CN121130083A

CN121130083A - Application of FKBP5 inhibitor SAFit2 in the preparation of drugs for the prevention of post-traumatic stress disorder

Info

Publication number: CN121130083A
Application number: CN202511355046.XA
Authority: CN
Inventors: 李红; 吴宁; 马妍; 田萧羽; 韩笑; 李锦�
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2025-09-22
Filing date: 2025-09-22
Publication date: 2025-12-16

Abstract

This invention provides the application of the FKBP5 inhibitor SAFit2 in the preparation of drugs for preventing post-traumatic stress disorder (PTSD), belonging to the field of biopharmaceutical manufacturing technology. This invention constructs a mouse model of PTSD using FKBP5 gene knockout mice as experimental subjects. Test results show that FKBP5 knockout can alleviate PTSD symptoms. FKBP5 can be used as a therapeutic target for the development, screening, or preparation of therapeutic drugs for PTSD. Furthermore, this invention provides the application of the FKBP5 inhibitor SAFit2 in the preparation of drugs for the prevention of PTSD, offering a new direction for PTSD prevention.

Description

Application of FKBP5 inhibitor SAFit2 in preparation of medicine for preventing post-traumatic stress disorder

Technical Field

The invention belongs to the technical field of biological medicine manufacturing, and particularly relates to application of FKBP5 inhibitor SAFit2 in preparation of a medicine for preventing post-traumatic stress disorder.

Background

Post-traumatic stress disorder (post traumatic stress disorder, PTSD) is a neuropsychiatric disease closely related to traumatic events and psychological stress, and refers to a group of unusual and extraordinary threatening and catastrophic events such as battlefield environments, natural disasters, accidents, and the like, which are mainly caused by various mental behavioral abnormalities and psychological disorders, and have obvious effects on individuals, so that the occupational capacity and social functions of the individuals are impaired, and (or) a durable response is caused. PTSD has three characteristic manifestations, wound re-experience, emotional numbness and evasion behavior, and sustained high-alertness symptoms. PTSD is a major global health problem and there is a great need for methods to provide effective treatments.

FKBP5 is a macromolecular immunoaffinity protein, and is involved in the formation and regulation of hormone-receptor complexes, and can also be involved in multiple cell signaling pathways to regulate cellular functions, such as regulating sugar metabolism and energy metabolism, and involved in cytoskeletal formation and microtubule folding, and plays an important role in regulating the structure and function of nerve cells. SAFit2 is FKBP5 specific antagonist capable of reducing paclitaxel-induced mechanical hypersensitivity in mice and improving paclitaxel-induced neuropathic pain by reducing peripheral sensitivity and addressing neuroinflammation. The SAFit also reduces alcohol consumption in men with stress, but does not reduce alcohol consumption in women.

However, there is no report on the treatment of PTSD with FKBP5 or SAFit.

Disclosure of Invention

Accordingly, the present invention aims to provide the application of FKBP5 inhibitor SAFit2 in preparing medicaments for preventing post-traumatic stress disorder.

The invention provides application of FKBP5 as a target in developing, screening or preparing a medicament for treating post-traumatic stress disorder.

Preferably, the FKBP5 reduces the behavioral symptoms of post-traumatic stress disorder by negative regulation.

The invention provides application of FKBP5 serving as a target in preparation of a reagent for post-traumatic stress disorder prognosis evaluation.

The invention provides an application of an agent for inhibiting the expression or biological function of FKBP5 genes or FKBP5 proteins in preparing medicaments for preventing post-traumatic stress disorder.

Preferably, the agent comprises an sgRNA or gene knockout system that knocks out the FKBP5 gene, an siRNA that reduces the expression level of the FKBP5 gene, or an antibody that inhibits the biological function of the FKBP5 protein.

Preferably, the nucleotide sequence of the sgRNA of the FKBP5 gene is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2.

The invention provides application of FKBP5 inhibitor SAFit2 in preparation of a medicament for preventing post-traumatic stress disorder.

Preferably, SAFit further comprises derivatives in salt form.

Preferably, the dosage form of the medicine comprises at least one of tablets, capsules, drops, injection powder and injection liquid.

Preferably, the mass percentage of SAFit% to 90% of SAFit% of the medicine.

The invention provides application of FKBP5 as a target in developing, screening or preparing a medicament for treating post-traumatic stress disorder. Experiments prove that the FKBP5 gene or protein can be knocked out to effectively avoid the occurrence of post-traumatic stress disorder. Experiments show that the time spent by FKBP5 knockout mice is obviously reduced compared with that of wild mice, and meanwhile, both open field experiments and elevated plus maze experiments show that the anxiety behavior of the FKBP5 knockout mice is obviously reduced by establishing a PTSD mouse model and testing the behavior change of the PTSD mouse model by adopting a behavioural score, an open field experiment and an elevated plus maze experiment. The above results demonstrate that FKBP5 knockout can reduce the occurrence of PTSD. Therefore, the experiment proves that FKBP5 can be used as a treatment target point for developing, screening or preparing the treatment medicament for the post-traumatic stress disorder, and a new way is provided for the clinical treatment of the post-traumatic stress disorder.

