The present invention relates to a method for treating bone loss diseases by inhibiting receptor activator of nuclear factor-KB ligand (RANKL)-induced osteoclast differentiation.
Bone is a living organ that is continuously being reshaped in a process called remodeling. In this process, cells called osteoclasts resorb bone tissues whereas cells called osteoblasts deposit new bone tissue. Osteoblasts can become trapped within the bone matrix they secrete, promoting their differentiation into osteocytes. These cells are thought to play an important role in sensing bone load. Under high loading conditions, osteoblasts increase bone mass, whereas under low loading conditions, osteoclasts remove bone tissue, optimizing its structure. Therefore, osteoclasts are closely related to bone loss or decreased bone density.
People may have bone loss or decreased bone density due to physiological factors or other external factors, which leads to osteoporosis. The signs of osteoporosis may include fracture, back pain, limited ability to act (even unable to move). Currently the treatment methods for osteoporosis include nutrition improvement, exercise, medication, etc. Regarding medication, drugs for clinical use comprise: estrogen or estrogen analogues, drugs for treatment of osteoporosis induced by glucocorticoid hormones, diphosphate, or calcitonin, etc. However, all these drugs described above have side effects, for example, diphosphate may cause nausea, indigestion, diarrhea, or constipation, and calcitonin may cause facial flushing, frequent urination, nausea, or itchy skin. Most of all, the drugs described above are unable to suppress the generation of osteoclasts cells, thus are unable to effectively cure osteoporosis.
Receptor activator of nuclear factor-KB ligand (RANKL) is a critical factor to induce the differentiation of osteoclasts from bone marrow-derived macrophages (BMM).
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The present invention relates to a method for treating a bone loss disease, condition, or disorder in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of a composition comprising thrombomodulin lectin-like domain (TMD1).
The object of the present invention is to treat a disease, condition, or disorder associated with bone loss. In the present invention, receptor activator of nuclear factor-KB ligand (RANKL)-induced osteoclast differentiation is inhibited to achieve therapeutic effect of osteoporosis. The thrombomodulin (TM) used in the present invention is a naturally occurring enzyme. The thrombomodulin lectin-like domain (the first domain of TM, TMD1) can be formulated into a protein drug for inhibiting activation of RANKL, thereby achieve treatment of osteoporosis by inhibition of osteoclast differentiation.
Using the model of RANKL-induced osteoclast differentiation in RAW264.7 cells, the present invention shows for the first time that supplementation of TMD1 significantly attenuates the osteoclast differentiation and bone resorption in a dose-dependent manner. In summary, as shown in the illustrated model in
Therefore, the present invention provides a method for treating a bone loss disease, condition, or disorder in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of a composition comprising thrombomodulin lectin-like domain (TMD1).
In an embodiment, the method inhibits osteoclast differentiation in the subject.
In a further embodiment, the method inhibits activation of receptor activator of nuclear factor-κB ligand (RANKL) in the subject.
In an embodiment, the bone loss disease, condition, or disorder includes but not limited to osteoporosis or rheumatoid arthritis.
In an embodiment, the subject is a mammal. In a further embodiment, the subject is a human.
The examples below are non-limiting and are merely representative of various aspects and features of the present invention. Therefore, the method of the present invention should not be limited to only for diagnosing neurodegenerative disease.
LysMcre/TMflox/flox mice with myeloid specific TM deletion were generated as previously reported. Compared with their controls (TMflox/flox mice), TM expression in the myeloid linage (macrophages and neutrophils), but not in other tissues and organs, was suppressed in LysMcre/TMflox/flox mice. Mice lacking the lectin-like domain of TM (TMLeD/LeD mice) and their controls (TMWT/LeD mice) were a gift from Dr. Conway. These B6-background mice were maintained in a pathogen-free animal facility at the Center of the National Cheng Kung University (NCKU). All animal protocols were approved by the Animal Care Committee of NCKU. Bilateral ovariectomy (OVX), as described previously, was performed at 8 weeks of age.
Human PBMCs were separated from whole blood using a Ficoll-Paque PLUS (GE Healthcare, Brussels, Belgium) gradient based on the manufacturer's instructions. Mouse bone marrow macrophages/monocytes (BM:Ms) were isolated from the femur and tibia (6 to 12 weeks old) as described previously with some modifications. Briefly, isolated total bone marrow cells were incubated overnight in complete Minimum Essential Medium (Alpha modification; α-MEM). Then, nonadherent cells were collected and mononuclear cells were prepared using a Ficoll-Hypaque (GE Healthcare, Piscataway, N.J., USA) density gradient centrifugation. The BM:Ms were evenly scattered across the interface between Ficoll-Hypaque and medium.
