Patent Application
20100333217
The present disclosure relates to a method for screening a composition for preventing or treating an osteoporosis and a metabolic bone disease using a TALLYHO/JngJ (hereinafter referred to as TH) mouse, and more particularly, to a method for screening a compound effective in preventing and treating a disease caused by abnormalities of a bone metabolism including an osteoporosis, through assessment of a bone regeneration activity, such as acceleration of osteoblast differentiation, inhibition of osteoclast differentiation, variation of cytokine in a serum, as well as a bone mineral density and a bone mineral content in a femur, using a TH mouse instead of a generally used ovariectomized animal.
With the development in medical science and genetic engineering, it is estimated that the elderly people over the age of 60 will comprise 25 percent of the population in the 21st century. Accordingly, research and development on senile diseases are growing in social and pharmaceutical industrial importance. Osteoporosis is a representative senile disease, and the number of patients suffering from the osteoporosis significantly increases particularly in industrial nations with a large number of elderly people.
A bone tissue in a human body is a dynamic organ formed by bone remodeling such as bone formation of osteoblast and bone resorption of osteoclast, which is unceasingly repeated throughout life. Balance between the bone formation and the bone resorption allows the bone mineral content in the bone tissue and the function of the bone tissue to keep their normal state. The bone tissue is necessary to maintain a life because it serves as a supporting tissue for supporting human body and serves to preserve important organs and hematopoietic hepatocytes so as to provide blood cells. The osteoporosis is quickly developed by a fracture due to bone loss which is caused by removal of calcium from the bone which is occurred by the balance between the bone resorption and the bone formation is broken to accelerate the bone resorption. According to a previous research report, recently, even young persons are attacked with the osteoporosis due to lack of calcium intake caused by an unbalanced diet and the like, as well as elder persons over the age of 65 susceptible to a senile osteoporosis and middle-aged women susceptible to a postmenopausal osteoporosis due to lack of sex hormone. The dangers of the osteoporosis are increased in persons suffering from hypertension, hyperlipidemia, diabetes, liver disease, renal failure, thyroid disease, cancer, or sexual dysfunction, and persons taking steroids or stomach and bowel medicines for a long time, persons taking much alcohols, tobaccos or coffees, persons taking much meats, persons taking little exercises, skinny persons, sedentary workers, persons had an operation on the stomach, persons suffering from lumbago, arthritis or myalgia, long supine persons, persons susceptible to fatigue, and the like. However, the mechanism thereof is not well known yet.
A typical experimental method for developing a therapeutic agent for the osteoporosis is as follows. First, a material for inhibiting activation of the osteoclast or promoting activation of the osteoblast is selected. Then, the efficacy of the material is assessed by measuring the amount of recovered bone mineral content and the amount of recovered bone strength, using an animal developing symptoms of the osteoporosis similar to human beings. A general animal model currently used for the osteoporosis utilizes animals where bone loss was induced through an artificial ovariectomy or a naturally aged SAMP-6 mouse to administer the developed agents thereto and then determine the efficacy of the medicine.
However, current ovariectomized osteoporosis animals require ovariectomy operations for every experiment, thereby increasing experiment time and cost and increasing experimental error due to the failure of the operation. Furthermore, aged models such as SAMP-6 need to be taken care of for a long time in the laboratory and may cause the experimental error. Meanwhile, recently, advanced pharmaceutical companies decided that it is necessary to resolve the problems, such as safety, absortionability and price, of the existing osteoporosis medicines to develop a new competitive osteoporosis medicine. Accordingly, they are making huge investments in researches for finding a new initial target point and in developments of a new medicine using the same, and they are taking interest in the combination therapeutic strategies (combination of an anti-resorptive agent and an anabolic agent). That is, they determined that it is the time to establish the research strategy for developing a compound for inhibiting the activation of the osteoclast and promoting the activation of the osteoblast at the same time, which is safe and has excellent absorptionability, by utilizing new initial target points and a variety of new approaches.
The TH mouse was recently established by Jackson Laboratory, USA, through selective breeding of transformed animals developing diabetes by type 2 diabetes model due to a new Darwin gene. The TH mouse was reported in 2001 that only male mice develop diabetes between 10 week ages and 14 week ages, and they show abdominal obesity although it is not serious. However, there has been no report with respect to the osteoporosis.
An aspect of the present invention provides an effective screening method using a TH mouse as a new disease animal model for developing a therapeutic medicine for osteoporosis caused by a variety of factors such as sex hormone, aging, inflammation, diabetes and the like.
The inventors found that a TH mouse develops osteoporosis, and thus found that it is possible to screen an ideal osteoporosis medicine, i.e., a therapeutic agent for improving a bone mineral density or a composition for preventing or treating a metabolic bone disease using a mechanism for causing the osteoporosis and the TH mouse.
