Use of pp'-dde and its homologues for the preparation of a medicament for the treatment of osteoporosis

Abstract

This invention provides the application of pp'-DDE in the preparation of drugs for treating osteoporosis. Using pp'-DDE and its homologues (DDT; op'-DDT; pp'-DDD, etc.) as pharmacological components, it promotes the formation of osteoblast mineralization nodules by increasing the expression of molecules such as Ocn, Alp1, and Sp7 in osteoblasts, thereby increasing the thickness of cortical bone or femur, improving the symptoms of osteoporosis, and achieving the purpose of treating osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of pp'-DDE in the treatment of osteoporosis-related diseases. Background Technology

[0002] Osteoporosis is a common chronic disease among the elderly. Bone homeostasis is mainly balanced by the bone formation of osteoblasts and the bone resorption of osteoclasts. Osteoporosis occurs because the activity of osteoclasts exceeds that of osteoblasts. Currently, most clinically available drugs for osteoporosis are bone resorption inhibitors, which are insufficient to address the fundamental problem of osteoporosis in the elderly. A few bone formation-promoting drugs, such as parathyroid hormone, have drawbacks such as joint pain, decreased bone density after discontinuation, and potential hypercalcemia. Therefore, finding drug delivery sites that can enhance osteoblast function and increase bone formation is an important aspect of research.

[0003] pp'-DDE, full name pp'-dichlorodiphenyl-dichloroethylene, also known as 44'-DDE, has the molecular formula C14H8Cl4, a molecular weight of 318.0, and CAS number 72-55-9. This invention demonstrates that low doses of pp'-DDE can promote osteoblast differentiation, increase cortical bone thickness in wild-type adult mice, and alleviate estrogen deficiency-induced osteoporosis. Summary of the Invention

[0004] This invention provides an anti-osteoporosis drug with a low concentration of pp'-DDE as the active ingredient.

[0005] The present invention relates to the use of pp'-DDE in the preparation of anti-osteoporosis drugs. The resulting anti-osteoporosis drugs can be formulated with auxiliary components that do not affect the drug's efficacy, such as carriers, excipients, and flavoring agents, based on common knowledge in the pharmaceutical field. Since the drug is effective through oral administration, injection, subcutaneous implantation, etc., the dosage form can be diverse, including but not limited to powders, granules, tablets, capsules, pills, solutions, suspensions, or injections. The structure of the pp'-DDE is shown below:

[0006]

[0007] A second aspect of this invention is the use of pp'-DDE in the preparation of formulations that increase cortical bone thickness, wherein the formulation incorporates excipients, such as carriers, excipients, and flavoring agents, based on common knowledge in the pharmaceutical field, without affecting the efficacy of the drug. Since the drug is effective via oral administration, injection, subcutaneous implantation, etc., the dosage form can be diverse, including but not limited to powders, granules, tablets, capsules, pills, solutions, suspensions, or injections. The structure of the pp'-DDE is shown below:

[0008]

[0009] A third aspect of this invention is the application of pp'-DDE in the preparation of formulations that increase femoral bone thickness, wherein the formulation incorporates excipients, such as carriers, excipients, and flavoring agents, based on common knowledge in the pharmaceutical field, without affecting the efficacy of the drug. Since the drug is effective via oral administration, injection, subcutaneous implantation, etc., the dosage form can be diverse, including but not limited to powders, granules, tablets, capsules, pills, solutions, suspensions, or injections. The structure of the pp'-DDE is shown below:

[0010]

[0011] A fourth aspect of this invention is the application of pp'-DDE in the preparation of osteoblast-promoting mineralization nodules, wherein the formulation incorporates excipients, such as carriers, excipients, and flavoring agents, based on common knowledge in the pharmaceutical field, without affecting the efficacy of the drug. Since the drug is effective via oral administration, injection, subcutaneous implantation, etc., the dosage form can be diverse, including but not limited to powders, granules, tablets, capsules, pills, solutions, suspensions, or injections. The structure of the pp'-DDE is shown below:

[0012]

[0013] In one specific embodiment, the application is achieved by increasing the expression of Ocn in osteoblasts; in another specific embodiment, the application is achieved by increasing the expression of Alp1 in osteoblasts; and in yet another specific embodiment, the application is achieved by increasing the expression of Sp7.

