CN107296808B

CN107296808B - Application of compound epothilone B in preparation of medicine for repairing corneal nerve trauma

Info

Publication number: CN107296808B
Application number: CN201610237382.9A
Authority: CN
Inventors: 李志杰; 董栋; 王寒晴; 肖程聚; 薛芸霞
Original assignee: Jinan University
Current assignee: Guangzhou Jinan University Science Park Management Co ltd; Guangzhou Youchen Biotechnology Co ltd
Priority date: 2016-04-15
Filing date: 2016-04-15
Publication date: 2020-09-04
Anticipated expiration: 2036-04-15
Also published as: CN107296808A

Abstract

The invention discloses application of a compound epothilone B in preparation of a medicine for repairing corneal nerve trauma, and belongs to the field of biological medicines. Pharmacodynamic experiments prove that the mouse subjected to corneal trauma is used as an experimental object, and EpoB is found to be capable of remarkably increasing the area of corneal nerves of the mouse and is close to a normal level. The compound EpoB can promote the polymerization of tubulin and inhibit the depolymerization of microtubules by stabilizing the combination of tubulin, improve the concentration of tubulin, promote the repair and reconstruction of nerve injury and maintain the normal structure and function of cornea. The EpoB has better treatment effect on corneal neuropathy and functional damage caused by refractive surgery, corneal transplantation, herpes simplex keratitis, xerophthalmia, diabetes, long-term use of eye drops and the like, and can relieve the eye symptoms of patients; the medicine has small toxicity and good stability, and has important development and application prospects.

Description

Application of compound epothilone B in preparation of medicine for repairing corneal nerve trauma

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of a compound epothilone B (Epothilone B, EpoB) in preparation of a medicine for repairing corneal nerve trauma.

Background

Since 1830 Sohlemm discovered corneal nerves at the limbus, there has been a tremendous progress in corneal nerve research. Corneal tissue has no blood vessels, but contains abundant nerve fibers, is one of the most dense tissues of nerves in a human body, and plays an important role in providing nutrition metabolism and maintaining the normal structure and function of the cornea. Corneal sensation is the most prominent feature of corneal nerves and has a sharp sensory function. The corneal nerve belongs to a peripheral nerve and has incomplete regeneration capability, and although the corneal nerve can be slowly regenerated after trauma, normal nerve density and function are difficult to recover.

Surgical injury, ocular disease, and chronic ocular medications can cause corneal nerve abnormalities. Surgical injury is the direct destruction of the corneal nerve. The eye diseases are mostly keratitis, dry eye disease, diabetes: the density of central stromal nerves of the cornea with keratitis is obviously lower than that of a normal cornea, and obvious stromal nerve structural abnormality of the cornea exists; the corneal nerve number, curvature and nerve branches of patients with dry eye are increased; diabetes is a systemic metabolic disease, can affect all organs of the whole body, can also cause the abnormality of corneal nerves, and the number of stromal nerve fiber bundles of patients is obviously reduced, and the corneal sensitivity is also obviously reduced. Prolonged use of ocular drugs results in a reduction in the number and density of subcutaneous nerves on the cornea. The patients have great pain, but at present, no treatment method and measure which can accelerate corneal nerve repair and have good function recovery effect exists clinically.

The compound EpoB has the formula: c₂₇H₄₁NO₆S, molecular weight 507.68, EpoB, as a tubulin stabilizer, has mostly focused on its anti-cancer effects. At present, no relevant research report for accelerating corneal nerve trauma repair and functional recovery by using EpoB is reported.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of a compound EpoB in preparing a medicine for repairing corneal nerve trauma. In particular to the effect of a compound EpoB in preparing medicines for accelerating the repair of corneal neuropathy and functional damage caused by refractive surgery, corneal transplantation, herpes simplex keratitis, dry eye, diabetes, long-term use of eye drops and the like.

The purpose of the invention is realized by the following technical scheme:

the invention provides application of a compound EpoB in preparation of a medicine for repairing corneal nerve trauma.

The invention provides application of a composition containing a compound EpoB in preparing a medicine for repairing corneal nerve trauma.

The structural formula of the compound EpoB is as follows:

the medicine for repairing corneal nerve trauma is any dosage form suitable for clinical use, such as tablets, capsules, oral liquid, injection, powder injection, eye drops or eye ointment (or gel) and the like, and the tablets, capsules, oral liquid, injection or powder injection, the eye drops or the eye ointment (or gel) and the like prepared by adopting a micro-nano technology.

