Application of Daraladib in preparation of medicine for treating osteoporosis

Abstract

The invention discloses application of Daraladib in preparation of a medicine for treating osteoporosis, and belongs to the technical field of medicines. The invention specifically discloses an application of Daraladib or a pharmaceutically acceptable salt of Daraladib in preparation of a medicine for treating osteoporosis. The Daraladib can inhibit osteoclast differentiation and the like through a lipoxygenase pathway, so that the osteoporosis can be prevented or relieved, and a new medicine is provided for preventing and treating the osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of Darapladib in the preparation of drugs for treating osteoporosis. Background Art

[0002] Bone is the most important supporting organ of the human body and is in a continuous process of renewal and remodeling. The dynamic balance and coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption are the basis for maintaining normal bone mass and bone physiological functions. Osteoclasts are derived from bone marrow monocytes in the hematopoietic stem cell line and fuse and differentiate into multinucleated giant cells under the action of related stimulating factors. They are the only cells in the human body with bone resorption function and play a key role in maintaining bone remodeling and bone metabolism balance. Excessive activation of osteoclasts, which breaks the "remodeling-resorption" balance relationship of bone tissue, is an important cause of bone metabolic diseases such as osteoporosis. According to statistics, there are currently more than 200 million osteoporosis patients worldwide. Among women over 60 years old in China, the prevalence of osteoporosis is as high as 40-50%, and about 30-50% of them will experience osteoporosis-related fractures, seriously affecting the physical health and quality of life of the elderly and causing heavy economic pressure and social burden.

[0003] Inhibiting bone resorption is one of the main means for clinical osteoporosis at present. Common drugs include bisphosphonates (such as the bisphosphonate for treating osteoporosis disclosed in the publication number CN1161363C), calcitonins (such as the Chinese patent with the publication number CN118684757A discloses a long-acting calcitonin analog for the prevention and treatment of osteoporosis, Paget's disease, painful neuropathic osteoarthropathy, and malignant osteolysis), RANKL (receptor activator of nuclear factor-κB ligand) monoclonal antibody (denosumab) (such as the Chinese patent with the publication number CN118324912A discloses a stem cell exosome composition and its application in the preparation of drugs for treating osteoporosis, and the stem cell exosome composition includes stem cell exosomes and anti-RANKL antibodies), and strontium salts, etc. Currently, common drugs usually inhibit osteoclast differentiation and osteoclastic activity by regulating calcium and phosphorus metabolism and competitively binding to RANKL. However, the application of these drugs in clinical practice still faces many problems and conditions, such as the long-term efficacy is still unclear, atypical fractures and osteonecrosis of the jaw occur after long-term use, the risk of cardiovascular diseases increases, and there are adverse reactions in the digestive system, etc.

[0004] Phospholipase A2 (Lp-PLA2) itself is an enzyme that can catalyze the hydrolysis of the acyl group at the second position of phospholipid glycerol molecules. It is also the rate-limiting enzyme for the generation of bioactive substances such as arachidonic acid (AA), prostaglandins, and platelet-activating factor (PAF). The lipid mediators produced play a key role in the activation of membrane channels, information transmission, hemodynamics, and pathophysiological processes during inflammation and tissue damage, as well as in regulating intracellular and extracellular metabolism. Currently, there is no relevant report on the use of phospholipase A2 in the preparation of drugs for the treatment of osteoporosis. Summary of the Invention

[0005] The object of the present invention is to provide the use of Darapladib in the preparation of drugs for the treatment of osteoporosis, which can effectively prevent and treat or alleviate osteoporosis.

[0006] The present invention provides the following technical solutions: The use of Darapladib (Lp-PLA2 inhibitor) or a pharmaceutically acceptable salt thereof in the preparation of drugs for the treatment of osteoporosis.

[0007] In the present invention, Darapladib can inhibit osteoclast differentiation and other effects through the lipoxygenase pathway, thereby preventing and treating or alleviating osteoporosis. The chemical structural formula of Darapladib (CAS: 356057-34-6) is as follows: .

