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• W1976475315 abstract "Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo. Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo. Receptor activator of nuclear factor-κB ligand (RANKL) 2The abbreviations used are: RANKLreceptor activator of nuclear factor-κB ligandALPalkaline phosphataseBFR/BSbone formation rate/bone surfaceBMDbone mineral densityBV/TVbone volume/tissue volumeES/BSeroded surface/bone surfacehRANKLhuman soluble RANKLhuRANKL micehuman RANKL knock-in miceMabmonoclonal antibodyMARmineral apposition ratemRANKLmouse soluble RANKLN.Ob/BSosteoblast number/bone surfaceN.Oc/BSosteoclast number/bone surfaceOb.S/BSosteoblast surface/bone surfaceOc.S/BSosteoclast surface/bone surfaceOPGosteoprotegerinpNPPp-nitrophenyl phosphatepQCTperipheral quantitative computed tomographyPTHparathyroid hormonesRANKsoluble RANKsRANKLsoluble RANKLSXAsingle-energy x-ray absorptiometryTb.Thtrabecular bone thicknessTNFtumor necrosis factorTRAILTNF-related apoptosis-inducing ligandTRAPtartrate resistant acid phosphataseX-SSIx axis strength strain indexANOVAanalysis of variance. is a member of the tumor necrosis factor (TNF) superfamily that binds to its receptor RANK, which is expressed on osteoclasts and their progenitors (1Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3597-3602Crossref PubMed Scopus (3558) Google Scholar, 2Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4612) Google Scholar). The interaction of RANK with RANKL is required for osteoclast formation, differentiation, activation, and survival (3Nakagawa N. Kinosaki M. Yamaguchi K. Shima N. Yasuda H. Yano K. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1998; 253: 395-400Crossref PubMed Scopus (616) Google Scholar, 4Hsu H. Lacey D.L. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Kelley M.J. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3540-3545Crossref PubMed Scopus (1416) Google Scholar). Osteoprotegerin (OPG) is a natural decoy receptor for RANKL (5Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4332) Google Scholar, 6Tsuda E. Goto M. Mochizuki S. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Crossref PubMed Scopus (729) Google Scholar, 7Yasuda H. Shima N. Nakagawa N. Mochizuki S.I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; 139: 1329-1337Crossref PubMed Scopus (965) Google Scholar). Bone resorption is mediated by RANKL-RANK signaling, and excessive osteoclastic bone resorption plays a central role in the pathogenesis of age-related bone loss and microstructural deterioration, leading to fragility fractures (8Shevde N.K. Bendixen A.C. Dienger K.M. Pike J.W. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7829-7934Crossref PubMed Scopus (409) Google Scholar, 9Eghbali-Fatourechi G. Khosla S. Sanyal A. Boyle W.J. Lacey D.L. Riggs B.L. J. Clin. Invest. 2003; 111: 1221-1230Crossref PubMed Scopus (657) Google Scholar, 10Tanaka S. Nakamura K. Takahasi N. Suda T. Immunol. Rev. 2005; 208: 30-49Crossref PubMed Scopus (260) Google Scholar). receptor activator of nuclear factor-κB ligand alkaline phosphatase bone formation rate/bone surface bone mineral density bone volume/tissue volume eroded surface/bone surface human soluble RANKL human RANKL knock-in mice monoclonal antibody mineral apposition rate mouse soluble RANKL osteoblast number/bone surface osteoclast number/bone surface osteoblast surface/bone surface osteoclast surface/bone surface osteoprotegerin p-nitrophenyl phosphate peripheral quantitative computed tomography parathyroid hormone soluble RANK soluble RANKL single-energy x-ray absorptiometry