[0014] A sixth aspect of this invention provides the application of pp'-DDE in the preparation of reagents that promote the expression of molecules such as Ocn, Alp1, and Sp7 in osteoblasts. The formulation incorporates auxiliary components that do not affect the drug's efficacy, such as carriers, excipients, and flavoring agents, based on common knowledge in the pharmaceutical field. Since the drug is effective via oral administration, injection, or subcutaneous implantation, the dosage form can be diverse, including but not limited to powders, granules, tablets, capsules, pills, solutions, suspensions, or injections. The structure of the pp'-DDE is shown below:

[0015]

[0016] This invention relates to anti-osteoporosis drugs or pharmaceutical compositions using pp'-DDE and its homologues (DDT; op'-DDT; pp'-DDD, etc.) as the active ingredient. Specifically, any drug or pharmaceutical composition labeled as having pp'-DDE as the active ingredient and indicating osteoporosis as its indication is a patented anti-osteoporosis drug for which this invention seeks protection. This invention also seeks protection for the use of pp'-DDE in the preparation of anti-osteoporosis drugs. Any profit-making production of anti-osteoporosis drugs or anti-osteoporosis pharmaceutical compositions using pp'-DDE as the active ingredient falls within the scope of protection claimed by this invention. Attached Figure Description

[0017] Figure 1 Changes in cortical bone thickness in wild-type mice after pp'-DDE treatment, where A: 3D schematic diagram of cortical bone, B: Statistical analysis of cortical bone thickness.

[0018] Figure 2 Effects of pp'-DDE on femoral bone mass and bone thickness in OVX model mice, where A: 3D schematic diagram of femoral cancellous bone, B: mouse bone mass / tissue mass, and C: mouse bone thickness.

[0019] Figure 3 Effect of pp'-DDE on mineral nodule staining after primary osteoblast differentiation.

[0020] Figure 4 The effects of pp'-DDE on molecular signaling such as Ocn, Alpl, and Sp7 in primary osteoblasts. Detailed Implementation

[0021] The objects and functions of the present invention, as well as the methods for achieving these objects and functions, will be explained below with reference to exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in various forms. This specification is merely intended to help those skilled in the art to comprehensively understand the specific details of the invention.

[0022] Example 1: Effects of pp'-DDE on the skeleton of wild-type mice

[0023] 1. Laboratory animals:

[0024] C57BL / 6 mice, male, 6 weeks old, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice had free access to food and water, and their diet was provided by the Animal Experiment Center of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. From 6 weeks of age, the mice were administered pp'-DDE daily by gavage, along with a control group, for 8 weeks. After 8 weeks, the mice were sacrificed, and their femurs were harvested for micro-CT scans to assess bone mass.

[0025] 2. Experimental reagents:

[0026] pp'-DDE was purchased from Aladdin Reagents, catalog number D117408. Corn oil was purchased from Aladdin Reagents. Trizol was purchased from Invitrogen.

[0027] Experimental equipment: weighing scale, scissors, tweezers, gavage needle.

[0028] 3. Experimental steps:

[0029] 1) Animal gavage treatment:

[0030] Mice in the treatment group were administered pp'-DDE by gavage at 2 mg / kg body weight daily, while the control group was administered corn oil by gavage. Each mouse received 100 μl of pp'-DDE daily, with gavage performed at 10:00 AM each day and body weight measured. After 8 weeks of gavage, mice were sacrificed and their femurs were collected for bone mass and thickness analysis.

[0031] 4. Statistical Analysis:

[0032] Experimental results are expressed as mean ± SEM. Significance between different treatment groups was determined using two-way ANOVA. Results are as follows: Figure 1 As shown, Figure 1 The results showed that wild-type mice treated with pp'-DDE by gavage from 6 weeks of age for 8 weeks exhibited a significant increase in bone thickness. (Each group = 6-7, *p < 0.05).

[0033] Example 2: Effects of pp'-DDE on osteoporosis in a mouse OVX model

[0034] 1. Laboratory animals:

[0035] C57BL / 6 mice, female, 12 weeks old, weighing approximately 22 grams, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice had free access to food and water, and their diet was provided by the Animal Experiment Center of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0036] Mice underwent OVX surgery and sham surgery. In the surgical group, the ovaries were removed, while in the sham surgery group, a piece of fat approximately the size of the ovary was removed after opening the abdominal cavity, followed by suturing. Mice were housed individually after surgery and allowed to recover for 2 weeks. Subsequently, they were administered pp'-DDE via gavage, and the control group (corn oil) was given for 8 weeks. After 8 weeks, the mice were sacrificed, and femurs were harvested for micro-CT scans to assess bone mass.

[0037] 2. Experimental reagents:

[0038] pp'-DDE was purchased from Aladdin Reagent Company, product number D117408. Corn oil was purchased from Aladdin Reagent Company. 6% chloral hydrate and 70% alcohol.

[0039] 3. Experimental equipment: weighing scale, scissors, tweezers, 70% alcohol, medical cotton balls, surgical suture needles and thread, razor blades, and gavage needles.

[0040] 4. Experimental steps:

[0041] 1) Animal surgery and recovery:

[0042] Female C57BL / 6 mice were divided into four groups of 5-7 mice each. After shaving and disinfecting with alcohol, the abdominal cavity of each mouse was opened to expose the uterus and ovaries. In the surgical group, the ovaries were removed; in the sham-operated group, a piece of fat approximately the size of the ovary was removed from the side of the uterus. The sutures were then sutured, and all suture needles and sutures were disinfected with alcohol. Postoperatively, each mouse received a single injection of antibiotics to prevent infection. All mice were housed individually and received medication two weeks after recovery.

