CN112972654A

CN112972654A - Application of FGF21 in preparation of medicine for treating cranial nerve injury

Info

Publication number: CN112972654A
Application number: CN202110242702.0A
Authority: CN
Inventors: 龚方华; 张宏宇; 张健; 王德仲
Original assignee: Cixi Institute Of Biomedicine Wenzhou Medical University
Current assignee: Cixi Institute Of Biomedicine Wenzhou Medical University
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2021-06-18
Anticipated expiration: 2041-03-05
Also published as: CN112972654B

Abstract

The invention relates to an application of FGF21 in preparation of a medicine for treating cranial nerve injury. The invention discovers that FGF21 can inhibit the activity of acetylcholinesterase for the first time, reveals that the acetylcholinesterase has good prevention or treatment effect on senile dementia, and particularly can obviously shorten the escape latency of senile dementia rats in the Morris water maze experiment. Further, the present invention has found that the combination of FGF21 and donepezil can further enhance the activity of inhibiting acetylcholinesterase.

Description

Application of FGF21 in preparation of medicine for treating cranial nerve injury

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of FGF21 in preparation of a medicine for treating cranial nerve injury.

Background

Senile dementia can be classified into Alzheimer's Disease (AD) dementia, vascular dementia and the coexistence of them. However, AD is more common clinically, and most of brain dysfunction caused by organic injury of the brain degrades and intelligently damages the abilities of memory, understanding, judgment and control and the like, thereby seriously affecting the daily life of patients. The prevalence rate of the senile dementia in China is about 2% -5%, but the senile dementia tends to gradually increase with the age. At present, the annual new onset is about 1 percent, and the death rate of the senile dementia is the 4 th death rate of common diseases, and is only second to cardiovascular and cerebrovascular diseases, tumors and cerebral apoplexy. With the further development of the aging of the social population structure in China, the proportion of the elderly is getting larger, so that the prevention and treatment of the senile dementia disease become a serious social problem.

Based on the existing pathogenesis of the senile dementia, different approaches of drug treatment are proposed. In the 70 s the field was mainly focused on the relationship of acetylcholine to AD, while in the 80 s the effects of cholinesterase inhibitors on AD were mainly studied, and in the 90 s acetylcholine receptor agonists were implicated, including estrogens, anti-inflammatory analgesics, antioxidants and free radical scavengers, drugs inhibiting amyloid deposition, and calcium antagonists. Impairment of the cholinergic pathway is one of the well-recognized pathogenesis of AD. Cholinergic nerves are closely related to cognitive functions, and phenomena such as cholinergic neuron loss, acetylcholine level reduction, acetylcholine receptor function damage and the like are found in brains of AD patients. These phenomena can cause memory and other cognitive dysfunction in the patient. Acetylcholine is easily hydrolyzed by Acetylcholinesterase (ACh E), and can inhibit the activity of Acetylcholinesterase, increase the concentration of acetylcholine in synaptic cleft, and improve cognitive function of AD patients. Therefore, ACh E inhibitors are clinically useful in the treatment of AD.

The medicine for treating senile dementia mainly aims at improving cognitive function and delaying disease progression. The development and development of these drugs is becoming more and more popular, and new drugs are coming out endlessly, improving cognitive function and behavior to some extent, and improving cognitive function score. According to the pharmacological actions of nootropic drugs, the drugs can be classified into drugs acting on neurotransmitters, cerebral vasodilators, drugs promoting cerebral metabolism, etc., and the actions of the drugs are also crossed. The inhibition of cholinergic system can cause memory and learning decline, similar to the normal amnesia of the elderly. If central cholinergic activity is enhanced, learning and memory ability of the elderly can be improved. Thus, changes in the cholinergic system are closely related to the extent of impairment of cognitive function in AD, the so-called cholinergic hypothesis. The objective of cholinergic therapy is to promote and maintain the function of the remaining cholinergic neurons. Such drugs are primarily used in the treatment of AD.