The corneal nerve trauma comprises corneal neuropathy and function damage caused by refractive surgery, corneal transplantation, herpes simplex keratitis, dry eye, diabetes or long-term use of eye drops and the like.

Compared with the prior art, the invention has the following advantages and effects:

(1) the mouse subjected to corneal trauma is used as an experimental object, and EpoB is found to be capable of remarkably increasing the area of corneal nerves of the mouse and is close to a normal level.

(2) EpoB stabilizes corneal tubulin and allows for the growth of diffuse neural aggregates.

(3) EpoB can make corneal perception of rats recover basically 6 days after injury, while corneal perception of control group is difficult to recover.

(4) EpoB is used for preparing a corneal nerve injury repair medicine, has a good treatment effect on corneal nerve abnormality caused by surgical injury, eye diseases and long-term eye medication in a safe dosage range, and can relieve eye symptoms of patients.

(5) The compound EpoB can promote the polymerization of tubulin and inhibit the depolymerization of microtubules by stabilizing the combination of tubulin, improve the concentration of tubulin, promote the repair and reconstruction of nerve injury and maintain the normal structure and function of cornea. The EpoB has better treatment effect on corneal neuropathy and functional damage caused by refractive surgery, corneal transplantation, herpes simplex keratitis, xerophthalmia, diabetes, long-term use of eye drops and the like, and can relieve the eye symptoms of patients; the medicine has small toxicity and good stability, and has important development and application prospects.

Drawings

Fig. 1 is a graph showing the results of the change in the total corneal nerve area after corneal trauma in mice.

FIG. 2 shows corneal nerve repair after corneal trauma in mice.

FIG. 3 is a graph showing the results of the expression level of corneal neurotubulin after corneal trauma in mice.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.

Example 1 EpoB accelerated corneal nerve trauma repair in mice

1. Laboratory animal

C57/BL6 female mice of 8 weeks, the weight of which is 15-20 g, are purchased from the center of experimental animals in Guangdong province. The eyes of the animals were examined in the 24 mice before the experiment to ensure that the experimental mice had no eye disease, corneal defect and conjunctival injury.

2. Experimental drugs

EpoB was dissolved in DMSO (dimethyl sulfoxide) to prepare a solution with a concentration of 5mg/mL for use. EpoB stock solution is diluted by adding physiological saline at a ratio of 1:200, and the mice are injected with 0.1mg/mL of EpoB in the abdominal cavity, and the control mice are injected with the same amount of blank solvent in the abdominal cavity.

3. Experimental methods

20 normal non-ocular disease mice were screened and randomly divided into a trauma control group and an EpoB administration group. After each group of mice is subjected to corresponding administration operation, the central area of the cornea is marked by a trephine with the diameter of 2mm, and a golf knife mechanically scratches the epithelial cell layer of the cornea, so that the central area of the cornea of the mice is wounded to form a circular area with the diameter of 2 mm. After trauma, materials are taken every 24 hours to observe the repairing condition of corneal nerve (taking the relative density of corneal nerve as an index), statistical analysis is carried out, and the pharmacodynamics evaluation of epothilone B (EpoB) is carried out. And the expression quantity of the corneal nerve microtubulin is measured by adopting a western-blotting method.

4. Results of the experiment

The effects of EpoB on accelerated corneal nerve repair following corneal trauma in mice are shown in figures 1 and 2: figure 1 shows that at 24h after corneal trauma in mice, the relative corneal nerve density of mice given EpoB treatment began to be higher than that of trauma control mice, and the corneal nerve density continued to increase for 5 days. Although the corneal nerve density of the mice in the trauma control group is also increased, the recovery rate is far lower than that of the mice in the EpoB administration group. The results in fig. 2 show that at day 5 after corneal nerve trauma, corneal nerve density of the EpoB-administered group was significantly higher than that of the control group, and was substantially restored to a normal level, and nerve restoration at the corneal center of the control group was not significant. The results in FIG. 3 show that tubulin in corneal nerve is obviously reduced after corneal nerve injury, and the tubulin expression is obviously increased after EpoB is administrated in abdominal cavity. The results show that EpoB can obviously improve the corneal nerve tubulin expression quantity, increase the area of damaged corneal nerve and accelerate the corneal nerve trauma repair.