Cells (RAW264.7, PBMCs, or BM:Ms) were cultured in 96-well plates (1×105 cells/well). RANKL and M-CSF were added to stimulate osteoclast generation. Media were replenished every 2 days. On day 8, cells were fixed with 3% formaldehyde and were stained with tartrate acid phosphatase (TRAP) (Sigma, St. Louis, Mo., USA). TRAP-positive cells with three or more nuclei were counted as osteoclasts.
TRAP activity was measured using 4-NPP as substrate, based on the microplate assay method with modifications.A 50 μL sample was incubated with 150 μL of substrate consisting of 8 mM 4-NPP in 100 mM sodium acetate buffer containing 500 mM sodium tartrate at 37° C. for 40 min with pH 5.0. The reaction was terminated by the addition of 50 μL 3 M NaOH. Absorbance was measured at 405 nm in a microplate reader (SPECTRAmax™ 340; Molecular Devices, Palo Alto, Calif., USA).
Cells were lysed and western blot analysis was performed as previously described. Approximately 50 μg of total protein was separated in a 10% sodium dodecyl sulfate-polyacrylamide gel and was transferred onto a polyvinylidene difluoride membrane. After probing with a primary and a secondary antibody, the signal was detected using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).
Bone resorption activities were determined by the bone resorption assay kit (Cosmo Bio. Co. Ltd., Tokyo, Japan) based on the manufacturer's instructions. In brief, RAW264.7 cells were incubated on fluoresceinated calcium phosphate-coated plates with M-CSF (30 ng/ml) and RANKL (30 ng/ml) in the presence or absence of various concentrations of rTMD1 for 7 days. The RANKL-induced bone resorption activity was evaluated by measuring the fluorescence intensity of the conditioned medium with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
All μCT analyses were consistent with the current guidelines.29 Bone samples from all groups were imaged using a SkyScan-1076 Micro-CT System (Skyscan, Belgium). For trabecular bone analysis, the μCT scanner was operated at 45 kV, 220 μA, 0.4μ rotation step, 0.5 mm aluminum filter, and a scan resolution of 18 pin/pixel. The following parameters were measured: total bone volume (TV, mm3), trabecular bone volume (BV, mm3), and trabecular bone volume fraction (BV/TV, %).
Osteoclastogenesis in mouse RAW 264.7 cells and human PBMCs was induced using RANKL and M-CSF to evaluate the levels of TM protein during the process. Immunofluorescence staining revealed TM expression dramatically decreased as RAW 264.7 cells differentiated into osteoclast-like cells (
To investigate whether TM might be a negative regulator in osteoclastogenesis, macrophages from myeloid-specific transgenic mice were isolated. TRAP staining analysis showed that RANKL can dose-dependently induce osteoclastogenesis in macrophages from LysMcre/TMflox/flox and TMflox/flox mice. However, the cell areas in the LysMere/TMflox/flox mice were larger than those from TMflox/flox mice (
TM lectin-like Domain in Macrophages retards RANKL-induced Osteoclastogenesis
It has been shown that osteoblast-derived C-type lectin can inhibit osteoclast formation. To further investigative whether the lectin-like domain of TM might contribute to the inhibition of RANKL-induced osteoclastogenesis, primary macrophages from TMWT/LeD and TMLeD/LeD mice were examined. TRAP staining analysis showed that RANKL can induce osteoclastogenesis in primary cells from both TMWT/LeD and TMLeD/LeD mice. However, the cell area of some OCs in the TMLeD/LeD group appeared larger than in the TMWT/LeD group (
Since the release of HMGB1 was required for RANKL-induced osteoclastogenesis, the effects of TM deficiency on the production of extracellular HMGB1 were further evaluated. As shown in
Treatment with rTMD1 Inhibits Bone Resorption and Bone Loss
The effects of human rTMD1 on bone resorption in vitro and bone loss in vivo were investigated. Results of the bone resorption assay indicated that rTMD1 treatment inhibited RANKL-induced bone resorption in a dose-dependent manner (
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and uses thereof are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.
It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, which are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The present application claims priority to U.S. Provisional Appl. No. 62/288,430, tiled Dec. 29, 2016, incorporated herein by reference in its entirety.
Number | Date | Country | |
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62288430 | Jan 2016 | US |