Application of iMDK in treatment of osteoporosis diseases

Abstract

The invention discloses application of iMDK in treatment of osteoporosis diseases, and belongs to the field of biomedicine. Specifically, the invention provides an application of iMDK in preparation of a medicine for treating osteoporosis diseases. Compared with the prior art, the novel application of iMDK clinical medication expands the application field of iMDK diseases, and is small in dosage and small in side effect. In addition, the iMDK can significantly relieve bone mass loss caused by menopause (estrogen reduction). The preparation prepared from the iMDK or the pharmaceutically acceptable carrier thereof has the application prospect of the medicine for treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of iMDK in treating osteoporosis. Background Art

[0002] Osteoporosis is a common metabolic bone disease, especially in the elderly and postmenopausal women. The pathological mechanism of osteoporosis lies in the decrease of bone formation and increase of bone resorption, which leads to the decrease of bone density and increases the risk of fracture.

[0003] For the treatment of osteoporosis, there are some commonly used drugs in the prior art, such as osteoclast inhibitors, represented by Denosumab, which can reduce bone resorption and delay bone loss by inhibiting osteoclast activity. However, since this type of drug mainly acts on the inhibition of bone resorption. After drug withdrawal, bone resorption activity recovers rapidly, which may cause a rapid decrease in bone density and an increased risk of fracture. Another example is bone formation stimulating drugs, represented by Teriparatide, which increase bone mass by stimulating bone formation. However, this type of drug has more side effects and cannot be used for a long time. Similarly, this type of drug unilaterally stimulates bone formation to treat osteoporosis, and cannot simultaneously play a role in stimulating bone formation and inhibiting bone resorption.

[0004] In addition, although existing anti-osteoporosis drugs can effectively control the disease, their high treatment costs limit the popularity of these drugs in many economically underdeveloped areas, and the low accessibility of patients limits the maximization of treatment effects. These factors indicate that the current drugs for the treatment of osteoporosis still have certain limitations in terms of efficacy, safety and affordability.

[0005] iMDK, molecular formula is C 21 H 13 FN 2 O 2 S, molecular weight 376.4, its chemical structure is as follows:

[0006]

[0007] iMDK is a PI3K-AKT pathway inhibitor that can inhibit the growth factor MDK. It does not affect physiological properties such as cell proliferation and differentiation. iMDK is a white powder that is insoluble in water but soluble in alcohol and DMSO. However, in the prior art, there is a lack of further exploration and development of the application of iMDK in other diseases. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention proposes the use of iMDK in treating osteoporosis.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The first aspect of the present invention relates to the use of iMDK in preparing a medicament for treating osteoporosis.

[0011] Optionally, the iMDK is an injection.

[0012] Optionally, the dosage of iMDK is: 9 mg / kg / day.

[0013] The second aspect of the present invention relates to the use of iMDK in the preparation of a medicament for treating reduced P1NP expression level caused by menopause.

[0014] The third aspect of the present invention relates to a drug for treating osteoporosis, comprising iMDK.

[0015] A fourth aspect of the present invention relates to a method for constructing a mouse model, comprising the following steps:

[0016] C57 mice were divided into three groups: blank control, bilateral ovariectomy, and bilateral ovariectomy followed by intraperitoneal injection of iMDK;

[0017] Serum samples and femur samples were obtained from three groups of mice;

[0018] The serum samples were used to detect the level of P1NP in mouse serum, and the femur samples were used to obtain Micro-CT detection and paraffin sections;

[0019] Quantitative analysis of bone density, bone volume fraction, trabecular number and trabecular thickness of mice;

[0020] The effect of iMDK injection on osteoporosis was evaluated by mouse serum P1NP levels, Micro-CT detection, paraffin sections, and mouse bone density, bone volume fraction, trabecular number and trabecular thickness.

[0021] Optionally, the method for obtaining the serum sample comprises the following steps:

[0022] The head of the mouse is fixed, the neck is tightly grasped, and both sides of the neck are compressed to make the orbital venous plexus congested, and blood is obtained by puncturing the inner canthus of the eye. The blood is centrifuged and the supernatant is obtained to obtain the serum.

