CN111888463A - Thymosin beta 4 medicinal preparation for treating depression, preparation method and application thereof - Google Patents

Abstract

The invention discloses a thymosin beta 4 medicinal preparation for treating depression, a preparation method and application thereof, and belongs to the technical field of disease treatment. The invention mainly induces the mouse depression-like behavior by establishing a chronic social contusion stress model (CSDS), and the model has the advantages that the model simulates the social attributes in human chronic stress, and after the model is nasally administered with T beta 4 for 7 consecutive days, the depression level is evaluated by the behavioral tests such as Social Interaction Test (SIT), Tail Suspension Test (TST), sugar water preference test (SPT) and the like, and the result shows that the T beta 4 has the rapid antidepressant effect in the mouse and can reverse the CSDS-induced depression-like behavior within 7 days.

Description

Thymosin beta 4 medicinal preparation for treating depression, preparation method and application thereof

Technical Field

The invention relates to a new application of a peptide substance, belongs to the technical field of disease treatment, and particularly relates to a thymosin beta 4 pharmaceutical preparation for treating depression, a preparation method and an application thereof.

Background

The main indications of mental disease are the obvious and persistent abnormalities of cognitive, emotional, will and behavioral manifestations of the patient, which are difficult to be accepted by society in routine, and the serious impairment of social and mental functions, which can not maintain normal learning, work and life. Under the control of pathological cognition and emotion, aggressive behavior, self-mutilation or suicide can occur. Mental disorders have become a significant medical and social problem in today's society.

Clinically, drugs are the main means and method for the treatment of mental disorders. The mental disease treatment drugs mainly comprise antipsychotics, antidepressants, anxiolytics, mood stabilizers, sedative hypnotics and the like. The research and development of modern mental disease treatment drugs depend on the establishment of mental disease animal models, so that the research and establishment of new test methods and animal models can promote the development and application of mental disease drug treatment, strengthen the understanding of relevant mental disease pathogenesis, improve clinical treatment effects and improve prognosis and outcome. The existing treatment methods for depression mainly comprise traditional Chinese medicine treatment and western medicine treatment, but have the defects of long treatment course, multiple side effects and the like.

Recombinant human/murine/rat thymosin beta 4 is a 5.2kDa glycoprotein containing 45 amino acid residues. Can stimulate the stem cell maturation of central nervous system and improve the neurological function obviously. Thymosin beta 4(T beta 4) has multiple biological functions, and is closely related to tissue regeneration, remodeling, wound healing, actin balance maintenance, apoptosis, inflammation, vascular balance, hair follicle development, cornea and cardiac muscle repair. T β 4 promotes remodeling following CNS/PNS nerve injury. In the chronic phase following nerve injury or the appearance of clinical symptoms, T β 4 promotes neurovascular remodeling. Tss 4 is expressed in neural progenitor cells, is a neural repair molecule with neural repair function, and is involved in cell proliferation, migration, angiogenesis and axon remodeling. It has been found to be effective in reducing stroke, traumatic brain injury and multiple sclerosis models with recovery of lesion volume and function. However, no studies have reported the antidepressant effect of T β 4.

Disclosure of Invention

In order to solve the problems of long treatment course, large side effect and the like of the existing antidepressant drugs, the invention discloses a thymosin beta 4 pharmaceutical preparation for treating depression, a preparation method and application thereof, and researches show that the behavior of mental diseases induced by a subject through a chronic social frustration stress model is reversed after a certain dose of the thymosin beta 4-containing pharmaceutical preparation is taken.

In order to achieve the purpose, the invention discloses a thymosin beta 4 pharmaceutical preparation for treating depression, which comprises effective dose of thymosin beta 4 and a carrier, wherein the carrier is one or two or more than two mixed auxiliary materials, and each 100 parts by mass of the pharmaceutical preparation comprises 12-48 parts by mass of thymosin beta 4, and the balance of the auxiliary materials.

Furthermore, each 100 mass parts of the pharmaceutical preparation comprise 20-40 mass parts of thymosin beta 4.