The invention provides application of FKBP5 inhibitor SAFit2 in preparation of a medicament for preventing post-traumatic stress disorder. The invention takes wild mice as objects, and the medicines for preventing and treating SAFit are respectively administered before and after the modeling of the posttraumatic stress disorder, and the results show that the preventive administration of SAFit2 before the modeling can improve the stiff behavior symptoms and anxiety behavior of PTSD, namely inhibit the occurrence and development of PTSD, while the administration after the modeling does not achieve the purpose of treating PTSD for SAFit 2. Therefore, the invention provides a preventive drug for post-traumatic stress disorder, which can effectively prevent the occurrence of post-traumatic stress disorder.

Drawings

FIG. 1 shows the effect of FKBP5 knockout on PTSD, A. Construction strategy of FKBP5 knockout mice (KO mice), B. Sequencing result of FKBP5 gene knockout of KO mice, C. Immunoblotting (Western Blot) experiment to verify FKBP5 knockout result, D. Proportional result of PTSD dead time of mice detected at different time points after modeling, E is anxiety related behavior result detected by open field experiment, F. Anxiety related behavior result detected by overhead cross maze experiment, G. Summary of experimental scheme and result;

FIG. 2 is a graph showing the experimental flow, A. The experimental flow is schematically shown, B. The brain section of the mouse shows the position of the lateral ventricle buried pipe, C. The behavior of the mouse PTSD is detected at each time point after modeling, and the change result of the stiff time proportion is detected;

Fig. 3 is a diagram of experimental flow of a result of drug effect evaluation of SAFit on mice after stress, b. The result of detection of anxiety-related behavior of mice by elevated plus maze is enough to detect changes in the ratio of time to time of the behaviours of PTSD of mice at each time point after modeling, d. A summary of the experimental scheme and results.

Detailed Description

In the present invention, the FKBP5 preferably reduces the behavioral symptoms of post-traumatic stress disorder by negative regulation. The behavioral symptoms of the post-traumatic stress disorder preferably include stiff time and/or anxiety behavior.

In one embodiment of the invention, the stiffness time is preferably determined using a mouse fear behavior box. The anxiety behavior is preferably tested by adopting open field experiments and overhead plus maze experiments, and the anxiety behavior is evaluated by taking the time of the middle area and the percentage of the time of entering the open arm as indexes respectively. Experiments show that compared with wild mice, the mice with FKBP5 genes or proteins knocked out have obviously reduced stiffness time, and meanwhile, the test results of open field experiments and overhead plus maze experiments show that the time of the middle region and the percentage of time of entering the open arm are obviously increased, which indicates that the FKBP5 genes or proteins reduce anxiety behaviors through negative regulation and reduce the traumas of post-traumatic stress disorder.

In view of the fact that downregulation of FKBP5 gene or protein expression can reduce the trauma of post-traumatic stress disorder, the invention provides application of FKBP5 serving as a target in preparation of a reagent for post-traumatic stress disorder prognosis evaluation.

In the present invention, the reagent preferably includes a primer for detecting the expression level of FKBP5 gene or an antibody for detecting the expression level of FKBP5 protein. The method for detecting the expression level of FKBP5 gene is preferably qPCR method. The method for detecting the expression level of FKBP5 protein is preferably an immunoblotting (WB) method.

In the method for prognosis evaluation of post-traumatic stress disorder, when the expression level of FKBP5 gene or protein is obviously reduced compared with that before treatment, the method indicates that the post-traumatic stress disorder prognosis has good curative effect, otherwise, indicates that the post-traumatic stress disorder prognosis has poor curative effect, and increases the treatment measures.

In the present invention, the agent preferably comprises sgRNA or gene knockout system for knocking out FKBP5 gene, siRNA for reducing expression level of FKBP5 gene or antibody for inhibiting biological function of FKBP5 protein. The gene knockout system preferably comprises a CRISPR/Cas system or a Cre/loxP knockout system. In one embodiment of the present invention, the nucleotide sequence of the sgRNA of the knockout FKBP5 gene is preferably as shown in SEQ ID NO. 1 and/or SEQ ID NO. 2. FKBP5 knockout mice were constructed using CRISPR/Cas9 system.

In the present invention, the prevention of post-traumatic stress disorder is preferably a reduction in anxiety behavior and an increase in behavioral scores of the patient.