[0023] Optionally, the method for obtaining a femur sample comprises the following steps:

[0024] The mice were killed by cervical dislocation, and the skin and muscles at the proximal end of the mouse lower limbs were cut to fully expose the femur and hip joint. The femur was separated and taken out intact, and the muscles and other soft tissues were removed, and fixed and preserved with 4% PFA.

[0025] Beneficial effects of the present invention:

[0026] The present invention discloses a new use of iMDK, or provides a new treatment idea for osteoporosis, with a small dosage and few side effects. iMDK can significantly alleviate bone loss caused by menopause (estrogen reduction). Specifically, the present application experimentally verifies that iMDK can increase the formation of cancellous bone, thereby increasing bone density. On the other hand, the present application also discloses that iMDK can reduce the number of TRAP-positive osteoclasts, inhibit bone resorption, and thereby increase bone density. It can be seen that iMDK can act on osteoporosis through the above-mentioned dual mechanism, thereby exerting a more comprehensive and lasting therapeutic effect than existing drugs. This dual mechanism may make it have higher efficacy and safety in the process of treating osteoporosis, especially for patients with refractory osteoporosis. In addition, iMDK, as a small molecule compound, has the advantages of easy synthesis and low cost. Therefore, the preparation prepared by iMDK or its pharmaceutically acceptable carrier has the application prospect of osteoporosis drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings.

[0028] Figure 1 Enzyme-linked immunosorbent assay was used to detect the level of serum type 1 collagen carboxyl terminal peptide (P1NP) in each group of mice.

[0029] Figure 2 Micro-CT scans were performed on the lower end of the femur of mice in each experimental group and three-dimensional images were reconstructed.

[0030] Figure 3 The bone density, bone volume fraction, trabecular number, trabecular thickness and trabecular spacing of each experimental mouse were quantitatively analyzed by three-dimensional images reconstructed by Micro-CT scanning. Figure 3 A in the figure is the bone density of mice in each experimental group. Figure 3 B in the figure is the bone volume fraction of mice in each experimental group. Figure 3 C in the figure is the trabecular thickness of mice in each experimental group. Figure 3 D in the figure is the number of trabeculae in each experimental group of mice. Figure 3 E in the figure is the trabecular spacing of mice in each experimental group.

[0031] Figure 4 H&E staining results of bone tissue sections of mice in each group.

[0032] Figure 5 TRAP staining results of bone tissue sections of mice in each group. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In some embodiments of the present invention, an experimental method for exploring the effect of iMDK on osteoporosis is disclosed, including the following contents.

[0035] 1. Materials

[0036] 1. Experimental Animals

[0037] C57BL / 6J mice, 18 females, 8 weeks old, weighing 20-23 g, SPF grade, provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Laboratory Animal Production License No. (SCXK(Su)2023-0009).

[0038] During the experiment, the temperature in the feeding room was 20±3.2℃, the relative humidity was 65-75%, and the animals were free to eat and drink.

[0039] 2. Drugs and reagents:

[0040] 2.1 Reagents

[0041] iMDK, white powder, purity ≥95%, 1 mg / bottle, provided by Shanghai Taoshu Biotechnology Co., Ltd., product number T9460, stored in a refrigerator at -80℃ away from light. The structural formula of iMDK is:

[0042]

[0043] P1NP ELISA kit, provided by Shanghai ELISA Biotechnology Co., Ltd., product number is ml057938-2.

[0044] 2.2 Instruments

[0045] Micro-CT scanner (micro-CT), model: SkyScan1174v2, instrument number: 16P03141, provided by the School of Public Health, Soochow University.

[0046] ELISA reader (multi-function microplate reader), model: Tecan M200PRO, manufactured in Austria, provided by the Experimental Center of the Second Affiliated Hospital of Soochow University.

[0047] 2. Experimental Methods

[0048] 1. Modeling and drug administration

[0049] The experimental groups were as follows: control group, bilateral ovariectomy group (OVX group) and iMDK treatment group (OVX+iMDK group), with 6 mice in each group.

[0050] Control group: 8-week-old C57 mice were used as controls.

[0051] OVX group: 8-week-old bilaterally ovariectomized mice were used for type I (postmenopausal) osteoporosis model.

[0052] OVX+iMDK group: 8-week-old bilaterally ovariectomized mice were given intraperitoneal injection of iMDK at a dose of 9 mg / kg / day, 3 times a week for 8 weeks.

[0053] 2. The mice in each group were sampled

[0054] Collection of serum samples: Orbital blood sampling method was used. The mouse was pressed on the mouse cage with the left hand. The mouse's head was fixed with the thumb and index finger. The neck was tightly grasped and the two sides of the neck were compressed to make the eyeball protrude and the orbital venous plexus congested. The blood collection tube was held in the right hand and inserted from the inner canthus of the eye at a 45° angle to the mouse's face. The mouse's body was fixed and the hind limbs were pressed. The fingers were relaxed and the blood collection tube was adjusted to allow the blood to flow out smoothly. After the blood collection was completed, the neck pressure was immediately removed and the blood collection device was pulled out. The bleeding was immediately stopped by pressing. The blood sample tube was gently shaken and placed on ice for 30 minutes. The tube was centrifuged at 4°C, 3000rpm, for 20 minutes. The supernatant was taken and centrifuged again at 4°C, 4000rpm for 10 minutes. The supernatant was a relatively pure serum sample for the detection of P1NP levels.

[0055] Collection of mouse femur specimens: The mice were killed by cervical dislocation, the skin and muscles of the proximal lower limbs of the mice were cut open, the femur and hip joint were fully exposed, the femur was carefully separated, the femur was completely removed, and the muscles and other soft tissues were removed, and the femur was fixed and preserved with 4% PFA. The left femur was used for Micro-CT detection, and the right femur was used to make paraffin sections for H&E staining and tartrate-resistant acid phosphatase (TRAP) staining.

[0056] 3. According to the animal experiment results of iMDK in treating type I osteoporosis and the clinical use of iMDK, the present invention can use iMDK for subcutaneous injection at 9 mg / kg / day when treating type I osteoporosis.

[0057] 3. Experimental Results

[0058] 1. iMDK can effectively restore P1NP levels in OVX mice

[0059] In this example, the serum P1NP levels of OVX group and OVX+iMDK mice were detected by enzyme-linked immunosorbent assay (ELISA). As shown in the figure, the P1NP level of OVX+iMDK group mice was higher than that of OVX group (P<0.05); this result indicates that iMDK can effectively restore the low P1NP level caused by OVX.

[0060] 2. The bone mass of mice decreased after bilateral ovariectomy. iMDK can partially restore the bone mass loss caused by ovariectomy in mice.

[0061] like Figure 2 As shown in the figure, using Micro-CT to scan the lower end of the femur of the above three groups of experimental mice and perform three-dimensional reconstruction and quantitative analysis of bone microstructure parameters can accurately describe bone density and bone microstructure, thereby determining the degree of osteoporosis. Three-dimensional image reconstruction showed that compared with the control group, the number of trabeculae in the OVX group mice was significantly reduced and thinner. Compared with the OVX group, after iMDK intervention, the trabeculae in the OVX+iMDK group increased and thickened significantly.

[0062] In order to further verify the results of the three-dimensional images, this example quantitatively analyzed the bone density, bone volume fraction, trabecular number, trabecular thickness and trabecular separation of each experimental mouse. Figure 3 As shown in the figure, compared with the control group, the bone density, bone volume fraction, trabecular thickness and trabecular number of mice in the OVX group were significantly reduced (P<0.05). Compared with the OVX group, the bone density, bone volume fraction, trabecular thickness and trabecular number of the femur of mice in the OVX+iMDK group were significantly increased (P<0.05). The above results show that the quality of cancellous bone can be significantly improved after iMDK intervention. Therefore, it is concluded that iMDK is effective in treating osteoporosis.

[0063] 3. After bilateral ovariectomy, the mice had reduced cancellous bone and increased osteoclasts. iMDK can partially restore the reduced cancellous bone and increased osteoclasts caused by ovariectomy in mice.

[0064] like Figure 4 As shown in the figure, H&E staining was performed on the bone tissue sections of the experimental mice. The results showed that the cancellous bone of the bilaterally ovariectomized mice (OVX) was significantly reduced compared with the control group, and the cancellous bone of the OVX+iMDK group mice was increased compared with the OVX mice. This result shows that iMDK can effectively rescue the cancellous bone reduction caused by OVX. Figure 5 As shown, the number of TRAP-positive osteoclasts in the bone tissue of OVX group mice increased significantly, while the number of TRAP-positive osteoclasts in the bone tissue of OVX+iMDK group mice decreased compared with the OVX group.

[0065] According to the animal experiment results of iMDK in treating type I osteoporosis, when implementing the technical scheme of the present invention to treat type I osteoporosis, the injection dose of iMDK can be changed according to other relevant factors such as the administration method, the patient's age, weight and severity of the disease.

[0066] The experimental results of the above examples show that in animal experiments, the OVX osteoporosis mouse model suffers from severe bone loss, and the bone mass of the mice is restored by injecting an appropriate amount of iMDK into the OVX mice.

[0067] iMDK was used to intervene in the type I osteoporosis animal model and found to have a certain anti-osteoporosis effect. This discovery will have a significant impact on the research and treatment of type I osteoporosis and has certain potential clinical practical value, opening up new clinical uses for iMDK. This embodiment uses intraperitoneal injection.

[0068] In the above technical solution, the iMDK pharmaceutical composition can be prepared into a preparation in the form of a solution, suspension, emulsion, lyophilized product or sterile powder by adding common pharmaceutical auxiliary ingredients to iMDK with a purity of more than 95% (mass percentage).

[0069] It should also be noted that the present invention and embodiments do not imply that iMDK is used as the only pharmaceutical ingredient or active ingredient in practical applications. In other embodiments of the present invention, iMDK can also be combined with other pharmaceutical ingredients (such as ingredients that can treat osteoporosis in the prior art) or excipients to form a pharmaceutical composition.

[0070] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0071] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Use of iMDK in the preparation of a drug for treating osteoporosis.

2. The use of IMDK in treating osteoporosis according to claim 1, characterized in that: The iMDK is an injection.

3. The use of IMDK in treating osteoporosis according to claim 1, characterized in that: The iMDK dosage is: 9 mg / kg / day.

4. Use of iMDK in the preparation of a drug for treating reduced P1NP expression levels caused by menopause.

5. A drug for treating osteoporosis, characterized in that: Including iMDK.

6. A method for constructing a mouse model, characterized in that: The following steps are involved: C57 mice were divided into three groups: blank control, bilateral ovariectomy, and bilateral ovariectomy followed by intraperitoneal injection of iMDK; Serum samples and femur samples were obtained from three groups of mice; The serum samples were used to detect the level of P1NP in mouse serum, and the femur samples were used to obtain Micro-CT detection and paraffin sections; Quantitative analysis of bone density, bone volume fraction, trabecular number and trabecular thickness of mice; The effect of iMDK injection on osteoporosis was evaluated by mouse serum P1NP levels, Micro-CT detection, paraffin sections, and mouse bone density, bone volume fraction, trabecular number and trabecular thickness.

7. The method for constructing a mouse model according to claim 6, characterized in that: The method for obtaining the serum sample comprises the following steps: The head of the mouse is fixed, the neck is tightly grasped, and both sides of the neck are compressed to make the orbital venous plexus congested, and blood is obtained by puncturing the inner canthus of the eye. The blood is centrifuged and the supernatant is obtained to obtain the serum.

8. The method for constructing a mouse model according to claim 6, characterized in that: The method for obtaining the femur sample comprises the following steps: The mice were killed by cervical dislocation, and the skin and muscles at the proximal end of the mouse lower limbs were cut to fully expose the femur and hip joint. The femur was separated and removed intact, and the muscles and other soft tissues were removed, and then fixed and preserved with 4% PFA.

9. The method for constructing a mouse model according to claim 6, characterized in that: The amount of iMDK injected intraperitoneally was 9 mg / kg / day.

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