[0043] 2) Animal gavage treatment:

[0044] Both the surgical and sham-operated mouse groups were administered corn oil control (DMSO dissolved in physiological saline) and pp'-DDE (2 mg / kg, dissolved in corn oil) by gavage, respectively. Gavage was performed at 10:00 AM every day, and body weight was measured. After 8 weeks of gavage, the mice were sacrificed, and femurs were collected for bone mass and thickness scanning analysis.

[0045] 5. Statistical Analysis:

[0046] Experimental results are expressed as mean ± SEM. Significance between different treatment groups was determined using two-way ANOVA. Results are as follows: Figure 2 As shown. Figure 2The results showed that after 8 weeks of gavage administration of pp'-DDE, the OVX true surgery group treated with pp'-DDE showed a significant anti-osteoporosis effect in OVX osteoporosis model mice, with a significant increase in bone mass / tissue mass and cancellous bone number (6-7 per group, *p<0.05).

[0047] The results showed that pp'-DDE could alleviate osteoporosis in mice caused by ovarian absence, and the ratio of bone mass to tissue mass and the number of cancellous bones were preserved, which were significantly higher than those in the OVX osteoporosis model group.

[0048] Example 3: Effect of pp'-DDE on primary osteoblast differentiation

[0049] 1. Laboratory animals:

[0050] Newborn C57BL / 6 fetal mice. Provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0051] 2. Experimental drugs:

[0052] pp'-DDE was purchased from Aladdin Reagents, catalog number D117408. Culture medium was purchased from Corning, and other chemical reagents were purchased from Sigma-Aldrich.

[0053] 3. Experimental apparatus:

[0054] Cell culture plates, cell culture dishes, pipettes, scissors, forceps, centrifuge, 50ml centrifuge tubes.

[0055] 4. Experimental steps:

[0056] 1) Primary cell isolation:

[0057] Primary osteoblasts were isolated from the skulls of newborn mice. The skulls were cut into 2 mm square fragments and digested with type I collagenase at 37 degrees Celsius. The digestion fluids from the first two digestions were discarded, while the digestion fluids from the last three digestions were collected and centrifuged to obtain primary cells, which were then cultured in α-MEM medium containing 10% fetal bovine serum.

[0058] 2) Cell differentiation and drug treatment:

[0059] Once the cells reached 100% confluence, they were treated with 10 μM pp'-DDE and differentiated. The differentiation medium was α-MEM medium containing 10% fetal bovine serum, supplemented with 10 μg / ml ascorbic acid and 10 mM β-glycerophosphate. Alizarin red staining was performed 14 days after osteogenic differentiation to stain the mineralized nodules in the cells. The degree of mineralization was determined by observing the color intensity after staining. The results are shown below. Figure 3As shown in the figure: After 14 days of treatment with 10 μM pp'-DDE and subsequent cell differentiation, the mineralized nodules (red) in the treatment group were significantly higher than those in the control group without drug treatment.

[0060] 3) Expression of molecular signaling such as Ocn, Alp1, and Sp7 in cells:

[0061] Cells were harvested with Trizol at different time points after differentiation (days 0, 7, and 14). RNA was extracted and reverse transcribed. The expression levels of molecular signals such as Ocn, Alp1, and Sp7 were detected using RT-PCR. The results are as follows: Figure 4 As shown: Results: 10 μM pp'-DDE significantly increased the expression of molecular signaling molecules such as Ocn, Alp1, and Sp7 in primary osteoblasts after 0, 7, and 14 days. Results are as follows. Figure 4 As shown: Results: 10 μM pp'-DDE significantly increased the expression of molecular signaling molecules such as Ocn, Alp1, and Sp7 in primary osteoblasts after 0, 7, and 14 days. The results indicate that pp'-DDE enhances primary osteoblast differentiation.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of pp'-DDE in the preparation of anti-osteoporosis drugs, characterized in that, It appears as a white solid crystal or white powder, with the chemical formula C. 14 H8Cl4, with a molecular weight of 318.0, has the following structure for pp'-DDE: 。 2. The use according to claim 1, characterized in that, pp'-DDE can increase cortical bone thickness.

3. The use according to claim 1, characterized in that, pp'-DDE can increase femoral bone thickness.

4. The use according to claim 1, characterized in that, pp'-DDE can promote osteoblast mineralization nodules.

5. The use according to claim 4, characterized in that, It appears as a white solid crystal or white powder, with the chemical formula C. 14 H8Cl4, with a molecular weight of 318.0, is used to enhance the expression of Ocn in osteoblasts.

6. The use according to claim 4, characterized in that, It appears as a white solid crystal or white powder, with the chemical formula C. 14 H8Cl4, with a molecular weight of 318.0, is used to enhance the expression of Alp1 in osteoblasts.

7. The use according to claim 4, characterized in that, It appears as a white solid crystal or white powder, with the chemical formula C14H8Cl4 and a molecular weight of 318.

0. Its intended use is achieved by increasing the expression of Sp7 in osteoblasts.

Images