Exemplary embodiments of the present invention provide a method for screening an agent for treating or preventing an osteoporosis and a metabolic bone disease, including:
1) a step of administering a candidate material for an agent for treating or preventing the osteoporosis and the metabolic bone disease to a male TH mouse;
2) a step of measuring an index related to the osteoporosis and the metabolic bone disease from the male TH mouse administered with the candidate material of the step 1); and
3) a step of comparing the index of the male TH mouse administered with the candidate material and that of a control group which is not administered with the candidate material to determine whether the candidate material varies the index with significance.
Hereinafter, the present invention will be described in detail.
An exemplary embodiment provides a method for screening an agent for treating or preventing an osteoporosis and a metabolic bone disease using a TH mouse.
The weights and the feed intakes of the TH mouse and the control mouse were measured, and the result told us that the feed intake of the TH mouse was greater than that of the control mouse, and the weight of the TH mouse was increased with significance (see
The femurs and the skulls of the TH mouse and the control mouse were analyzed, and the result told us that the femur of the male TH mouse was reduced with significance in a bone mineral density and a bone mineral content compared with the control mouse, and the skull of the 8-week-old male TH mouse was reduced in the bone mineral density and the bone thickness compared with the control mouse.
The bone marrow was separated from the femur of the TH mouse and then the differentiations of the osteoblast and the osteoclast were induced. As a result, giant multinucleate cells were observed in the osteoblast culture group, and they were identified as osteoclasts (see
The expression level of the genes related to the differentiation of the osteoblast and the osteoclast was examined. An osteoprotegerin (OPG) playing an important role in the bone formation was reduced in the TH mouse. However, a receptor activator of NF-κ B ligand (RANKL) causing the bone resorption was significantly increased in the TH mouse. In addition, IL-6, which is one of cytokines playing an important role in the bone loss was increased in the TH mouse (see
Further, in order to determine whether the lack of the bone differentiation in the TH mouse is a posteriori or a priori, the shape of the osteoblast separated from a skull of a 1-day-old mouse was examined, and the variation of the expression of the genes related to the bone differentiation was also examined. The examination on the expression of the genes related to the differentiation of the osteoblast and the differentiation transcription genes in the cell, which is cultured for 8 days, showed a significant decrease in the gene expression of the transcription factors, such as Fra2, NF-AT1, JunD, and Fos, which control the differentiation of the osteoblast (see
From the above mentioned results, it can be seen that the male TH mouse of an age of 4 weeks shows features similar to the ovariectomized mouse model.
Hence, the inventors further investigated the effects of alendronate and parathyroid hormone (PTH), which showed the effect of bone formation in the ovariectomized mouse model, in the male TH mouse. The result was similar to the ovariectomized osteoporosis model mouse (see
As a result, it can be concluded that the male TH mouse can be used usefully as a new animal model in the development of the osteoporosis medicine.
When using the TH mouse, the artificial ovariectomy operation is not required because of the bone loss induced by the excessive amount of IL-6 in the blood serum and the problem of the complex bone-related gene expression. In addition, the ratio of OPG to RANKL, which is a representative feature of the osteoporosis, is significantly small. Therefore, the male TH mouse can be used as a useful natural disease model in the development of the new medicine for treating and preventing diseases such as a pre- or post-menopausal osteoporosis of women, a senile osteoporosis, a osteoporosis of men, an osteoporosis after a variety of implantations, an osteoporosis after a variety of surgeries including a heart valve surgery and a gastrectomy, a secondary osteoporosis caused by an osteomalacia and a steroid, and an inflammation including an osteoarthritis.
Another exemplary embodiment provides a screening method for an agent for treating or preventing an osteoporosis and a metabolic bone disease, including:
1) a step of administering a candidate material for an agent for treating or preventing the osteoporosis and the metabolic bone disease to a male TH mouse;
2) a step of measuring an index related to the osteoporosis and the metabolic bone disease from the male TH mouse administered with the candidate material of the step 1); and
3) a step of comparing the index of the male TH mouse administered with the candidate material and that of a control group which is not administered with the candidate material to determine whether the candidate material varies the index with significance.
The candidate material includes, for example, a peptide, a protein, a nonpeptide compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal texture extract, or a blood plasma. Such a compound may be a new compound or a well-known compound. The candidate material may form a salt thereof. The salt of the candidate material includes an acid (e.g., an inorganic acid) or a base (e.g., an organic acid, etc) which is physiologically acceptable, and is preferably a physiologically acceptable acid-added salt. For example, a salt of the inorganic acid (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc), or a salt of the organic acid (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, bezenesulfonic acid, etc) may be used as the salt.
The administration method for the candidate material may be appropriately selected according to the symptoms of the experiment animal, the characteristics of the candidate material, and the like. The administration method includes, for example, oral administration, intravenous injection, swabbing, subcutaneous administration, intracutaneous administration, intraperitoneal administration or the like. In addition, the dose of the candidate material may also be appropriately selected according to the administration method, the characteristics of the candidate material, and the like.
