents osteoclast formation and osteoclastic bone resorption by inhibiting the RANKL-RANKL receptor interaction. MATERIALS AND METHODS: In this review, we would like to summarize our experimental results on signal transduction that regulates the expression of RANKL and OPG. RESULTS: Using OPG gene-deficient mice, we have demonstrated that OPG and sclerostin produced by osteocytes play an important role in the maintenance of cortical and alveolar bone. In addition, it was shown that osteoclast-derived leukemia inhibitory factor (LIF) reduces the expression of sclerostin in osteocytes and promotes bone formation. WP9QY (W9) is a peptide that was designed to be structurally similar to one of the cysteine-rich TNF-receptortype-I domains. Addition of the W9 peptide to bone marrow culture simultaneously inhibited osteoclast differentiation and stimulated osteoblastic cell proliferation. An anti-sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) antibody inhibited multinucleated osteoclast formation induced by RANKL and macrophage colony-stimulating factor (M-CSF). Pit-forming activity of osteoclasts was also inhibited by the anti-Siglec-15 antibody. In addition, anti-Siglec-15 antibody treatment stimulated the appearance of osteoblasts in cultures of mouse bone marrow cells in the presence of RANKL and M-CSF. CONCLUSIONS: Bone mass loss depends on the RANK-RANKL-OPG system, which is a major regulatory system of osteoclast differentiation induction, activation, and survival.

Siglec-15-targeting therapy protects against glucocorticoid-induced osteoporosis of growing skeleton in juvenile rats.

Effective treatment of juvenile osteoporosis, which is frequently caused by glucocorticoid (GC) therapy, has not been established due to limited data regarding the efficacy and adverse effects of antiresorptive therapies on the growing skeleton. We previously demonstrated that sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) targeting therapy, which interferes with osteoclast terminal differentiation in the secondary, but not primary, spongiosa, increased bone mass without adverse effects on skeletal growth, whereas bisphosphonate, a first-line treatment for osteoporosis, increased bone mass but impaired long bone growth in healthy growing rats. In the present study, we investigated the efficacy of anti-Siglec-15 neutralizing antibody (Ab) therapy against GC-induced osteoporosis in a growing rat model. GC decreased bone mass and deteriorated mechanical properties of bone, due to a disproportionate increase in bone resorption. Both anti-Siglec-15 Ab and alendronate (ALN) showed protective effects against GC-induced bone loss by suppressing bone resorption, which was more pronounced with anti-Siglec-15 Ab treatment, possibly due to a reduced negative impact on bone formation. ALN induced histological abnormalities in the growth plate and morphological abnormalities in the long bone metaphysis but did not cause significant growth retardation. Conversely, anti-Siglec-15 Ab did not show any negative impact on the growth plate and preserved normal osteoclast and chondroclast function at the primary spongiosa. Taken together, these results suggest that anti-Siglec-15 targeting therapy could be a safe and efficacious prophylactic therapy for GC-induced osteoporosis in juvenile patients.

Siglec-15-targeting therapy increases bone mass in rats without impairing skeletal growth.

The treatment of juvenile osteoporosis has not been established due to a lack of data regarding the efficacy and adverse effects of therapeutic agents. The possible adverse effects of the long-term use of antiresorptive therapies on skeletal growth in children is of particular concern. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is an immunoreceptor that regulates osteoclast development and bone resorption, and its deficiency suppresses bone remodeling in the secondary spongiosa, but not in the primary spongiosa, due to a compensatory mechanism of osteoclastogenesis. This prompted us to develop an anti-Siglec-15 therapy for juvenile osteoporosis because most anti-resorptive drugs have potential adverse effects on skeletal growth. Using growing rats, we investigated the effects of an anti-Siglec-15 neutralizing antibody (Ab) on systemic bone metabolism and skeletal growth, comparing this drug to bisphosphonate, a first-line treatment for osteoporosis. Male 6-week-old F344/Jcl rats were randomized into six groups: control (PBS twice per week), anti-Siglec-15 Ab (0.25, 1, or 4 mg/kg every 3 weeks), and alendronate (ALN) (0.028 or 0.14 mg/kg twice per week). Treatment commenced at 6 weeks of age and continued for the next 6 weeks. Changes in bone mass, bone metabolism, bone strength, and skeletal growth during treatment were analyzed. Both anti-Siglec-15 therapy and ALN increased bone mass and the mechanical strength of both the femora and lumbar spines in a dose-dependent manner. Anti-Siglec-15 therapy did not have a significant effect on skeletal growth as evidenced by micro-CT-based measurements of femoral length and histology, whereas high-dose ALN resulted in growth retardation with histological abnormalities in the growth plates of femurs. This unique property of the anti-Siglec-15 Ab can probably be attributed to compensatory signaling for Siglec-15 inhibition in the primary spongiosa, but not in the secondary spongiosa. Thus, anti-Siglec-15 therapy could be a safe and effective for juvenile osteoporosis.