In the present invention, SAFit2 preferably further includes derivatives in salt form. The dosage form of the medicine preferably comprises at least one of tablets, capsules, drops, injection powder and injection liquid. The mass percentage of SAFit-90%, 10-80%, 15-70%, 20-60%, 30-50%, and 40% of SAFit% of the medicine is preferable. The preparation method of the medicine is not particularly limited, and the preparation method of the medicine known in the art can be adopted.

In the present invention, the method of administering the drug is preferably to administer the drug in advance before the wound irritation occurs.

The use of the FKBP5 inhibitor SAFit2 provided by the present invention in the preparation of a medicament for the prevention and/or treatment of post-traumatic stress disorder is described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Efficacy evaluation experiment of FKBP5 inhibitor SAFit2 in preventing and treating post-traumatic stress disorder

1. The material method comprises the following steps:

Construction of FKBP5 Gene knockout mice

FKBP5 Knockout (KO) mice were constructed using CRISPR/Cas9 technology. The gRNA sequence was designed, gRNA1:5'-UUGGACCCUAUCUUUAGCAA-3' (SEQ ID NO: 1), gRNA2:5'-CAGGAGACUGUCAUGCCGGA-3' (SEQ ID NO: 2). 100 ng/. Mu.L of Cas9 and 50 ng/. Mu.L of gRNA (gRNA 1: gRNA 2=1:1, mass ratio) were simultaneously microinjected into fertilized eggs of C57BL/6JGpt mice. F0 mice are obtained by transplanting fertilized eggs, and positive mice with FKBP5 knockdown are confirmed by PCR and sequencing. Stable F1 generation mice FKBP5 KO model mice were obtained by mating F0 generation positive mice with C57BL/6JGpt mice.

2. Pre-clock model modeling method

After one week of postsurgical mice were acclimatized, continuous intermittent electric shock molding was performed on day 1 and day 2. The electric shock program is totally 7 min, the front 3 min is the adaptation period, the rear 4 min is discontinuous electric shock, the current is 0.8 mA, the electric shock duration is 10 s, the interval is 10 s, 12 cycles are total, and the environment in the electric shock box is a dim environment. Contextual fear tests were performed on days 3, 8, and 15, i.e., mice were placed in a shock box to freely explore 5min and were not given shocks, and the "stiff behavior" of the animals in 5min was recorded as indicative of fear response. After each mouse test was completed, the box was wiped with 70% alcohol to eliminate the odor left by the previous experimental animal. Control group mice were also explored in the mouse fear behavior box for 7 min, but were not given a shock. The frigidity time was measured using a mouse fear box and the open field experiment and the elevated plus maze experiment were performed on the tenth day.

3. Open field experiment (Open FIELD TEST)

An experimental method for evaluating animal's behavior and tension in new and different environment by open field experiment. The open field experimental device is a real box with an open top and made of a white polyethylene plate with the length of 50cm multiplied by 50cm, and the top is connected with a video analysis device. The experimental mice were placed in the experimental room to adapt to the environment at least 30 minutes in advance before the start of the experiment, and were kept quiet until the mice were finished with the experiment, reducing stress. After the experiment is started, the mice are placed in the middle area from one corner of the experiment box, free to move for 5 minutes and video is shot in the whole course. After the test of each mouse is finished, 70% ethanol is required to be sprayed and the excrement and the movement track in the open field box are wiped off, and the next mouse experiment can be carried out after the test is volatilized. After the experiment is finished, the time and the distance percentage, the total distance and the like of the mice entering the middle area are statistically analyzed to be used as indexes for evaluating anxiety degree.

4. Overhead plus maze test (elevated plus maze, EPM)

The elevated plus maze comprises two closed arms 30 cm x 5cm x 15 cm and two open arms 30 cm x 5cm, a central area 5cm x 5cm, and a distance of 50 cm from the ground. The mice were placed in the central region of the elevated plus maze with their heads facing the open arm, allowing them to explore freely 5min a, and the four limbs of the mice all entered one arm as once. The number of times of entering the arm and closing the arm and the time of entering the arm in the mice 5min are recorded and analyzed by Visutrack software, and the statistical indexes are the total number of times of entering the arm, the percentage of times of entering the arm and the percentage of time of entering the arm.

Total number of arm entries = number of arm entries + number of arm entries closed formula I;

Percentage of number of arm-opening times = number of arm-opening times/total number of arm-opening times x 100% formula II;

Percentage of time to open arm = time to open arm/total time x 100% formula III.