Further, the auxiliary material is phosphate buffered saline solution.

Further, the pharmaceutical preparation is a liquid preparation, and preferably contains thymosin β 4 in an amount of 30 parts by mass per 100 parts by mass of the liquid preparation.

In addition, the invention also discloses a preparation method of the pharmaceutical preparation, which comprises the steps of blending thymosin beta 4 and a carrier and preparing the required preparation. Preferably, the liquid formulation is prepared according to the invention.

In order to better realize the technical purpose of the invention, the invention also discloses the application of the pharmaceutical preparation in preparing drugs for treating the depression of mammals.

Further, specifically, the method comprises the step of nasally administering a pharmaceutical preparation containing thymosin beta 4 to a mammalian subject, wherein the administration amount is as follows: 12-48 mg thymosin beta 4 per day for 7 consecutive days;

the tested mammal is obtained by screening through establishing a chronic social frustration stress model.

Further, the method comprises the step of comparing the experimental behaviors of the mammal to be tested before and after administration to evaluate the drug effect of the thymosin beta 4-containing drug preparation;

after 7 days of continuous administration, the behavior of the subject mammal was reversed in depression induced by a model of chronic social frustration stress.

Further, the experimental behavior for evaluating the mammalian body to be tested includes one or any two or more of social interaction experiment, tail suspension experiment and sugar water preference experiment.

Further, the subject mammal is a mouse.

Has the advantages that:

the invention induces the depression-like behavior of the mouse by establishing a chronic social frustration emergency model (CSDS). Evaluating the depression level through the behavioral tests such as Social Interaction Test (SIT), Tail Suspension Test (TST), sugar water preference test (SPT) and the like, screening out depressed rats, randomly dividing the rats into a PBS group, 12mg/kg T beta 4, 24mg/kg T beta 4 and 48mg/kg T beta 4(n is 8-10), carrying out nasal administration for 7 days every day, carrying out social behavior tests on the 1 st, 3 th and 7 th days, and simultaneously carrying out tail suspension and sugar water preference tests on the 7 th day to evaluate the depression level. As a result, T β 4 was nasally administered without significantly increasing the residence time in the social area after day 1 compared to PBS administration; after day 3, 24mg/kg and 48mg/kg T β 4 administration significantly increased social residence time (24mg/kg T β 4 group: P <0.05, 48mg/kg T β 4 group: P < 0.01); after day 7, nasal administration of multiple doses of tss 4(12mg/kg, 24mg/kg, 48mg/kg) all showed antidepressant-like effects, i.e. significantly increased residence time in social area (P <0.01), the 48mg/kg tss 4 group significantly decreased immobility time in TST (P <0.01), improved reduction of sugar water preference rate (P < 0.01). Therefore, the thymosin beta 4-containing pharmaceutical preparation designed by the invention has a rapid antidepressant effect in mice, and can reverse CSDS-induced depressive behavior within 7 days.

Drawings

FIG. 1 is a schematic diagram of a social interaction site and a chronic social frustration process, wherein FIG. 1A is the social interaction site and FIG. 1B is the chronic social frustration process;

FIG. 2 is a graph of depressed rat results obtained after the chronic social frustration stress model of the present invention;

FIG. 3 is a diagram of social interaction trajectories after a model of chronic social frustration stress in accordance with the present invention, wherein

FIG. 3A is a diagram of a depression group social interaction trajectory; FIG. 3B is a diagram of resistant group social interaction trajectories;

FIG. 4 is a graph of the analysis of social interaction results after different doses of Tss 4 were administered continuously for different periods of time according to the present invention, wherein FIG. 4A is after 1 day of administration, FIG. 4B is after 3 days of continuous administration, and FIG. 4C is after 7 days of continuous administration;

FIG. 5 is a graph of the percent time to failure of tail suspension after 7 days of continuous administration of Tss 4 at various doses as contemplated by the present invention; wherein, fig. 5A is a schematic view of mouse tail suspension, and fig. 5B is a test result;

figure 6 is a graph of the results of the carbohydrate preference test following 7 days of continuous administration of different doses of tss 4 designed according to the present invention.