Example 2 EpoB accelerated recovery of corneal nerve function in rats

1. Laboratory animal

Healthy male 8-week-old SD rats 30 with body weights of about 250 + -20 g were purchased from the center of laboratory animals in Guangdong province. Animals were examined for both eyes within 24 hours prior to the experiment, and animals without eye irritation symptoms, corneal defects, or conjunctival damage were used for the experiment.

2. Experimental drugs

SR8278 (available from sigma, usa) was dissolved in an appropriate amount of DMSO, and soybean oil for injection was added to prepare SR8278 oily eye drops containing 0.2% of drug.

3. Establishment of animal model for corneal nerve injury of rat

Rats are anesthetized with fast dormancy (purchased from Jilin province Hua mu animal health products Co., Ltd.), a ring incision with the diameter of 3mm and the depth of 0.5mm is drilled at the center of the cornea by a core cornea trephine with the diameter of 3mm under a microscope, and the injury to the posterior elastic layer and the endothelial layer below the ring incision is avoided, so that the rat corneal nerve injury animal model is prepared. The damaged area was visualized by corneal fluorescein staining and the anterior segment analysis system photographed and analyzed the size of the damaged area.

4. Treatment of rat corneal nerve injury

20 normal non-ocular rats were randomly divided into a trauma control group and an EpoB administration group. In the EpoB administration group, EpoB oily eye drops (the EpoB oily eye drops are soybean oil containing 0.2 percent of EpoB) are dropped for 3 times a day; in the wound control group, the blank solvent is dropped into the eye for 3 times a day by adopting equal measurement.

5. Rat corneal nerve sensitivity assay

Corneal perception of rats after anesthesia was measured using a home-made nylon wire corneal perception determinator on

days

2, 4, 6, and 8 after molding. The nylon wire of the corneal perception determinator was gently touched to the center of the cornea of the eye to be examined, starting from 150 mm. In normal corneal perception, the nylon filaments bend and may immediately appear reflective, transient or perceptible. If no temporal reflex or no perception occurs, the corneal perception disappears. If the transient ocular reflex is slow or the perception is insensitive, the nylon wire is reduced from 150mm to 10mm in turn (the interval is 10mm), the acting force of the nylon wire is calculated through a standard curve, the perception sensitivity of the cornea of the rat is reflected by the magnitude of the acting force, and the result is recorded. Corneal perception determinator standard curve determination: the nylon wires are sequentially reduced from 150mm to 10mm in a gradual mode (the interval is 10mm), the nylon wires are lightly touched with an analytical balance, the force required by the nylon wires with different lengths to bend is measured, and a standard curve is drawn by taking the lengths as horizontal coordinates and the acting force as vertical coordinates.

6. Results of the experiment

The acting force of the EpoB administration group at 2 days, 4 days and 6 days is obviously higher than that of the wound control group, and the results are shown in a table 1. Corneal perception of the EpoB-administered group substantially recovered almost 6 days after injury, whereas corneal perception of the trauma-controlled group recovered slowly.

TABLE 1 corneal perception assay in rats (N)

Time of day	Wound control group	EpoB administration group
			2 days	0.1137±0.0152	0.0864±0.0131*
4 days	0.0986±0.0215	0.0465±0.0312***
			6 days	0.0725±0.0132	0.0059±0.0033***
8 days	0.0558±0.0158	0.0062±0.0048***

*p<0.1，***p<0.005

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The application of the compound epothilone B in the preparation of the medicine for repairing corneal nerve trauma.

2. The application of the composition containing the compound epothilone B in the preparation of the medicine for repairing corneal nerve trauma.

3. Use according to claim 1, characterized in that:

the medicament for repairing the corneal nerve trauma is any one preparation form suitable for clinical use.

4. Use according to claim 3, characterized in that:

the dosage form is tablet, capsule, oral liquid, injection, eye drop or eye ointment.

5. Use according to claim 4, characterized in that:

the dosage form is a tablet, a capsule, oral liquid, injection, eye drops or eye ointment prepared by adopting a micro-nano technology.

6. Use according to claim 1, characterized in that:

the corneal nerve trauma comprises corneal neuropathy and functional damage caused by refractive surgery, corneal transplantation, herpes simplex keratitis, dry eye, diabetes or long-term use of eye drop medicaments.

7. Use according to claim 2, characterized in that:

8. Use according to claim 7, characterized in that:

9. Use according to claim 8, characterized in that:

10. Use according to claim 2, characterized in that: