阅读说明：本技术 齐墩果酸及其衍生物在制备促进骨折愈合的药物中的应用 (Application of oleanolic acid and derivatives thereof in preparation of medicines for promoting fracture healing ) 是由 舒冰 董玉峰 王拥军 杨骏杰 赵永见 张岩 赵东峰 王晶 张�浩 程韶 张伟强 于 2019-10-29 设计创作，主要内容包括：本发明涉及齐墩果酸及其衍生物的医药用途,具体公开了齐墩果酸及其衍生物在制备促进骨折和/或骨质疏松性骨折愈合的药物中的应用。本发明确证不同浓度齐墩果酸及其衍生物的局部干预,具有促进骨形成和提升骨量的作用,可以显著促进骨折部位的愈合和修复,且高剂量齐墩果酸及其衍生物也无明显肝肾毒性。本发明可用于医学和兽医学应用,使用途径包括注射、缓释技术或者支架材料中复合齐墩果酸及其衍生物等局部给药方式,亦可与其它补充活性成分、试剂、药物或激素一起施用。(The invention relates to medical application of oleanolic acid and derivatives thereof, and particularly discloses application of oleanolic acid and derivatives thereof in preparation of a medicine for promoting fracture and/or osteoporotic fracture healing. The invention confirms the local intervention of oleanolic acid and derivatives thereof with different concentrations, has the effects of promoting bone formation and bone mass, can obviously promote the healing and repair of fracture parts, and has no obvious hepatotoxicity or hepatotoxicity due to high dose of oleanolic acid and derivatives thereof. The invention can be used for medical and veterinary applications, and the application route comprises local administration modes of injection, slow release technology or composite oleanolic acid and derivatives thereof in stent materials, and the like, and can also be used together with other supplementary active ingredients, reagents, medicaments or hormones.)

1. Application of oleanolic acid and its derivatives in preparation of medicine for promoting fracture and/or osteoporotic fracture healing is provided.

2. Use according to claim 1, characterized in that oleanolic acid is a compound of formula (I).

3. The use according to claim 1, wherein the oleanolic acid derivative is an oleanolic acid derivative A-H having the following molecular structure in formula (II) by introducing carboxyl group, hydroxyl group and double bond:

a is C ═ O at the 11 position, and a double bond at the 12 and 13 positions;

b is hydroxyl at 11 position, and CH at 12 and 13 positions respectively₂；

C is a double bond at 11 and 12 positions, a double bond at 13 and 18 positions;

d is 11-position CH₂12-position C ═ O, 13-position>CH；

E is 11-position CH₂The 12-hydroxy group;

f is a double bond at the 9 and 11 positions, and C at the 12 position is O;

g is a double bond at the 9-and 11-positions, CH at the 12-position₂13 bit>CH；

H11 and 12 are respectively CH₂13 bit>CH。

4. Use according to any one of claims 1 to 3, characterized in that oleanolic acid and any one of its derivatives are used as a pharmaceutical intervention for promoting fracture healing and repair.

5. Use according to claim 4, characterized in that oleanolic acid and its derivatives are combined with pharmaceutically acceptable excipients and/or carriers suitable for pharmaceutical use to make a medicament for medical and veterinary use.

6. The use as claimed in claim 4, wherein the intervention mode of oleanolic acid and its derivatives is topical administration, including topical injection, various topical sustained release techniques, or composite oleanolic acid or its derivatives in a scaffold material.

7. The use as claimed in claim 6, wherein the concentration of oleanolic acid and its derivatives intervenes at a concentration of 5 μ M to 20 μ M.

Technical Field

The invention relates to medical application of oleanolic acid and derivatives thereof, in particular to application of local administration of oleanolic acid and derivatives thereof as a local treatment method for repairing bone fracture caused by various reasons.

Background

The fracture belongs to very common clinical diseases, and in view of the characteristics of bone tissues, the healing time is long, and complications such as muscular atrophy, venous thrombosis, weighing pneumonia and the like can be caused by long-term bed rest and braking, and the condition is particularly prominent in the elderly; more seriously, due to the basic diseases of the elderly, menopause, renal insufficiency, intestinal diseases and the like and the influence of various factors, fracture healing and repair are delayed, so that the whole course of disease is prolonged remarkably, the complication incidence rate is increased greatly, and the elderly patients suffering from other serious basic diseases are even fatally dangerous. In the aspect of treatment, at present, the treatment is mainly surgical treatment, old patients and patients suffering from other serious basic diseases even cannot receive surgery, and the drug treatment mainly aims at the basic diseases, but medicines capable of promoting the healing and repair of fracture parts are rare. Therefore, there is an urgent clinical need to develop drugs that can promote fracture healing and repair, thereby shortening the course of treatment and reducing complications.

At present, no research report is found about the effect of oleanolic acid and derivatives thereof in promoting fracture healing.

Disclosure of Invention

The invention mainly aims to develop a medicine capable of promoting fracture healing, thereby shortening the treatment course and reducing complications, and further provides a treatment method capable of effectively promoting fracture healing.

In order to achieve the aim, the inventor conducts related research and finds that the oleanolic acid and the derivatives thereof can effectively promote fracture healing after being locally administered, and the efficacy can be used for treating fracture caused by various reasons and/or osteoporotic fracture.

The oleanolic acid disclosed by the invention is a compound shown as a formula (I).

The oleanolic acid derivative is an oleanolic acid derivative A-H which introduces functional groups such as carboxyl, hydroxyl, double bonds and the like and has the following molecular structure in a formula (IV):

a is C ═ O at the 11 position, and a double bond at the 12 and 13 positions;

b is hydroxyl at 11 position, and CH at 12 and 13 positions respectively₂；

C is a double bond at 11 and 12 positions, a double bond at 13 and 18 positions;

d is 11-position CH₂12-position C ═ O, 13-position>CH；

E is 11-position CH₂The 12-hydroxy group;

f is a double bond at the 9 and 11 positions, and C at the 12 position is O;

g is a double bond at the 9-and 11-positions, CH at the 12-position₂13 bit>CH；

H11 and 12 are respectively CH₂13 bit>CH。

It is a further object of the present invention to provide a method for the use of oleanolic acid and derivatives thereof in promoting healing of bone fractures and/or osteoporotic bone fractures.

The present invention is based on the discovery that oleanolic acid and derivatives thereof can promote fracture healing. Oleanolic acid and any one of its derivatives can be used as medicine intervention, combined with pharmaceutically acceptable excipient and/or carrier suitable for medicinal use, and made into medicine for medical and veterinary use; local administration is adopted, including local injection, various local slow release technologies, or composite oleanolic acid or derivatives thereof in a scaffold material; the local intervention concentration of oleanolic acid and its derivatives is 5-20 μ M.

Drawings

FIG. 1 is a graph of local dry prognosis of oleanolic acid and its derivative A, B with 14 days of renal HE staining. FIG. 1 shows that, after oleanolic acid and its derivative A, B are locally dried, no obvious tissue structural abnormality is found in mouse kidney.

FIG. 2 is a graph of HE staining of liver 14 days after partial dry prognosis of oleanolic acid and its derivative A, B. FIG. 2 shows that, after the local drying of oleanolic acid and its derivative A, B, no obvious histological abnormality of mouse liver was found.

Fig. 3 is a three-dimensional reconstruction picture of lumbar vertebra parts 14 days after local intervention of oleanolic acid and its derivative A, B. Fig. 3 shows that the lumbar vertebra bone mass of the model group is decreased, which indicates that the osteoporosis model is successfully established, and that the local dry prognosis of oleanolic acid and derivatives A, B thereof is increased, and the lumbar vertebra bone mass of each group is increased.

FIG. 4 is a three-dimensional reconstruction diagram of a fracture site 14 days after local intervention of oleanolic acid and derivatives A-G thereof. FIG. 4 shows that the cortical bone of the fracture part of the model group is discontinuous and still has obvious fracture lines, and after oleanolic acid and derivatives A-G thereof are intervened for 14 days, the cortical bone of the fracture part of most intervened groups is continuous, and the bone repair is remarkably accelerated.

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples. These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

In the following examples of the present invention, experimental methods not specifically identified, namely HE staining and MiCT bone scanning, are common experimental methods in the art.