trabecular bone thickness tumor necrosis factor TNF-related apoptosis-inducing ligand tartrate resistant acid phosphatase x axis strength strain index analysis of variance. In addition to regulation of bone homeostasis, RANKL also plays an important role in the immune system, cancer metastasis, and differentiation of mammary gland stem cells (11Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett 3rd, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 12Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1938) Google Scholar, 13Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2857) Google Scholar, 14Oyajobi B.O. Anderson D.M. Traianedes K. Williams P.J. Yoneda T. Mundy G.R. Cancer Res. 2001; 61: 2572-2578PubMed Google Scholar, 15Jones D.H. Nakashima T. Sanchez O.H. Kozieradzki I. Komarova S.V. Sarosi I. Morony S. Rubin E. Sarao R. Hojilla C.V. Komnenovic V. Kong Y.Y. Schreiber M. Dixon S.J. Sims S.M. Khokha R. Wada T. Penninger J.M. Nature. 2006; 440: 692-696Crossref PubMed Scopus (640) Google Scholar, 16Akiyama T. Shimo Y. Yanai H. Qin J. Ohshima D. Maruyama Y. Asaumi Y. Kitazawa J. Takayanagi H. Penninger J.M. Matsumoto M. Nitta T. Takahama Y. Inoue J. Immunity. 2008; 29: 423-437Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 17Asselin-Labat M.L. Vaillant F. Sheridan J.M. Pal B. Wu D. Simpson E.R. Yasuda H. Smyth G.K. Martin T.J. Lindeman G.J. Visvader J.E. Nature. 2010; 465: 798-802Crossref PubMed Scopus (539) Google Scholar). These studies suggest that RANKL may have further unknown functions in vivo. To investigate these possible functions, OPG and soluble RANK (sRANK) have been used in previous studies (4Hsu H. Lacey D.L. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Kelley M.J. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3540-3545Crossref PubMed Scopus (1416) Google Scholar, 5Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4332) Google Scholar, 6Tsuda E. Goto M. Mochizuki S. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Crossref PubMed Scopus (729) Google Scholar, 7Yasuda H. Shima N. Nakagawa N. Mochizuki S.I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; 139: 1329-1337Crossref PubMed Scopus (965) Google Scholar, 18Dougall W.C. Glaccum M. Charrier K. Rohrbach K. Brasel K. De Smedt T. Daro E. Smith J. Tometsko M.E. Maliszewski C.R. Armstrong A. Shen V. Bain S. Cosman D. Anderson D. Morrissey P.J. Peschon J.J. Schuh J. Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1197) Google Scholar, 19Capparelli C. Morony S. Warmington K. Adamu S. Lacey D. Dunstan C.R. Stouch B. Martin S. Kostenuik P.J. J. Bone Miner. Res. 2003; 18: 852-858Crossref PubMed Scopus (93) Google Scholar, 20Ominsky M.S. Kostenuik P.J. Cranmer P. Smith S.Y. Atkinson J.E. Osteoporos. Int. 2007; 18: 1073-1082Crossref PubMed Scopus (51) Google Scholar). However, pharmacokinetic studies have shown that these recombinant proteins are not maintained in serum for a long period, which makes it necessary to inject the proteins into animals frequently, e.g. every day to inhibit RANKL in vivo. OPG also binds to the TNF-related apoptosis-inducing ligand (TRAIL) and inhibits TRAIL functions (21Emery J.G. McDonnell P. Burke M.B. Deen K.C. Lyn S. Silverman C. Dul E. Appelbaum E.R. Eichman C. DiPrinzio R. Dodds R.A. James I.E. Rosenberg M. Lee J.C. Young P.R. J. Biol. Chem. 1998; 273: 14363-14367Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar, 22Truneh A. Sharma S. Silverman C. Khandekar S. Reddy M.P. Deen K.C. McLaughlin M.M. Srinivasula S.M. Livi G.P. Marshall L.A. Alnemri E.S. Williams W.V. Doyle M.L. J. Biol. Chem. 2000; 275: 23319-23325Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), although the affinity of sRANK for soluble RANKL (sRANKL) is lower than that of OPG (3Nakagawa N. Kinosaki M. Yamaguchi K. Shima N. Yasuda H. Yano K. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1998; 253: 395-400Crossref PubMed Scopus (616) Google Scholar, 4Hsu H. Lacey D.L. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Kelley M.J. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3540-3545Crossref PubMed Scopus (1416) Google Scholar, 23Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Goto M. Mochizuki S.I. Tsuda E. Morinaga T. Udagawa N. Takahashi N. Suda T. Higashio K. Bone. 1999; 25: 109-113Crossref PubMed Scopus (228) Google Scholar). Therefore, OPG and sRANK may not be suitable for specific inhibition of RANKL in vivo. Denosumab, a fully human anti-RANKL-neutralizing monoclonal antibody (Mab), has recently been approved in Europe and the United States for treatment of osteoporosis and in the United States for prevention of skeletal related events in patients with bone metastases from solid tumors. Because denosumab is not cross-reactive with rodent RANKL, its evaluation in preclinical studies was performed in cynomolgus monkeys or human RANKL knock-in mice (huRANKL mice), in which exon 5 in mouse rankl was replaced with that in human RANKL (24Kostenuik P.J. Nguyen H.Q. McCabe J. Warmington K.S. Kurahara C. Sun N. Chen C. Li L. Cattley R.C. Van G. Scully S. Elliott R. Grisanti M. Morony S. Tan H.L. Asuncion F. Li X. Ominsky M.S. Stolina M. Dwyer D. Dougall W.C. Hawkins N. Boyle W.J. Simonet W.S. Sullivan J.K. J. Bone Miner. Res. 2009; 24: 182-195Crossref PubMed Scopus (325) Google Scholar). However, there were several abnormalities in huRANKL mice, including a decreased osteoclast number, increased trabecular bone mineral density (BMD), and a reduced osteoblast surface, compared with normal mice, and these abnormalities reduce the suitability of these mice for analysis of RANKL inhibition with an anti-RANKL-neutralizing Mab such as denosumab (24Kostenuik P.J. Nguyen H.Q. McCabe J. Warmington K.S. Kurahara C. Sun N. Chen C. Li L. Cattley R.C. Van G. Scully S. Elliott R. Grisanti M. Morony S. Tan H.L. Asuncion F. Li X. Ominsky M.S. Stolina M. Dwyer D. Dougall W.C. Hawkins N. Boyle W.J. Simonet W.S. Sullivan J.K. J. Bone Miner. Res. 2009; 24: 182-195Crossref PubMed Scopus (325) Google Scholar, 25Gerstenfeld L.C. Sacks D.J. Pelis M. Mason Z.D. Graves D.T. Barrero M. Ominsky M.S. Kostenuik P.J. Morgan E.F. Einhorn T.A. J. Bone Miner. Res. 2009; 24: 196-208Crossref PubMed Scopus (175) Google Scholar, 26Hofbauer L.C. Zeitz U. Schoppet M. Skalicky M. Schüler C. Stolina M. Kostenuik P.J. Erben R.G. Arthritis Rheum. 2009; 60: 1427-1437Crossref PubMed Scopus (107) Google Scholar, 27Pierroz D.D. Bonnet N. Baldock P.A. Ominsky M.S. Stolina M. Kostenuik P.J. Ferrari S.L. J. Biol. Chem. 2010; 285: 28164-28173Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Parathyroid hormone (PTH) is the only bone anabolic agent that is currently used for treatment of osteoporosis in humans. The precise mechanisms through which PTH increases bone formation in vivo are unknown, but previous studies have shown that osteoclasts are required for the bone anabolic effect of PTH (27Pierroz D.D. Bonnet N. Baldock P.A. Ominsky M.S. Stolina M. Kostenuik P.J. Ferrari S.L. J. Biol. Chem. 2010; 285: 28164-28173Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 28Koh A.J. Demiralp B. Neiva K.G. Hooten J. Nohutcu R.M. Shim H. Datta N.S. Taichman R.S. McCauley L.K. Endocrinology. 