[0008] The pharmaceutically acceptable salt is phosphate.

[0009] The effective amount of Darapladib or a pharmaceutically acceptable salt thereof in the drug is 100 - 400 nmol / L.

[0010] The drug includes a pharmaceutically acceptable carrier.

[0011] The pharmaceutically acceptable carrier includes excipients, lubricants, disintegrants, fillers, binders, osmotic pressure regulators, diluents, absorption promoters, surfactants, or adsorption carriers.

[0012] Preferably, one or more of excipients such as starch or water; one or more of lubricants such as glycerol or magnesium stearate, talc, calcium stearate or polyethylene glycol, etc.; one or more of disintegrants such as microcrystalline cellulose, agar, calcium carbonate or sodium bicarbonate, etc.; one or more of fillers such as starch or lactose, etc.; one or more of binders such as pregelatinized starch, dextrin, cellulose derivatives, alginates, gelatin or polyvinylpyrrolidone, etc.; one or more of osmotic pressure regulators such as glucose, sucrose, sorbitol or mannitol, etc.; diluents such as water, etc.; absorption promoters such as quaternary ammonium compounds, etc.; surfactants such as cetyl alcohol, etc.; adsorption carriers such as kaolin or saponite clay, etc.; in addition, other adjuvants such as one or more of flavoring agents or sweetening agents, etc. can also be added to the pharmaceutical composition.

[0013] The dosage form of the drug is tablet, capsule, injection, drop, pill, powder or oral liquid.

[0014] The osteoporosis is osteoporosis caused by estrogen deficiency, osteoporosis caused by aging, osteoporosis caused by chronic inflammation, osteoporosis caused by hormone drugs or osteoporosis caused by obesity.

[0015] Specifically, the osteoporosis can be a bone metabolic disease characterized by bone mass reduction, destruction of the fibrous structure of bone tissue, and then increased bone fragility and easy fracture caused by the relative activation of osteoclasts due to estrogen reduction after menopause in women, or it can also be osteoporosis caused by other factors such as aging, chronic inflammation, hormone drugs or obesity.

[0016] Pharmacological experiments on the prevention and treatment of osteoporosis in ovariectomized mice by Darapladib in the present invention show that Darapladib can significantly prevent and treat bone mass reduction caused by estrogen reduction, indicating its effect of preventing and treating osteoporosis, so it can be used to prepare drugs for preventing and treating osteoporosis; thus confirming the new activity and new use of Darapladib.

[0017] Compared with the prior art, the present invention has the following excellent effects: the present invention not only provides a new therapeutic use of Darapladib, but also provides a new drug for preventing and treating osteoporosis. Description of the Drawings

[0018] Figure 1 It is a bone mass detection diagram for the prevention and treatment of estrogen-reduced osteoporosis by Darapladib in Example 1; Figure 2 It is the statistical result of bone mass detection for the prevention and treatment of estrogen-reduced osteoporosis by Darapladib in Example 1; Figure 3The inhibitory effect of Darapladib on the fusion and differentiation of macrophages into osteoclasts; Figure 4 The inhibitory effect of Darapladib on the expression of related genes during osteoclast differentiation; Figure 5 The inhibitory effect of Darapladib on the expression of related proteins during osteoclast differentiation. Detailed implementation manners

[0019] The present invention will be further described below in conjunction with the detailed implementation manners. These implementations are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0020] As used herein, the term "acceptable" means that a pharmaceutical ingredient or active ingredient does not have an excessive harmful effect on the health of the general treatment target.

[0021] As used herein, the term "prevention and treatment" includes preventing, alleviating, inhibiting or improving the symptoms or conditions of a disease; inhibiting the occurrence of complications; inhibiting the occurrence of a disease or symptoms, such as controlling the development of a disease or condition; alleviating a disease or symptoms; reducing a disease or symptoms; reducing the complications caused by a disease or symptoms, or preventing or treating the signs caused by a disease or symptoms. As used herein, after administration of a certain compound or pharmaceutical composition, a certain disease, symptom or condition can be improved, especially the severity thereof can be improved, the onset can be delayed, the progression of the disease can be alleviated, or the duration of the disease can be reduced. Whether administered regularly or temporarily, continuously or intermittently, it can be attributed to or related to the situation of administration.