Examples

1. Preparation of a mouse osteoporosis fracture model:

the mouse is placed into a sealed box connected with a respiratory anesthesia machine, the anesthesia machine is started, the head of the mouse is plugged into a mask interface of the anesthesia machine after the mouse is anesthetized, the mouse is in a prone position, the two lateral iliac posterior superior spines of the mouse are positioned, about 1cm above the posterior iliac bones and about 1cm beside the spines are used for central skin preparation and disinfection. Making a longitudinal incision about 1cm above the posterior iliac crest, centering on spinal column, making the incision about 2cm long, cutting skin, separating hypodermis, selecting muscular layer 1cm beside spinal column at both sides, making a longitudinal incision 0.5cm, and exposing ovary at both sides (the shape is red cauliflower, and irregular nodular follicle can be seen on the surface). The ovaries and fallopian tubes were lifted with forceps, and the ovaries were excised by ligating the surrounding tissue and a portion of the fallopian tubes with silk. The incision and skin are sutured after the tissue is retrieved. The sham group was operated the same as the ovariectomized group except that no ligation was performed and no ovaries were removed. After the wound is sutured, the eye ointment is smeared on the wound to prevent infection.

The mouse is put into the airtight box of connecting the anesthesia machine of breathing, starts the anesthesia machine, waits to fill in the face guard interface of anesthesia machine with the mouse head after the mouse anesthesia, with mouse left hind limb skin preparation. Taking a longitudinal incision on the outer side of the left calf, wherein the incision is about 2cm, separating skin, subcutaneous tissues and fascia, and exposing the middle section of the tibia; meanwhile, a small opening with the diameter of about 2mm is arranged at the outer side of the knee joint, and the patella and the patellar ligament are exposed. The length of about 1.5cm is driven into the tibia platform from the front by a 1ml syringe needle along the long axis of the tibia, the needle is cut by rongeur after slightly withdrawing the needle, the middle section of the tibia is cut by an ophthalmic scissors, the needle is inserted for intramedullary fixation, and the cut muscle, subcutaneous tissue and skin are sutured.

2. Pharmaceutical formulation and intervention

A suspension of 5-20 μ M oleanolic acid and its derivatives A-G was prepared using 0.5% sodium nitrate solution. Injecting 100 μ l into micro osmotic pump, and locally slowly releasing for 1.1 μ l/hr. Only 100. mu.l of 0.5% sodium nitrate solution was injected into the mini-osmotic pumps of the surgical and model groups.

The lateral longitudinal incision of the left calf, approximately 2cm, was taken, the skin, subcutaneous tissue and fascia were separated, and the mid-tibia was exposed (the above steps can be performed together during the modeling of the fracture model). The discharge end of the medicine conveying pipe is inserted along the tibia, so that the discharge port of the medicine conveying pipe reaches the slow-release part. And cutting the epidermis at the lower edge of the left rib of the mouse to prepare a transverse incision about 0.6cm, and blunt separating the subcutaneous fascia from the incision along the rib towards the spine by using vascular forceps to form a space with the size of about 1.5 x 0.8cm capsule. 100ul of solution was filled in the osmotic pump and the pump was implanted in the prepared subcutaneous space. The input end of the drug delivery tube is punctured to the direction of the drug release rod through a 50ml injection needle, and the input end of the drug delivery tube is cut to reach a proper length and is connected with the drug release rod. The incision was quickly closed after cleaning.

3. Results of the experiment

3.1 examination of liver and Kidney toxicity

After A, B local (20 μ M) intervention of oleanolic acid and its derivatives for 14 days, mice were sacrificed, and the kidneys and livers of the mice were taken, frozen sections were prepared, and HE (hematoxylin and eosin) staining was performed. The local dry prognosis of the oleanolic acid and the derivative A, B thereof is shown, no obvious tissue structure abnormality is found in the kidney and the liver of the mouse, and the traditional Chinese medicine composition has no obvious adverse reaction and toxicity (as shown in figures 1 and 2).

3.2 three-dimensional reconstruction of bone tissue

The oleanolic acid and the derivatives A-H thereof are locally (5 mu M-20 mu M) intervened for 14 days, the mice are killed, the lumbar vertebrae and the injured tibia of the mice are taken, fixed by 10% neutral formalin, and then miCT scanning and bone three-dimensional reconstruction are carried out. The results show that the lumbar vertebra bone mass of the model groups is reduced, which prompts the successful establishment of the osteoporosis model, the local dry prognosis of the oleanolic acid and the derivative A, B thereof, and the lumbar vertebra bone mass of each group is increased (as shown in figure 3).

Three-dimensional reconstruction of fractured tibia also found that the method can accelerate healing of feather quill at the fracture site of the mouse (as shown in fig. 4): the cortical bone of the fracture part of the model group is discontinuous and still has obvious fracture lines, and after the intervention of oleanolic acid and the derivatives A-H thereof for 14 days, the cortical bone of the fracture part of most intervention groups is continuous, and the bone repair is obviously accelerated.