Impaired osteoclast differentiation and function and mild osteopetrosis development in Siglec-15-deficient mice.

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a cell surface receptor for sialylated glycan ligands. Recent in vitro studies revealed upregulated Siglec-15 expression in differentiated osteoclasts and inhibition of osteoclast differentiation by anti-Siglec-15 polyclonal antibody, demonstrating Siglec-15 involvement in osteoclastogenesis. To discern the physiological role of Siglec-15 in skeletal development and osteoclast formation and/or function in vivo, we generated Siglec-15-deficient (siglec-15(-/-)) mice and analyzed their phenotype. The siglec-15(-/-) mice developed without physical abnormalities other than increased trabecular bone mass in lumbar vertebrae and metaphyseal regions of the femur and tibia, causing mild osteopetrosis. Histological analyses demonstrated that the number of osteoclasts present on the femoral trabecular bone of the mutant mice was comparable to that of the wild-type mice. However, urinary deoxypyridinoline, a systemic bone resorption marker, decreased in the siglec-15(-/-) mice, indicating that impaired osteoclast function was responsible for increased bone mass in the mutant mice. In addition, the ability of bone marrow-derived monocytes/macrophages from the siglec-15(-/-) mice to differentiate into osteoclasts was impaired, as determined in vitro by cellular tartrate-resistant acid phosphatase activity in response to the receptor activator of nuclear factor-κB ligand or tumor necrosis factor-α. These results reveal the importance of Siglec-15 in the regulation of osteoclast formation and/or function in vivo, providing new insights into osteoclast biology.

Inhibition of experimental colorectal cancer and reduction in renal and gastrointestinal toxicities by copper-indomethacin in rats.

PURPOSE: To evaluate, for the first time, the efficacy of copper-indomethacin in the inhibition of aberrant crypt foci formation using the azoxymethane-induced adenocarcinoma model, to examine cell viability in the HCT-116 colorectal cancer cell line, gastrointestinal permeability, mitochondrial oxidative damage, and renal toxicity in rat models. METHODS: Azoxymethane-induced adenocarcinoma rats were dosed with indomethacin and copper-indomethacin for 28 days and aberrant crypt foci were evaluated. HCT-116 colorectal cancer cells were exposed to indomethacin and copper-indomethacin at 0-250 microg/mL (0-698 microM for indomethacin, and 0-147 microM for copper-indomethacin), and cell viability was measured. Acute gastrointestinal toxicity was measured using gastrointestinal permeability markers, gastrointestinal ulceration and bleeding, and measurement of an acute-phase protein haptoglobin. Effects of acute and chronic administration of indomethacin and copper-indomethacin on urinary electrolyte concentrations were examined. RESULTS: Both indomethacin and copper-indomethacin resulted in a significant reduction in aberrant crypt foci in azoxymethane-treated rats. In parallel, high concentrations of indomethacin and copper-indomethacin also reduced cell viability in HCT-116 colorectal cancer cells. However, copper-indomethacin was considerably safer in all measures of gastrointestinal toxicity compared to indomethacin. In addition, indomethacin reduced urinary electrolytes at an ulcerogenic dose of 10 mg/kg acutely and chronically at 3.0 mg/kg for 28 days, whereas copper-indomethacin at equimolar doses of indomethacin affected urine electrolytes after acute dosing but not after chronic dosing for 28 days. CONCLUSIONS: Copper-indomethacin has both gastrointestinal and renal sparing properties while maintaining efficacy in experimental adenocarcinoma.

Direct high-performance liquid chromatographic analysis of D-tocopheryl acid succinate and derivatives.

A method of analysis of a Vitamin E derivative D-tocopheryl acid succinate (TS) in biological fluids and commercially available products is necessary to study the kinetics of in vitro and in vivo metabolism, tissue distribution, and content uniformity. A simple and inexpensive high-performance liquid chromatographic method was developed for the direct determination of D-tocopheryl acid succinate in commercially available products, rat serum, and rat tissues. This method can also be applied to the determination of 15 Vitamin E derivatives. Rat serum (0.1 ml) was extracted with sodium dodecyl sulfate, ethanol, hexane, and then dried under nitrogen gas after addition of the internal standard, DL-alpha-tocopherol acetate. Separation was achieved on a C18 column with UV detection at 205 nm. The calibration curve for D-tocopheryl acid succinate was linear ranging from 0.025 to 100 microg/ml. The mean extraction efficiency was >92%. Precision of the assay was <5% (CV), and was within 5% at the limit of quantitation (0.025 microg/ml). Bias of the assay was lower than 5%, and was within 5% at the limit of quantitation. The assay was applied successfully to the serum and tissue distribution of D-tocopheryl acid succinate in rats, various Vitamin E derivatives, and content uniformity in commercially available products containing D-tocopheryl acid succinate.

Pharmacokinetics and tissue distribution of d-alpha-tocopheryl succinate formulations following intravenous administration in the rat.

Unlike d-alpha tocopherol (T), d-alpha tocopheryl succinate (TS) has the unique ability to selectively kill tumor cells while protecting normal tissue from toxic oxidative stress. The pharmacokinetics of TS and the serum and tissue disposition of TS were studied in male Sprague-Dawley rats to delineate formulation dependent disposition between TS administered as the Tris salt (TS-T) (a liposomal formulation) or as the free acid (TS-FA) dissolved in polyethylene glycol (PEG) 400. The pharmacokinetics of TS was studied after single intravenous (i.v.) equimolar doses of 124 mg/kg TS-T and 100 mg/kg of TS-FA. Serial blood samples were collected via a catheter inserted into the right jugular vein and serum samples were analysed for TS and T levels using a reverse phase HPLC method. Terminal tissue samples were also collected at 24 and 48 h. After an acute i.v. dose of TS-T, serum AUC, t(1/2), Cl and V(d) of TS were 2601.0 +/- 351.7 microg h/ml, 9.98 +/- 1.02 h, 0.049 +/- 0. 0073 l/h/kg and 0.7 +/- 0.14 l/kg (mean +/- SD), respectively. The acute i.v. administration of TS-FA (PEG formulation) yielded results similar to those observed for TS-T, with a serum AUC, t(1/2), Cl and V(d) of 2553.3 +/- 166.4 microg h/ml, 9.83 +/- 0.86 h, 0.039 +/- 0.0027 l/h/kg and 0.56 +/- 0.09 l/kg (mean +/- SD), respectively. Distribution into tissues and a low Cl was apparent, with the highest concentrations of TS in the liver and lung, regardless of formulation. As expected, baseline endogenous concentrations of T were present in both groups, with a net increase in T levels, occurring as TS was hydrolysed to T, which slowly peaked in serum between 7-8 h post-dose. Intravenous TS administration, regardless of formulation, also resulted in significant T accumulation in all tissues examined, which was especially abundant in the liver and lung. Likewise, there was a lack of significant effect of formulation on the pharmacokinetics and tissue distribution of TS. The only significant formulation difference was a small but significant increase in serum T and liver T levels in the TS in PEG formulation group. These results indicate that TS may be especially useful for the targeted delivery of T and TS to the lung and liver for anti-oxidant and anti-cancer activity.