[0022] As used herein, the term "pharmaceutically acceptable" herein refers to a substance, such as a carrier or diluent, that does not cause the loss of the biological activity or properties of the compound and is relatively non-toxic. For example, when a substance is administered to an individual, it does not cause unwanted biological effects or interact with any components it contains in a harmful manner. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, glucose, glycerol or ethanol, and the like and combinations thereof. In many cases, it is preferably to include an osmotic pressure regulator in the composition, such as one or more of sugars, polyols such as mannitol, sorbitol, sorbitol or sodium chloride, etc. The pharmaceutically acceptable carrier may also contain a small amount of adjunct substances, such as one or more of wetting agents or emulsifiers, preservatives or buffers, etc.

[0023] In terms of specific use, Darapladib of the present invention can be used alone or in combination with many other chemical substances. Whether these chemical substances have biological activity or a function of treating diseases, the auxiliary functions include, such as, synergistic amplification, antagonism or alleviation of the side effects of Darapladib, etc. These chemical substances include one or more of pharmaceutically acceptable carriers, foods, natural products, chemically synthesized drugs, human medications, etc.; preferably include one or more of pharmaceutically acceptable carriers or foods, etc.; more preferably a pharmaceutically acceptable carrier.

[0024] In the following examples, the Darapladib used is a white powder with a content > 99.89% and a pharmaceutical grade. Only the test contents that are significantly significant in the present invention will be briefly described in the following invention process.

[0025] Example 1 Prevention and treatment of estrogen-deficient osteoporosis by Darapladib (I) Animal grouping and treatment Healthy 12-week-old female C57BL / 6 mice, with a body weight of 20 - 25 g, were provided by the Experimental Animal Center of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The animals were raised and observed for 2 weeks before the experiment and randomly divided into 3 groups, among which: sham operation group, with a total of 6 C57BL / 6 mice; ovariectomy group, with a total of 6 C57BL / 6 mice, and the ovariectomy group was given intraperitoneal injection treatment with phosphate buffered saline; Darapladib group, with a total of 6 C57BL / 6 mice, and was given intraperitoneal injection treatment with Darapladib at 20 mg / kg three times a week. Darapladib was dissolved in 10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline to prepare a mixed solution, and 100 μL was administered by intraperitoneal injection. The drug was administered three times a week from the day of surgery to the end of the 8th week after surgery.

[0026] (II) Ovariectomy of experimental animals For each group of experimental mice, after intraperitoneal injection of pentobarbital anesthesia, surgical operations were performed. The sham operation group of mice was given sham operation treatment without removing the bilateral ovaries. For the ovariectomy group and the Darapladib treatment group, through the dorsal skin approach, the peritoneal cavity was entered at the mid-level of the bilateral lumbar vertebrae, and the bilateral ovaries were ligated and removed, and the peritoneum, skin, etc. were sutured layer by layer. After the operation, the corresponding treatment group was given intraperitoneal administration of the drug.

[0027] (III) Bone mass detection Each group of mice was euthanized 8 weeks after surgery. The bilateral femoral tissues were harvested and fixed with 4% paraformaldehyde at room temperature for 2 days. The left femurs of the mice in each group after fixation were scanned by Micro CT with a resolution of 9 μm. The scanning parameters were: voltage 70 kV and current 80 μA. 150 layers starting from the growth plate area were selected as the region of interest for data analysis and three-dimensional reconstruction. The analysis indexes included: relative bone volume of trabecular bone, trabecular bone thickness, and trabecular bone number.

[0028] (IV)Statistical analysis The data were expressed as mean ± standard deviation. The results were statistically analyzed using the SPSS 19 software package, and P < 0.05 was considered statistically significant.

[0029] (V)Experimental results See Figure 1 , 8 weeks after ovariectomy in 12-week-old female mice, the relative bone volume of trabecular bone in the ovariectomy group (HFD) was significantly lower than that in the sham operation group (CD). The relative bone volume of trabecular bone in the femurs of mice in the Darapladib treatment group (HFD + Darapladib) was significantly higher than that in the ovariectomy group. It was proved that Darapladib had a significant preventive and therapeutic effect on osteoporosis caused by estrogen deficiency. Figure 2 In, group 0 was the sham operation group, group 1 was the ovariectomy group, and group 2 was the Darapladib treatment group; compared with group 1, **p < 0.01.

[0030] (VI)Experimental conclusion Darapladib can prevent and treat osteoporosis mainly manifested by bone mass reduction caused by estrogen deficiency, and has the effect of preventing and treating osteoporosis.

[0031] Example 2 Inhibitory effect of Darapladib on osteoclast differentiation in vitro (I)Extraction of mouse bone marrow-derived macrophages Healthy 6-week-old male C57BL / 6 mice were provided by the Experimental Animal Center of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The mice were euthanized, and the bilateral femurs and tibiofibulas were dissected. The bone marrow cavities were rinsed with phosphate buffer solution. The obtained cell mixture was filtered through a 0.4-μm filter and centrifuged, and then inoculated into a 96-well culture plate with α-MEM medium containing 10 ng / mL macrophage colony-stimulating factor (MCSF) and 10% fetal bovine serum (FBS).

[0032] (II)Osteoclast induction After the mouse bone marrow-derived macrophages to be extracted adhered to the wall, the osteoclast induction medium containing 25 ng / mL MCSF and 50 ng / mL RANKL was replaced, and at the same time, they were supplemented with Darapladib at 100, 200, and 400 nanomoles per liter. The medium was changed once every 2 days.

[0033] (III) Osteoclast Staining After 5 days of osteoclast induction of macrophages, they were fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing 3 times with phosphate buffer, tartrate-resistant acid phosphatase (TRAP) staining was performed and observed under a microscope.

[0034] (IV) Experimental Results See Figure 3 , after 5 days of osteoclast induction, a large number of fused and multinucleated osteoclasts differentiated from macrophages untreated with Darapladib, while the number of mature osteoclasts differentiated from macrophages treated with Darapladib was significantly reduced.

[0035] (V) Experimental Conclusion Darapladib can inhibit osteoclast differentiation in vitro and has the effect of preventing and treating osteoporosis.

[0036] Example 3 Darapladib Inhibits the Expression of Related Genes in Osteoclast Differentiation (I) Extraction of Mouse Bone Marrow-Derived Macrophages Healthy 6-week-old male C57BL / 6 mice were provided by the Experimental Animal Center of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The mice were euthanized, and the bilateral femurs and tibiofibulas were dissected. The bone marrow cavities were rinsed with phosphate buffer. The obtained cell mixture was filtered through a 0.4-micron filter and centrifuged, and then inoculated into a 12-well culture plate with α-MEM medium containing 10 ng / mL macrophage colony-stimulating factor (MCSF) and 10% fetal bovine serum (FBS).

[0037] (II) Osteoclast Induction After the mouse bone marrow-derived macrophages to be extracted adhered to the wall, the osteoclast induction medium containing 25 ng / mL MCSF and 50 ng / mL RANKL was replaced, and at the same time, they were supplemented with Darapladib at 200 and 400 nanomoles per liter for 2 days.

[0038] (III) Ribonucleic Acid (RNA) extraction and quantitative polymerase chain reaction (qPCR) detection.

[0039] After 2 days of osteoclast induction in macrophages, RNA was extracted according to the instructions of the Kangwei Century Ultra-Pure RNA Extraction Kit (product number CW0581), as described below. Add 1 mL of TRizon Reagent to each well and mix evenly. After lysing at room temperature for 5 minutes, add 200 μL of chloroform, invert and mix evenly, and let stand for 5 minutes. Centrifuge at 4 °C for 10 minutes at a speed of 12,000 revolutions per minute. Aspirate the supernatant and mix it with 70% ethanol at a volume ratio of 1:1, then pass it through a silica-based adsorption column. After washing the adsorption column multiple times, elute the RNA with enzyme-free water (RNase-Free water). After extracting the RNA, perform reverse transcription using the Kangwei Century Reverse Transcription Kit (product number CW2569). The reverse transcription product was diluted 1:20 with DEPC water and then subjected to qPCR detection using the Yisheng qPCR reagent (product number 11201ES03). The primer sequences used are shown in Table 1.