2005; 146: 4584-4596Crossref PubMed Scopus (97) Google Scholar). To investigate the effects of RANKL inhibition on bone mass and other features in normal mice, we prepared an anti-mouse RANKL-neutralizing Mab (OYC1) and established a novel mouse osteopetrotic model with high bone mass induced by administration of OYC1 to normal mice. In this study, we characterized OYC1 and established a method for long term neutralization of RANKL in vivo in normal mice, in which a single injection of OYC1 neutralized RANKL activity for 4 weeks. We examined the effect of OYC1 on bone mass and showed the utility of OYC1 for evaluating the bone anabolic effect of PTH. Two hybridoma-producing mouse RANKL Mabs (clones OYC1 and OYC2) were subcloned from hybridoma kindly provided by Dr. Okumura (Juntendo University School of Medicine) and manufactured by Oriental Yeast Co. (29Kamijo S. Nakajima A. Ikeda K. Aoki K. Ohya K. Akiba H. Yagita H. Okumura K. Biochem. Biophys. Res. Commun. 2006; 347: 124-132Crossref PubMed Scopus (50) Google Scholar). Recombinant human OPG-Fc and mouse soluble RANKL (sRANKL) were purchased from R&D Systems. PTH(1–34) and calcein were purchased from Sigma. Other reagents were purchased from Nacalai Tesque, Inc. (Japan). Five-week-old female C57BL/6N mice were purchased from Charles River Inc. and acclimated for 1 week under standard laboratory conditions at 24 ± 2 °C and 40–70% humidity. Mice were treated according to the institutional ethical guidelines for animal experimentation and safety. To establish the effect of the mRANKL Mabs on bone mass, the neutralizing antibody (OYC1) and non-neutralizing control antibody (OYC2) were administered intraperitoneally to 6-week-old female mice (n = 5) three times per week for 2 weeks. Calcein was injected twice subcutaneously for labeling on days 10 and 13. At 12 h after the last administration, femurs were extirpated and fixed with 70% ethanol. To determine the suboptimal dose of OYC1 for increasing the BMD, various doses (0.5, 1, 1.5, 5, and 15 mg/kg) of OYC1 or vehicle (PBS) were injected subcutaneously in 6-week-old female mice (n = 5) once on day 0. Blood samples and both femurs were obtained on day 14, and the femurs were fixed with 70% ethanol. To examine the time course of the effect of OYC1, 5 mg/kg OYC1 or PBS was administered subcutaneously to 6-week-old female mice (n = 5–6) on day 0. The mice were sacrificed on days 4, 7, 14, and 28, and sera and femurs were obtained on these days. To examine the early part of the time course in more detail, 5 mg/kg OYC1 or PBS was administered subcutaneously to 6-week-old female mice (n = 5–6) on day 0. The mice were sacrificed on days 1–4, and sera and femurs were obtained on these days. To examine the utility of the RANKL-neutralizing model, we tested whether PTH could induce bone formation in OYC1-treated mice. OYC1 (5 mg/kg) or PBS was injected once in 6-week-old female mice (n = 5). After 4 days, PTH (160 μg/kg) or PBS was injected subcutaneously daily for 2 weeks in these mice. The mice treated with PTH after transient neutralization of RANKL with OYC1 were sacrificed on day 18, and sera, femurs, and tibias were collected. BMDs were determined in fixed right femurs by single energy x-ray absorptiometry (SXA, DCS-600EX; Aloka, Japan). The femurs were divided into 20 equal regions from distal (region 1) to proximal (region 20), and the BMD of each region was measured by SXA. In some experiments, BMD was measured by peripheral quantitative computed tomography (pQCT, XCT Research SA+, Stratec Medizintechnic, Pforzheim, Germany) using a voxel size of 0.08 × 0.08 × 0.46 mm. Image analysis was carried out using integrated XCT 2000 software version 6.00f. Total BMD at 0.8 or 1.0 mm from the growth plate is shown due to the marked increase of BMD after injection of OYC1. Cortical bone was defined based on BMD >690 mg/cm3 at the femoral metaphysis. Cortical area (mm2), cortical thickness (mm), periosteal perimeter of the cortical bone (in mm), endosteal perimeter of the cortical bone (in mm), x axis strength-strain index (in mm3), y axis strength-strain index (in mm3), and polar strength-strain index (in mm3) were measured. Three-dimensional digital images of femurs were reconstructed by micro-CT analysis at 0.2–1.2 mm from the growth plate using a ScanXmate-A080 (Comscan Tecno). Analysis of micro-CT images was performed with TRI/3D-Bon (Ratoc System Engineering Co., Japan). Fixed and undecalcified femurs were embedded in glycol methacrylate or methyl methacrylate and 3–5-μm sections in the proximal region and distal femoral metaphysis of each femur were cut longitudinally and stained with toluidine blue O. Some samples were stained by the Villanueva method. Histomorphometry was performed with an image analysis system (Ostoplan II, Carl Zeiss, New York) linked to a light microscope. Histomorphometric measurements were made at ×400 magnification in the secondary spongiosa area. The bone volume/tissue volume (BV/TV, %), trabecular bone thickness (Tb.Th, μm) osteoclast surface/bone surface (Oc.S/BS, %), eroded surface/bone surface (ES/BS, %), osteoclast number/bone surface (N.Oc/BS, 1/mm), osteoblast surface/bone surface (Ob.S/BS, %), osteoblast number/bone surface (N.Ob/BS, 1/mm), the mineral apposition rate (MAR, μm/day), bone formation rate/bone surface (BFR/BS, mm3/mm2/year) were calculated and expressed according to standard formulas and nomenclature (30Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4899) Google Scholar). For TRAP staining, 3-μm sections in the proximal metaphysis of the undecalcified tibia were cut longitudinally and stained with TRAP. Serum levels of calcium and ALP were measured according to the manufacturers' instructions using a calcium C assay and a LabAssay ALP (Wako, Japan) (p-nitrophenyl phosphate (pNPP) assay), respectively. Serum mouse osteocalcin, TRAP-5b, and intact PTH were measured by enzyme-linked immunosorbent assay (ELISA) according to manufacturers' instructions (Biomedical Technologies, MA; Immunodiagnostic Systems, AZ; and Immutopics, CA; respectively). The serum level of OYC1 was measured by ELISA. Recombinant mRANKL was coated on an ELISA plate (Nunc) at 2.5 nm, and the serum antibody titer was detected by HRP-conjugated rat IgG2a (Zymed Laboratories Inc.). Each value was determined by absorbance at 450 nm using a microplate reader. Results are expressed as means ± S.D. for all data. Significance of differences was determined by ANOVA with a Dunnett's test or Student's t test. p < 0.05 was considered significant. To characterize the anti-mRANKL Mabs, OYC1 and OYC2, a binding assay was performed by direct ELISA. Both antibodies recognized recombinant mouse sRANKL (mRANKL), including the extracellular domain from Arg-72 to Asp-316 (supplemental Fig. 1A). Neither OYC1 nor OYC2 bound to mouse TRAIL (data not shown). OYC1 neutralized mRANKL-induced osteoclastogenesis in a dose-dependent manner. OYC1 (0.2 μg/ml) and human OPG-Fc (0.1 μg/ml) completely suppressed osteoclast formation induced by 5 nm mRANKL, but OYC2 did not do so (supplemental Fig. 1B). OYC1 did not bind to or inhibit human sRANKL (hRANKL, supplemental Fig. 1B, and data not shown). Morphological examination of cells cultured in the presence of mRANKL with or without OYC1 confirmed the complete inhibition of formation of TRAP-positive multinucleated cells (supplemental Fig. 1C). These results indicate that OYC1 is a neutralizing Mab and OYC2 is a non-neutralizing Mab. To investigate the effect of the Mabs in vivo, 15 mg/kg OYC1 or OYC2 or vehicle (PBS) were administered intraperitoneally to mice three times per week for 2 weeks (Fig. 1A). OYC2 showed no effect on BMD in femurs of the treated mice, whereas OYC1 markedly increased the BMD, especially in the distal metaphysis, compared with OYC2 and vehicle. In contrast, OYC1 had little effect on BMD in the femoral diaphysis (Fig. 1A). Photomicrographs of toluidine blue O staining of femurs of the OYC1-treated mice also indicated a marked increase in bone mass in the distal femoral metaphysis (Fig. 1B). Histomorphometry revealed marked decreases in Oc.S/BS, N.Oc/BS, and ES/BS (Fig. 1C) and also marked decreases in Ob.S/BS, MAR, and BFR/BS in the secondary trabecular area in femurs in OYC1-treated mice (Fig. 1C). Because OYC2 exhibited no effect on bone mass (Fig. 1, A–C), we used vehicle (PBS) as a negative control in the following experiments. To establish a simple method for increasing bone mass, various doses (0.5, 1, 1.5, 5, and 15 mg/kg) of OYC1 were injected subcutaneously once in female mice, and femurs and sera were obtained for analysis after 2 weeks. SXA and pQCT analyses showed that the total BMD in the femurs was augmented significantly at doses of 5 and 15 mg/kg OYC1 (Fig. 2A and data not shown). Micro-CT analysis of the femurs confirmed marked augmentation of bone mass at 5 and 15 mg/kg OYC1 (Fig. 2B). Histomorphometric analysis of the distal femoral metaphysis in mice given 5 and 15 mg/kg OYC1 also showed significant decreases in Ob.S/BS, Oc.S/BS, N.Oc/BS, and ES/BS (Fig. 2C). Consistent with these results, serum TRAP-5b and ALP decreased to similar levels at 5 and 15 mg/kg OYC1 (Fig. 2D). There was no difference in the serum calcium concentration at any dose of OYC1 (data not shown). Pharmacokinetic studies showed rapid clearance of OYC1 at doses of 0.5 and 1.5 mg/kg but significantly higher serum concentrations of OYC1 of >40 and >100 μg/ml at 5 and 15 mg/kg, respectively (Fig. 2D). Denosumab has been characterized as a drug with long term efficacy in clinical trials (31Bekker P.J. Holloway D.L. Rasmussen A.S. Murphy R. Martin S.W. Leese P.T. Holmes G.B. Dunstan C.R. DePaoli A.M. J. Bone Miner. Res. 2004; 19: 1059-1066Crossref PubMed Scopus (671) Google Scholar). The concentration of denosumab in serum remained high for a week after injection in hRANKL knock-in mice (24Kostenuik P.J. Nguyen H.Q. McCabe J. Warmington K.S. Kurahara C. Sun N. Chen C. Li L. Cattley R.C. Van G. Scully S. Elliott R. Grisanti M. Morony S. Tan H.L. Asuncion F. Li X. Ominsky M.S. Stolina M. Dwyer D. Dougall W.C. Hawkins N. Boyle W.J. Simonet W.S. Sullivan J.K. J. Bone Miner. Res. 2009; 24: 182-195Crossref PubMed Scopus (325) Google Scholar). To examine the duration of efficacy of OYC1, we examined the time course of the effects of OYC1 in mice by measuring total BMD, and the concentrations of serum TRAP-5b, ALP, and OYC1 between days 4 and 28 (Fig. 3, A–C). OYC1 significantly elevated femoral total BMD in a time-dependent manner from days 4 to 28 after a single subcutaneous injection (Fig. 3A). Expansion of the dense bone structure in the trabecular area was accompanied by augmentation of total BMD, which was sustained until day 28 (Fig. 3, A and B). Surprisingly, the concentrations of OYC1 remained at >50 μg/ml in serum until day 14 and at >10 μg/ml until day 28 (Fig. 3C). Maintenance of the high OYC1 concentration resulted in a continued increase in bone mass daily until day 28 (Fig. 3, A and B). To confirm this long term effect of OYC1, biomarkers of bone turnover were examined. OYC1 markedly decreased serum TRAP-5b and ALP levels on day 4, and these levels then remained constant until day 28 (Fig. 3C). Interestingly, the TRAP-5b level in the OYC1-treated mice was almost undetectable between days 4 and 28 (Fig. 3C). In contrast, the ALP level in the treated mice was reduced to half the level of the vehicle between days 4 and 28 (Fig. 3C). To examine the time course of the effects of OYC1 in more detail in the early period, sera and femurs were collected on days 1–4. Analyses showed that OYC1 significantly increased total BMD in the trabecular area on day 2 and then increased BMD in a time-dependent manner, consistent with micro-CT imaging data (Fig. 4B). Surprisingly, a marked decrease of serum TRAP-5b was observed on day 1, with no change in ALP apparent on day 1 (Fig. 4C). The significant decrease of ALP following that of TRAP-5b indicated down-regulation of bone turnover. Concentrations of OYC1 in serum declined from day 1 to day 4 but were still >90 μg/ml on day 4 (Fig. 4C). To investigate whether the serum TRAP-5b level reflected the conditions of osteoclasts, histological sections of the trabecular bone in tibia of the OYC1-treated mice were stained for TRAP (Fig. 4D). Consistent with the decrease in serum TRAP-5b, there were less TRAP-positive cells (osteoclasts) in the trabecular bone in the OYC1-treated mice on day 1, and only a few osteoclasts were observed in the trabecular bone on day 4. Optimization of the dose and time course effects of OYC1 was performed with the goal of establishing a method for making an osteoclast-depleted mouse model. The above results indicate that single administration of 5 mg/kg OYC1 is suitable for depletion of osteoclasts from days 4 to 28. To examine the bone anabolic effect of PTH in the osteoclast-depleted mice, PTH was injected daily for 2 weeks into mice that had been treated with OYC1 for 4 days. pQCT analysis revealed that administration of PTH and OYC1 increased the total BMD in trabecular bone independently and synergistically (PBS, 465 ± 24; PTH, 607 ± 8; OYC1, 634 ± 40; PTH + OYC1, 719 ± 14 mg/cm3) (Fig. 5A). In contrast, the total BMD in cortical bone was augmented in the PTH groups (PTH and PTH + OYC1) but not with OYC1 treatment alone (PBS, 981 ± 7; PTH, 1001 ± 7; OYC1, 978 ± 17; PTH + OYC1, 1013 ± 15 mg/cm3) (Fig. 5B). The robust depletion of osteoclasts did not affect the anabolic effect of PTH on trabecular and cortical bones. PTH and OYC1 also tended to increase the cortical area, cortical thickness, and polar-SSI, indicating a synergistic effect on bone strength (Table 1). Micro-CT images confirmed the marked increases in trabecular bone mass in mice treated with PTH or OYC1 alone and the synergistic increase with treatment with PTH + OYC1 (Fig. 5C).TABLE 1Effects of PTH, OYC1, and PTH + OYC1 on cortical bone indices in treated micePBSPTHOYC1PTH + OYC1Cortical area0.665 ± 0.016 mm20.752 ± 0.028 mm2ap < 0.01 versus PBS (ANOVA).0.700 ± 0.040 mm2bp < 0.05 versus PBS (ANOVA).0.778 ± 0.021 mm2ap < 0.01 versus PBS (ANOVA).Cortical thickness0.314 ± 0.012 mm0.326 ±" @default.
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• W1976475315 title "Increased Bone Mass in Mice after Single Injection of Anti-receptor Activator of Nuclear Factor-κB Ligand-neutralizing Antibody" @default.
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