Detailed Description

The invention discloses a thymosin beta 4 medicinal preparation for treating depression, a preparation method and application thereof, and particularly relates to a thymosin beta 4-containing medicinal preparation for evaluating the medicinal effect of the thymosin beta 4-containing medicinal preparation by establishing a chronic social frustration emergency model to induce depression-like behaviors of a subject and carrying out experimental behaviors of the subject before and after administration. The results found that the behavior of the subject in mental illness induced by the acute model of chronic social frustration was reversed.

The invention discloses a thymosin beta 4 medicinal preparation for treating depression, which comprises thymosin beta 4 with effective dose and a carrier, wherein the carrier is one or two or more than two auxiliary materials, each 100 parts by mass of the medicinal preparation comprises 12-48 parts by mass of thymosin beta 4, and the balance is the auxiliary materials. Preferably, the thymosin beta 4 is contained in an amount of 20-40 parts by mass per 100 parts by mass of the pharmaceutical preparation.

Wherein the carrier is phosphate buffer saline solution, and the pharmaceutical preparation is in the form of liquid. And further preferably contains thymosin beta 4 in an amount of 30 parts by mass per 100 parts by mass of the liquid preparation.

The pharmaceutical formulations contemplated by the present invention may be conveniently prepared in unit dosage form and using any of the methods well known in the art of pharmacy. Specifically, the active compound thymosin ss 4 is homogeneously and intimately admixed with phosphate buffered saline and formulated into the desired liquid formulation.

Secondly, the pharmaceutical preparation prepared by the invention has better application in the preparation of the drugs for treating the depression of mammals.

In order to better explain the present invention, the following further through each embodiment detailed description of the specific experimental process;

example 1-depressed mice were obtained by chronic social frustration stress experiments;

the embodiment obtains the mouse with the depression-like behavior by establishing a chronic social frustration stress model (CSDS), the model can simulate the depression of human beings caused by frustration in social life, and the model has the advantages of strong repeatability, easiness in operation and the like. The specific model building process is as follows:

1. 6-week-old C57BL/6J mice were randomly divided into test and control groups. For the test group of mice, the CSDS phase lasted for 10 days, carried out daily at the same time period, e.g., 9:00-11:00 in the morning. The test group C57BL/6J mouse is used as an invader to be placed into a 5-month-old male CD-1 mouse cage, so that the mouse is physically attacked for 10min, and the attack duration and effectiveness are ensured.

2. After the frustration treatment was completed, the C57BL/6J and CD-1 mice were separated using a transparent, perforated barrier, ensuring that the C57BL/6J mice were no longer attacked, but were kept in sensory contact, such as olfactory and visual contact, i.e., exposure to social frustration pressure. After 24h, the C57BL/6J mouse was removed and placed in another CD-1 mouse cage, and the procedure described above for step 1 was repeated, and so on. The social interaction site selected by the invention is shown in fig. 1A.

3. Within 10 days, each C57BL/6J mouse in the test group was challenged with a different CD-1 mouse daily and exposed to social frustration pressure, as shown in FIG. 1B. After the end of the last setback treatment, all C57BL/6J mice were housed in a single cage and used for behavioural testing 24h later.

4. The control group C57BL/6J mice were not treated and were fed simultaneously until the CSDS phase was over, and were behaviorally tested together with the test group mice.

All the above procedures ensure that all mice have access to water and food at will.

Example 2-screening of depressed rats;

c57BL/6J mice exposed to social frustration stress for a prolonged period of time develop a socially avoidable depression-like phenotype, in particular, the length of time that a socially frustrated C57BL/6J mouse has been exposed to strange mice is significantly shortened compared to normal C57BL/6J mice over the same period of time. The Social Interaction Test (SIT) can be used to evaluate the social avoidance behavior of mice, which is mainly classified into those in the absence of strange CD1 mice (called the nocarget stage) and those in the presence of strange CD1 mice (called the Target stage), and into CSDS susceptible mice (susceptable) and CSDS resistant mice (resilient) according to the ratio of the lengths of time that C57BL/6J mice enter the social contact area in the two test stages.