The brain metabolism activation medicine is also used for treating senile dementia, has more and complex effects, mainly expands cerebral vessels, increases the utilization of oxygen, glucose, amino acid and phospholipid by cerebral cortical cells, promotes the recovery of the cerebral cells, improves functional cerebral cells and further achieves the aim of improving memory. But due to AD at the onset, the degree of dementia, impairment of brain function, genetic factors and differences in responses that exist between individuals. The existing medicines for treating senile dementia comprise tacrine, donepezil, galanthamine, huperzine A, metabolism promoting medicines, antioxidants, amyloid protein resistance and the like

FGF21 was a novel member isolated from mouse embryos from Nishimura et al in 2000, and it was found that human FGF 2/gene is located on chromosome 19 and that the amino acid sequence of human FGF21 has 75% homology with mouse FGF 21. Fibroblast growth factors are a family of signaling proteins that act in a paracrine, endocrine, autocrine fashion, unlike the autocrine fashion of other members of the fibroblast growth factor family, which act in an endocrine fashion due to the lack of a binding domain, FGF 21. FGF21 is also expressed predominantly in liver, adipose and pancreatic, white and brown adipose tissue, skeletal muscle, thymus and endothelial cells, and is an important locus for its expression. Expression of FGF21 in the liver is controlled by peroxisome proliferator activator receptor a (PPARa), and expression of FGF21 in adipose tissue is controlled by peroxisome proliferator activator receptor (PPARy). In addition, FGF21 is also expressed in the heart and in the microvasculature. A series of researches show that FGF21 has certain influence on pathological processes of myocardial hypertrophy, atrial fibrillation, diastolic heart failure, hypertension, atherosclerosis, coronary heart disease, acute myocardial infarction and acute kidney injury. However, the prior art does not disclose that FGF21 has a alleviating effect on cranial nerve damage, particularly Alzheimer's disease, and the present invention has been completed based on this finding.

Disclosure of Invention

Based on the background technology, the technical problem to be solved by the invention is the new pharmaceutical application of FGF21, in particular to the application in preparing medicines for treating cranial nerve injury. In order to realize the purpose of the invention, the following technical scheme is adopted:

the invention relates to an application of FGF21 in preparation of a medicament for treating cranial nerve injury.

In a preferred embodiment of the present invention, the cranial nerve injury is senile dementia.

In a preferred embodiment of the invention, the medicament further comprises other agents that inhibit acetylcholinesterase activity.

In a preferred embodiment of the present invention, the drug inhibiting acetylcholinesterase activity is donepezil or a salt thereof.

In a preferred embodiment of the invention, the medicament further comprises pharmaceutically acceptable excipients, with or without other active ingredients.

In another aspect, the present invention relates to a medicament for treating cranial nerve injury, comprising FGF21 as an active ingredient.

In a preferred embodiment of the present invention, the medicament further comprises donepezil or a salt thereof; preferably, the weight ratio of FGF21 to donepezil or salt thereof is 1:1 to 1: 0.4; further preferably, the weight ratio is 1: 0.9-1:0.5. The invention is beneficial to improving the activity of inhibiting acetylcholinesterase and improving the treatment effect by reasonably determining the weight ratio of the two.

In a preferred embodiment of the invention, the medicament is an oral formulation. Such oral formulations include, but are not limited to, tablets, capsules, liquid formulations, and the like.

Advantageous effects

The invention discovers that FGF21 can inhibit the activity of acetylcholinesterase for the first time, reveals that the acetylcholinesterase has good prevention or treatment effect on senile dementia, and particularly can obviously shorten the escape latency of senile dementia rats in the Morris water maze experiment. Further, the present invention has found that the combination of FGF21 and donepezil can further enhance the activity of inhibiting acetylcholinesterase.

Drawings

FIG. 1: mean escape latency for FGF21 for senile dementia rats control, high dose treatment, low dose treatment and young rats control.

Detailed Description

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1: activity assay for FGF21 inhibition of acetylcholinesterase

(1) Dissolving FGF21 (purchased from Beijing Yi Qian Shen science and technology Co., Ltd.) in dimethyl sulfoxide (DMSO) to obtain a 5mg/mL mother solution, and diluting with PBS buffer solution to obtain test solution with mass concentration of 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.13mg/mL, 0.063mg/mL, 0.031mg/mL and 0.015 mg/mL;

(2) dissolving donepezil in dimethyl sulfoxide (DMSO) to obtain 10mg/mL mother solution, and diluting with PBS buffer solution to obtain test solutions with mass concentrations of 1000 μ M, 500 μ M, 250 μ M, 125 μ M, 62.5 μ M, 31.25 μ M, 15.625 μ M, 7.8125 μ M, 3.91 μ M, 1.95 μ M, and 0.98 μ M;

(3) respectively preparing acetylcholinesterase and butyrylcholinesterase into 4U/mL solution by using a protein stabilizer (1 ‰ BSA PBS solution); ATCI (iodothioacetylcholine) was made into 10mM solution with PBS; preparing 5mM DTNB (5, 5' -dithio (2-nitrobenzoic acid)) into a solution by PBS;

(4) the in vitro inhibitory activity of acetylcholinesterase was determined in 96-well plates by a modified Ellman colorimetric method, as follows: setting 3 multiple wells for each concentration, adding 59 mu LPBS, 1 mu L of a sample to be detected, 1 mu L of 4U/mL acetylcholinesterase and 20U/mLDTNB into each well, and placing a 96-well plate in an incubator at 37 ℃ for incubation for 20 minutes; then, 20 μ L of ATCI was added to each well and incubation was continued in an incubator at 37 ℃ for 20 minutes; and (3) measuring the absorbance value at 412nm by using a microplate reader, wherein the control group is the absorbance of adding the sample without adding acetylcholinesterase, the experimental group is the absorbance of adding the sample and acetylcholinesterase, the negative control is the absorbance of adding the acetylcholinesterase without adding the sample, and the blank group is the absorbance of not adding the sample and the acetylcholinesterase. Donepezil hydrochloride was used as a positive control.

Based on the above experiments, FGF21 has strong inhibitory activity on acetylcholinesterase, and the IC50 value is 1.3 μ g/mL, which is equivalent to donepezil (1.2 μ g/mL). IC (integrated circuit)₅₀The values are the concentrations corresponding to 50% inhibition of the sample to acetylcholine esterase.

Example 2: activity assay for co-inhibition of acetylcholinesterase by FGF21 and donepezil

Taking 5mg/mL FGF21 mother liquor and 10mg/mL donepezil mother liquor, preparing mother liquor with the mass ratio of FGF21 to donepezil being 1:1, 1:0.8, 1:0.6 and 1:0.4 in sequence, adding samples to be tested into a 96-well plate according to the mode of example 1, and measuring the FGF21 concentration corresponding to the acetylcholinesterase inhibition rate of each sample being 50%. The experimental results are shown in table 1 below.

Table 1: acetylcholinesterase IC₅₀Value of

Mass ratio of FGF21 to donepezil	IC50(μg/mL)
		1:0	1.3
1:1	0.44
		1:0.8	0.37
1:0.6	0.40
		1:0.4	0.7
0:1	1.2

Based on the above experiments, the combination of FGF21 and donepezil has a strong inhibitory activity against acetylcholinesterase, and compared to FGF21 or donepezil alone, the inhibitory activity can be significantly improved.

Example 2

The invention further adopts a natural aging cognitive disorder (senile dementia) animal model, and the animal model of senile dementia is obtained through natural aging of the animal. The method comprises the steps of adaptively feeding 60 rats aged 21-22 months for 10 days, enabling the animals to take water freely, controlling the room temperature to be 22-25 ℃, controlling the humidity to be 50% -70%, illuminating for 12 hours, and keeping the animals dark for 12 hours. Rats with cognitive impairment were screened by training in the Morris water maze mapping experiment.

The Morris water maze experiment is an experiment for forcing an animal to swim and searching an underwater platform, is mainly used for evaluating the spatial learning and memory capacity of the animal, provides an experiment index which is sensitive and reliable, is simple and convenient to operate, and is a classic experiment for testing and evaluating the senile dementia index of a rat. The water depth of the water maze is 50cm, the water temperature is controlled at 22-25 ℃, and a platform is arranged in the center of the pool. Milk powder is put into the pool and is fully and evenly mixed until the water is milk white, so that the rat can not identify the position of the platform through vision. Before formal test, the rat is put into water facing the wall of the pool to train the rat to find an escape platform, if the rat is found and stands on the platform for 3 seconds, the rat does not slide off, the training can be stopped, the time and the traveling distance of the rat reaching the platform are recorded, and the rat stays on the platform for 10 seconds, so that the rat learns and memorizes. The person who cannot be found can be guided to the platform after continuously recording for 120s and is left for 30 seconds to train the learning and memory of the person. Training is carried out 4 times every day, the next training is carried out at intervals of 15-30 seconds, water is filled from 4 different directions, and the training is continued for 4 days. And (5) performing formal test on the day, automatically recording the activity condition of the rat by a real-time image system, calculating the average time of the rat passing through the platform (central area) for the first time, and evaluating the learning and memory conditions of the animal. Taking the upper limit value of the range of 99% normal value of the average escape latency of young (4-month old) rats as a standard, and determining the old rats with the escape latency of more than 99% as the known obstacle old rats. Each rat was labeled so that the mean escape latency before dosing for each group of rats could be calculated after completion of the final trial.

A control group of senile dementia rats (no administration, gavage of an equal volume of physiological saline), a high dose treatment group (continuous wash gastric administration in an amount of 45 μ g of fgf21 administered per kg of rats, 2 times per day, for 8 weeks) and a low dose treatment group (continuous wash gastric administration in an amount of 25 μ g of fgf21 administered per kg of rats, 2 times per day, for 8 weeks); meanwhile, a control group of young rats (no drug administration, gavage of physiological saline of the same volume) is set. During the experiment, 10 rats per group were maintained. After 8 weeks of dosing, training was continued for another 5 days, recording the average time each group of animals looked for the platform in water each day, and the Morris water maze test was performed directly on day 6. The time for each group of animals to find the platform in water was determined and dosing was continued during the training period.

The results are shown in figure 1, and before administration, the senile dementia rat control group, the high dose treatment group and the low dose treatment group have no significant difference, and P is less than 0.001. After 8 weeks of administration, the training of the Morris water maze positioning experiment was performed again, and the senile dementia rats in the sixth day were significantly different from the control group, the high dose treatment group and the low dose treatment group, and showed dose dependence. The results show that: the FGF21 with high dose and low dose can improve the average escape latency of a rat group with senile dementia to a certain extent and improve the learning and memory ability of rats with senile dementia.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims

Use of FGF21 in the manufacture of a medicament for the treatment of cranial nerve injury.
2. The use according to claim 1, wherein the cranial nerve injury is senile dementia.
3. The use of claim 1, wherein the medicament further comprises an additional agent that inhibits acetylcholinesterase activity.
4. The use according to claim 3, wherein the agent that inhibits acetylcholinesterase activity is donepezil or a salt thereof.
5. The use according to any one of claims 1 to 4, wherein the medicament further comprises pharmaceutically acceptable excipients, with or without other active ingredients.
6. A medicament for treating cranial nerve injury, comprising FGF21 as an active ingredient.
7. The medicament of claim 6, further comprising donepezil or a salt thereof.
8. The medicament of claim 6, wherein the weight ratio of FGF21 to donepezil or salt thereof is 1:1 to 1: 0.4.
9. The medicament of claim 8, which is an oral formulation.
10. The medicament of claim 9, wherein the oral formulation is selected from the group consisting of tablets, capsules, and liquid formulations.