[0040] Table 1 Primer sequences related to the expression of osteoclast-related genes detected by fluorescence quantitative PCR .

[0041] (IV) Experimental results See Figure 4 . After 2 days of osteoclast induction, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene, macrophages without Darapladib treatment highly expressed osteoclast differentiation-related genes, such as nuclear factor of activated T cells 1 (NFATc1), proto-oncogene, AP-1 transcription factor subunit (c-Fos), and cathepsin K (CTSK). After treatment with different concentrations of Darapladib, the expression of osteoclast differentiation-related genes in macrophages increased compared with the non-induced group, but decreased compared with the simple osteoclast induction group. Figure 4 Among them: Group 0 was the group of unstimulated macrophages, Group 1 was the group of simply osteoclast-induced macrophages, Group 2 was the osteoclast induction group treated with 200 nM Darapladib, compared with Group 1, ***p < 0.001; Group 3 was the osteoclast induction group treated with 400 nM Darapladib, ***p < 0.001.

[0042] (V) Experimental conclusion Darapladib can inhibit the related expression of osteoclast differentiation genes in vitro and has the effect of preventing and treating osteoporosis.

[0043] Example 4 Darapladib inhibits the expression of related proteins in osteoclast differentiation (1) Extraction of mouse bone marrow-derived macrophages Healthy 6-week-old male C57BL / 6 mice were provided by the Experimental Animal Center of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The mice were euthanized, and the bilateral femurs and tibiofibulas were dissected. The bone marrow cavities were rinsed with phosphate buffer solution. The obtained cell mixture was filtered through a 0.4-μm filter and centrifuged, and then inoculated into a 12-well culture plate with α-MEM medium containing 10 ng / mL macrophage colony-stimulating factor (MCSF) and 10% fetal bovine serum (FBS).

[0044] (2) Osteoclast induction After the mouse bone marrow-derived macrophages to be extracted adhered to the wall, the osteoclast induction medium containing 25 ng / mL MCSF and 50 ng / mL RANKL was replaced, and at the same time, 100, 200, and 400 nanomoles per liter of Darapladib was supplemented and treated for 2 days.