1. The social interaction trial was performed 24h after the CSDS process described in example 1 above. The aggressive CD-1 mice need to be rescreened before the test starts and to ensure that the CD-1 mice have no contact and complete strangeness with the C57BL/6J mice tested.

2. And assembling social interaction places, and setting a video tracking system to automatically acquire all data. The tested C57BL/6J mice were acclimated to the test environment, e.g., red light conditions, in advance and isolated from other animals and noise sources.

3. First stage-No Target, C57BL/6J mice were placed in the field of social interaction, such as centered on the opposite side of the pen, allowing them to explore freely for 3min without CD-1 mice. After this phase, the C57BL/6J mice were returned to their primary cages until the second phase, noting that: the social interaction site does not need to be cleaned after the first stage.

4. Second stage-Target, the aggressive CD-1 mice were gently placed into mobile boxes and replaced with the tested C57BL/6J mice, allowing free exploration for 3min in the presence of CD-1 mice, and the manner of placement of the C57BL/6J mice in both stages was consistent.

5. After both phases were completed, the mice were returned to their home cages. And the equipment was sterilized with 75% alcohol before the next round of introduction of a new test C57BL/6J mouse.

6. Calculating the social interaction rate (SI ratio), wherein the mice with the social interaction rate lower than 100 percent belong to CSDS susceptible mice, and the mice with the social interaction rate higher than 100 percent belong to CSDS resistant mice, and the formula is as follows:

as can be seen from FIG. 2, 100C 57BL/6J mice had results of depressed mice and resistant mice obtained after social contusion stress, in which 11 mice died during the challenge, and finally 42 depressed mice and 47 resistant mice were obtained.

As can be seen from FIG. 3, the locus diagrams of depressed rats and resistant rats at no-target and target stages are shown. The residence time of depressed rats in the social area during the target phase is significantly shorter than that of no-target phase, and the opposite is true for resistant rats.

Example 3-thymosin beta 4 was instilled nasally;

tss 4 was dissolved in PBS at concentrations of 30, 60 and 120. mu.g/. mu.L, respectively, and Tss 4 was administered by nasal drip using a 10. mu.L pipette, as needed.

The categories for the selected depressed rats were: CSDS + PBS group (10 μ L PBS, 5 μ L in each nostril), CSDS +12mg/kg T β 4 group (10 μ L PBS, 5 μ L in each nostril); CSDS +24mg/kg Tbeta 4 group is dripped with 10 mu L of 60 mu g/mu L T beta 4, 5 mu L of each nostril; CSDS +48mg/kg Tss 4 group was instilled with 10. mu.L of 120. mu.g/μ L T ss 4, 5. mu.L per nostril.

Example 4-social interaction test to assess depression levels;

social interaction method as shown in example 2, mice given different doses of thymosin T β 4 were tested for social interaction at different times, and the optimal dose and time of administration were then explored. Fig. 4 is a graph of social interaction results after different doses of T β 4 were administered continuously for different periods of time, and it can be seen from fig. 4 that the control group and the T β 4(0, 12, 24 and 48mg/kg T β 4) group with different doses stayed in the social area at different administration conditions for different periods of time, and the results show that 12, 24 and 48mg/kg T β 4 all significantly (P <0.01) improved social fear of mice after the seventh day of administration.

Example 5-tail suspension test to assess depression levels;

the tail suspension test is to induce the "behavior despair state" of the mice by suspending the tail of the mice, and the immobility time of the test mice is recorded to evaluate the depression level thereof.

1. During the test, the tip 1/3 of the test C57BL/6J mouse was taped and suspended in a tail box with the head about 50cm from the bottom and facing the camera. The test is shown in FIG. 5A.

2. The absolute motionless time within 5min after suspension was recorded with a video analysis system.

The test results are shown in fig. 5B. Mice were given Tss 47 days later, the 24mg/kg and 48mg/kg Tss 4 dosing groups showed a significant decrease in percent immobility time compared to the PBS group in tail suspension experiments over a 5min period (P < 0.01).

Example 6-carbohydrate preference test to assess depression levels;

anhedonia is one of the core symptoms of depression, which can be more intuitively manifested in rodents as a reduction in consumption of, and a reduction in preference for, a solution of saccharin or sucrose. Sugar water preference test mice were evaluated for anhedonia levels by comparing their drinking amounts of sucrose solution and water over 24 h.

1. Before the official test, the training was tested and C57BL/6J mice were acclimated to use two drinking bottles for 24 h. Firstly, filling tap water in two drinking bottles, and replacing the drinking bottles with 1% of sucrose solution after 12 hours;

2. after the test is started, two drinking bottles are respectively filled with tap water and 1% sucrose solution and placed on two sides of a mouse cage, and the positions of the two drinking bottles are exchanged every 12 hours, so that the mice can freely take the tap water or the sucrose solution, and the position preference is avoided;

3. after 24h, the consumption of tap water and sucrose solution was recorded and the sugar water preference rate of the mice was calculated as follows: the sugar water preference rate is [ (sucrose consumption)/(water consumption + sucrose consumption) ] × 100%. Considering that the ratio is expressed as sugar water preference when the ratio is more than 70 percent, and losing the sugar water preference when the ratio is less than 65 percent;

4. in the test, the leakage of the drinking bottle is avoided as much as possible, and the accurate reading is ensured. All tested C57BL/6J mice were housed in a single cage and provided with sufficient water and food.

Sugar water preference results as shown in figure 6, sugar water preference rates of 12, 24 and 48mg/kg T β 4 group mice increased significantly (P <0.01) 7 days after T β 4 application.

As can be seen from the above examples, the pharmaceutical formulation comprising thymosin beta 4 designed by the invention has a rapid antidepressant effect in mice, and can reverse CSDS-induced depressive-like behavior within 7 days.

Claims (10)

1. The thymosin beta 4 pharmaceutical preparation for treating depression is characterized by comprising effective dose of thymosin beta 4 and a carrier, wherein the carrier is one or two or more than two auxiliary materials, and each 100 parts by mass of the pharmaceutical preparation comprises 12-48 parts by mass of thymosin beta 4, and the balance is the auxiliary materials.

2. The thymosin beta 4 pharmaceutical preparation for treating depression according to claim 1, comprising 20-40 parts by mass of thymosin beta 4 per 100 parts by mass of the pharmaceutical preparation.

3. Thymosin beta 4 pharmaceutical preparation for the treatment of depression according to claim 1 or 2, wherein said adjuvant is phosphate buffered saline.

4. The thymosin beta 4 pharmaceutical preparation for treating depression according to claim 3, wherein said pharmaceutical preparation is a liquid preparation and contains thymosin beta 4 in an amount of 30 parts by mass per 100 parts by mass of the liquid preparation.

5. A method of preparing the pharmaceutical formulation of claim 1, comprising blending thymosin β 4 with a carrier and formulating the desired formulation.

6. Use of a pharmaceutical formulation according to claim 1 for the manufacture of a medicament for the treatment of depressive behaviour in a mammal.

7. The use of claim 6, which comprises nasally administering to the subject mammal a pharmaceutical formulation comprising thymosin β 4 in amounts of: 12-48 mg daily, and continuously administering for 7 days;

the tested mammal is obtained by screening through establishing a chronic social frustration stress model.

8. The use of claim 7, further comprising evaluating the efficacy of a pharmaceutical formulation comprising thymosin β 4 by comparing the experimental behavior of said subject mammal before and after administration;

after 7 days of continuous administration, the behavior of the subject mammal was reversed in depression induced by a model of chronic social frustration stress.

9. The use according to claim 8, wherein the test for assessing the body of the subject mammal comprises one or any two or more of social interaction test, tail suspension test and sugar water preference test.

10. The use according to any one of claims 6 to 9, wherein the subject mammal is a mouse.

Images