The index related to the osteoporosis of the step 2) includes, for example, increase of the bone mineral density and the bone mineral content of the femur, increase of the thickness and the bone mineral density of the skull, increase of OPG or decrease of IL-6 in the blood serum, decrease of RANKL, increase of the expressions of Fra2, NF-AT1, JunD and Fos genes which are transcription factors for controlling the differentiation of the osteoblast, and increase of expressions of alkaline phosphatase and COLL I genes which are differentiation factors of the osteoblast.
By comparing the above mentioned indexes of the male TH mouse administered with the candidate material and the control mouse administered with no candidate material, it can be determined whether the candidate material affects the index or not. As such, it is possible to screen the agent for treating or preventing the osteoporosis and the metabolic bone disease.
The screening method using the TH mouse according to the present invention has the following effects in comparison to the screening method using the typical ovariectomized osteoporosis animal. The experiment procedure is simple and stable. The screening method using the TH mouse may also be used usefully in predicting indirect efficacy on the metabolic bone disease as well as the osteoporosis. The male TH mouse according to the present invention can be used as a new animal model in the development of the medicine for osteoporosis symptom caused by a variety of causes such as a senile osteoporosis, an osteoporosis due to an inflammation, an osteoporosis due to inherited components, as well as osteoporosis symptoms of menopausal women.
a) illustrates graphs of bone mineral densities (BMD) versus week ages of control mice (C57BL/6) and TH mice according to an embodiment of the present invention; and
Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings.
However, it should be understood that the description of the exemplary embodiments is merely illustrative and that it should not be taken in a limiting sense.
Control mice (C57BL/6) and TH mice (Jackson Laboratory, bar harbor, USA) were freely fed, and their weights were measured every week from 4 week age to 20 week age after delectation.
As shown in
Control mice (C57BL/6) and TH mice were freely fed, and their bone mineral densities were measured every week from 4 week age to 20 week age after delectation. The mice were anesthetized with Avertin (2,2,2-tribromoethanol) to keep them alive while measuring their bone mineral densities. Then, scanning was performed 400 times at a thickness of 45 using eXplore Locus micro-CT (GE Healthcare, USA) which is devised as a tomographic apparatus for small animals. Thereafter, the resulting images were reformed using Microview (GE Healthcare, USA) to obtain the final bone mineral density (BMD) and the final bone mineral content (BMC) (see
As shown in
This is probably because of the natural specific bone loss of the male TH mice. The TH mice showed the same symptoms as the ovariectomized mice without the artificial ovariectomy. Therefore, this result shows the possibility of the male TH mice as a new osteoporosis animal model.
Control mice (C57BL/6) and TH mice were fed freely. 8-week-old mice which showed bone loss based on male TH mice were anesthetized and then sacrificed to extract their skulls. Thereafter, the bone thicknesses and the bone mineral densities of the extracted skulls were measured as described in Example 2, using a tomographic apparatus (micro-CT) for animals.
As shown in
This example was performed to examine the possibility of TH mice as an osteoporosis animal model. Bone marrows were separated from control mice (C57BL/6) and TH mice, and then cells were cultured for 1 day. After removing floating cells, the cells adsorbed to an incubator were differentiated to osteoblasts for 8 days while replacing an alpha-MEM medium added with ascorbic acid and beta-glycerolphosphate which are osteoblast differentiation accelerators, and 10% bovine serum, with a new alpha-MEM medium every three days.
Using the plates cultured for 8 days, an alkaline phosphatase (ALP) staining and a tartrate-resistant acid phosphatase (TRAP) staining were performed to measure the differentiation degree of the osteoblast and the osteoclast, respectively. The ALP staining was performed as follows. The medium in the culture plate was removed and washing was performed twice with 1×PBS. The cells were fixed with 2% paraformaldehyde (Sigma, USA) at an ambient temperature for 10 minutes. The fixed cells were added with diazonium salt solution mixed with equivalent amounts of sodium nitrate solution (Sigma, USA) and FRV-alkaline phosphatase solution (Sigma, USA) and reacted at an ambient temperature for 20 minutes to examine the activity of the stained cells. The TRAP staining was performed as follows. Washing was performed twice with 1×PBS. 4% formalin solution (Sigma, USA) was added to fix the cells at an ambient temperature for 10 to 15 minutes. The TRAP staining solution (50 mM acetate buffer, 30 mM sodiotartrate, 0.1 mg/ml naphthol, 0.1% triton X-100, 0.03% fast red violet, Sigma, USA) was added to react with the cells at 37° C. in a darkness for 30 minutes to 1 hour. The activity was measured by counting only giant multinucleate cells merged with three or more nuclei to assess the differentiation amount of the osteoclast.