[0045] (3) Protein extraction and Western Blot detection Two days after the macrophages were induced into osteoclasts, proteins were extracted according to the instructions of BIO-RAD 4x Laemmli Sample Buffer reagent (product number #161-0737), as follows: 20 μL of TRizon Reagent, 60 μL of ddH2O, and 1 μL of DTT were added to each well and mixed evenly. After lysing at room temperature for 5 minutes, it was left standing for 5 minutes; scraped into a 1.5-mL EP tube and centrifuged at 100 °C in a metal bath for 10 minutes; then the following operations were carried out: 1. Assemble the 1.5-mm glass plate with the short glass plate, clamp it between the glass plates of the electrophoresis device without gaps, and fix it on the gel-casting rack; 2. Prepare 10% separating gel liquid (5.9 mL of ddH 2 O, 5 mL of polyacrylamide, 3.8 mL of Tris-HCl with pH = 8.8, 150 μL of 10% SDS) as needed, and then add 150 μL of 10% ammonium persulfate and 15 μL of TEMED, and gently stir and mix evenly; quickly add the separating gel liquid along the edge of the glass plate into the sandwich of the glass plates with a plastic pipette until reaching the appropriate position and then stop, and then add isopropanol or absolute ethanol to seal the separating gel, and let it stand at room temperature for 20 - 30 minutes; 3. Pour out the isopropanol or absolute ethanol, wash it with filter paper, and prepare the stacking gel liquid (4 mL of ddH 2O, 1 mL of polyacrylamide, 1 mL of Tris-HCl with pH = 6.8, 80 μL of 10% SDS), then add 60 μL of 10% ammonium persulfate and 7.5 μL of TEMED, mix well and add it to the sandwich of the glass plate. Insert a 15-well comb with a thickness of 1.5 mm into the middle of the glass plate and let it stand at room temperature for 20 - 30 minutes; 4. Gently pull out the comb, being careful not to damage the sample wells. Add 1× electrophoresis buffer to the electrophoresis tank to an appropriate liquid level; 5. Carefully use a pipette to add the protein samples in the same volume to the sample wells. The blank wells must be added with an equal volume of 1× loading buffer to prevent the diffusion of samples in adjacent lanes. Finally, add the protein marker; 6. Connect the positive and negative electrodes of the power supply. First, electrophorese at a constant voltage of 80 V until reaching the separating gel, then adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue is close to the bottom of the gel, and terminate electrophoresis in a timely manner; 7. Turn off the power supply, take out the glass plate, carefully remove the gel plate from the glass plate, place it on the filter paper soaked with transfer buffer, cover it with a PVDF membrane soaked with methanol on it. After completely expelling the air bubbles between the gel and the membrane, clamp it with a transfer clip and place it on the transfer device according to the electrode orientation. Connect the power supply, constant current of 300 mA, for 95 minutes; 8. After the transfer is completed, take out the PVDF membrane, face up, and place it in 5% skim milk for blocking for 1 - 2 hours, shaking slowly; 9. Then wash the skim milk with TBST, incubate with the corresponding primary antibody (dilution ratio 1:1000), and incubate overnight at 4°C; 10. Wash with TBST 5 times, 7 minutes each time; 11. Incubate with the HRP-labeled secondary antibody corresponding to the primary antibody at room temperature for 1 hour, and then wash with TBST 5 times, 7 minutes each time; 12. Use ECL luminescent solution to detect protein bands.

[0046] (IV) Experimental Results See Figure 5 As shown in [reference], after 2 days of osteoclast induction, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene, macrophages not treated with Darapladib highly expressed osteoclast differentiation-related proteins, such as nuclear factor of activated T cells 1 (NFATc1), proto-oncogene, AP-1 transcription factor subunit (c-Fos), and cathepsin K (CTSK). After treatment with Darapladib, the protein expression of osteoclast differentiation-related proteins in macrophages decreased compared with the simple osteoclast induction group.

[0047] (V) Experimental Conclusions Darapladib can inhibit the related expression of osteoclast differentiation protein in vitro and has the effect of preventing and treating osteoporosis.

[0048] Example 5 Preparation of Darapladib Tablets Preparation process: Dissolve hypromellose in an appropriate amount of water to make a 5% solution, and dissolve lactose in the hypromellose solution as a binder solution. Grind Darapladib through a 100-mesh sieve, mix it with microcrystalline cellulose and granulate with the binder. Add magnesium stearate to the obtained granules and mix evenly, and then press tablets with a tablet press to obtain Darapladib tablets.

[0049] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Darapladib or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating osteoporosis.

2. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt is phosphate.

3. The use according to claim 1, characterized in that: The effective amount of Darapladib or a pharmaceutically acceptable salt thereof in the drug is 100-400 nmol / L.

4. The use according to claim 1, characterized in that: The medicament includes a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable carrier includes an excipient, a lubricant, a disintegrant, a filler, a binder, an osmotic pressure regulator, a diluent, an absorption enhancer, a surfactant or an adsorption carrier.

6. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, capsule, injection, drop, pill, powder or oral solution.

7. The use according to claim 1, characterized in that: The osteoporosis is osteoporosis caused by estrogen deficiency, osteoporosis caused by aging, osteoporosis caused by chronic inflammation, osteoporosis caused by hormone drugs or osteoporosis caused by obesity.

8. The use according to claim 7, characterized in that: The osteoporosis is osteoporosis caused by estrogen deficiency in postmenopausal women.

Images