Consequently, as shown in
Considering that one of the major factors in the osteoporosis is the osteoclast activation faster than the osteoblast activation, it can be seen from the above mentioned result that the bone loss occurred in the TH mice.
After separating mRNA from the cell of
Reverse Transcription System (Promega, USA) was added with total RNA 1 μg, 10 mM dNTP 2 μl, 100 pmole Oligo dT 1 μl, 10×buffer 2 μl, 25 mM MgCl2 4 μl, RNase inhibitor 0.2 μl, AMV RTase 0.2 μl, and DEPC-treated water, and cultured at 42° C. for 60 minutes and at 72° C. for 10 minutes to prepare cDNA. Then, the reverse transcripted cDNA sample was amplified using primer pairs specific to respective target genes. The PCR was repeated 25-30 times after denaturation at 95° C. for 5 minutes. In specific, the PCR was performed under the condition of denaturation at 95° C. for 30 seconds, primer annealing at 55° C. (NPY1receptor), 56° C. (IL-6, IL-1beta, TNFalpha, TLR2, TLR4, OC, MMP-9, GAPDH), 57° C. (OPG), 58° C. (RANK), 60° C. (IGF1, IGF2, RANKL, NPY, NPY2receptor) and 62° C. (Leptin) for 30 seconds, and extension reaction at 72° C. for 30 seconds. The PCR product was electrophoresed in an 1.5% agarose gel and stained with GelRed. The result was photographed with GelDoc (BioRad, USA).
Resultantly, as shown in
In order to determine whether the lack of the bone differentiation in the TH mouse is a posteriori or a priori, skulls were separated from 1-day-old mice, and cultured for 8 days in alpha-MEM media added with betaglycerophosphate and ascorbic acid which are osteoblast differentiation induction factors. Using real time reverse transcription PCR (Real time PCR, Rotor-Gene 300 real-time DNA detection system; Corbett Research, Sydney, Australia), variations of the genes related to the bone differentiation were examined. Total RNA was separated and cDNA was synthesized as described in Example 5. To investigate the expression of each of the genes, 20 pmol of each primer listed in the Table 2 was mixed with cDNA and SYBR Green Master Kit (Stratagene, USA), and PCR was performed 40 times after the dinaturation at 94° C. for 10 minutes under the condition of denaturation at 94° C. for 40 seconds, primer annealing at 60° C. for 40 seconds, and extension reaction at 72° C. for 1 minute. A standard curve was obtained using glyceraldehyde-3-phosphate (GAPDH), and mRNA was obtained by subtracting Ct (threshold cycle) of each gene from GAPDH among calculated Ct values and calculating delta CT value with a formula, 2.0−(Ct). Finally, the expression levels of the mice were shown relatively based on the male TH mice.
Interestingly, as shown in
4-week-old male TH mice were orally administered with alendronate (CALBIOCHEM, USA), which is widely used as a osteoporosis medicine, with a concentration of 5 mg/kg, once a day for 4 weeks. Thereafter, the bone mineral density and the bone mineral content of the femur and the thickness and the bone mineral density of the skull were measured as described in Examples 2 and 3.
Resultantly, as shown in
Meanwhile, an accurate histomorphological scanning was carried out to observe the effect of the alendronate on recovering the bone loss in the femur of the 8-week-old TH mouse. The TH mice and B6 mice were sacrificed with carbon dioxide. The femurs thereof were extracted and scanned 400 times with a thickness of 27 μM using eXplore Locus micro-CT (GE Healthcare, USA) which is devised as a tomographic apparatus for small animals. The scanned images were accurately reformed using Microview (GE Healthcare, USA) to obtain final histomorphological images of the femurs.
Resultantly, as shown in
Meanwhile, after separating the blood serum, IL-6 level was measured using ELISA Kit (ALPCO diagnostics, USA). The measurement showed low IL-6 level in the mice group treated with the alendronate.
In order to observe the expression of the genes, cells were separated from the bone marrow and cultured for days. Thereafter, the variations of the genes were investigated through RT-PCR as described in Example 5. As shown in
From the above mentioned results, it can be seen that the male TH mouse can be used as a new animal model in developing the osteoporosis medicine.
8-week-old male TH mice were orally administered with parathyroid hormone (hPTH(1-34), Sigma, USA), which is started to be used as an osteoporosis medicine increasing the bone mineral content, with a concentration of 50 μg/kg, once a day for 4 weeks. Thereafter, the bone mineral density and the bone mineral content of the femur was measured as described in Example 2.
The result showed that, as shown in
From the above mentioned results, it can be seen that the male TH mouse can be used as a new animal model in developing the osteoporosis medicine.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2007-0098323 | Sep 2007 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2008/002286 | 4/23/2008 | WO | 00 | 3/26/2010 |