5. Lateral ventricle burial tube and administration

FKBP5 knockout mice and wild type mice were anesthetized with pentobarbital sodium at a dose of 70mg/kg and 0.1ml/10g, and the mice were intraperitoneally injected (intraperitoneal injection, i.p.). After the mice were fully anesthetized (the eversion disappeared), the head hair was removed by shaving Mao Qiti, moved to a brain stereotactic apparatus, the heads of the mice were fixed by using ear posts and tooth holders, and the head fixation was performed with light pressure on the heads of the mice without apparent shaking. The method comprises the steps of coating erythromycin eye ointment on the eyeballs of mice, disinfecting the scalp of the mice by using iodophor disinfectant, shearing off the scalp by using disinfected scissors, fully exposing the skull of the mice, wiping off periosteum on the surface of the skull by using a cotton stick dipped with physiological saline to expose bone gaps, and positioning until bregma areas and herringbone gaps are clear. Taking bregma as an origin and a herringbone seam as an end point, leveling front and back, and then leveling left and right by about 4.2mm, and then leveling left and right by about 2.3mm (the error of front, back, left and right is not more than 0.1 mm). After leveling, coordinates (AP: -0.3mm, ML: + -1.0 mm, DV: -2.5 mm) are selected with bregma points as origins, and the catheter is buried to a specified position by using a skull drill for punching. The catheter and the skull are coated with a small amount of strong quick-drying adhesive, the catheter is covered with dental cement, after the cement is hardened, the mouse is taken down from the positioning instrument, the catheter cap is covered, the mouse is put on an electric blanket to accelerate the awakening, and the mouse is put back into a raising cage after the awakening. The experiment can be started after 1 week of recovery of the mice. When the lateral ventricle is used for administration, a micro injection needle is connected with an injection inner tube through a section of hose for administration, and the needle is left for 2 minutes after the injection is finished.

6. Preparation of SAFit solution:

1) Mother liquor the concentration of mother liquor is 20 mg/mL, and the specific preparation method is to dissolve 2 mg drugs in 100 mu L DMSO.

2) The working solution has the concentration of 2 mg/mL, and the specific preparation method comprises the steps of taking 100 mu L of mother solution, adding 400 mu L of PEG300, uniformly mixing until the mixture is clear, adding 50 mu L of Tween 80, uniformly mixing until the mixture is clear, and adding 450 mu L of physiological saline.

3) Mixed solvent (Vehicle) without SAFit% DMSO, 40% PEG300, 5% Tween-80 and 50% physiological saline.

Results:

1. FKBP5 knockout can reduce PTSD occurrence

First, in order to clarify the effect of FKBP5 on PTSD, the present example constructed FKBP5 knockout mice (a in fig. 1), and sequencing results showed that FKBP5 gene was knocked out (B in fig. 1), immunoblotting (WB) experiments confirmed that KO mice did not express FKBP5 protein (C in fig. 1). Based on this, PTSD model was established between knockout mice and wild mice, and PTSD-related behavioral index and dead time of FKBP5 knockout mice were found to be improved (D and E in fig. 1). The above results demonstrate that FKBP5 knockout can reduce the occurrence of PTSD (G in fig. 1).

2. Delivery SAFit of 2 prior to electric shock molding can reduce PTSD occurrence

Proteins are widely expressed in mammals, particularly notably in the brain. The embodiment of the invention uses the specific inhibitor SAFit of FKBP5 to carry out lateral ventricle injection of a wild mouse, and discusses the administration time. The first dosing strategy was prophylactic (5 μl/day) before shock and its effect on PTSD development was observed (a, B in fig. 2). The mice PTSD-related behavioral index, the stiff time scale (C in fig. 2) and the elevated plus maze (D in fig. 2) were examined at various time points after molding. The results show that the pre-stress prophylactic administration SAFit of the drug can improve PTSD related symptoms, namely reduce the occurrence and development of PTSD.

3. The administration of SAFit2 after stress did not affect the occurrence of PTSD

The effect of D3-10 fear memory formation period following electric shock on the development of PTSD was observed by administering SAFit doses (2. Mu.L/day) to the patient (FIG. 3A). The mice PTSD-related behavioral index, the stiff time scale (B in fig. 3) and the elevated plus maze (C in fig. 3) were examined at various time points after molding. The results show that post-stress administration SAFit2 does not alleviate the symptoms associated with PTSD, i.e., SAFit2 is not effective in treating PTSD.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

The use of fkbp5 as a target in the development, screening or preparation of a medicament for the treatment of post-traumatic stress disorder.
2. The use according to claim 1, wherein FKBP5 reduces the behavioral symptoms of post-traumatic stress disorder by negative regulation.
Application of FKBP5 as a target in preparing a reagent for post-traumatic stress disorder prognosis evaluation.
4. Use of an agent that inhibits the expression or biological function of an FKBP5 gene or FKBP5 protein in the manufacture of a medicament for preventing post-traumatic stress disorder.
5. The use according to claim 4, wherein the agent comprises an sgRNA or gene knockout system for knocking out FKBP5 gene, siRNA for reducing expression level of FKBP5 gene or an antibody for inhibiting biological function of FKBP5 protein.
6. The use according to claim 5, wherein the nucleotide sequence of the sgRNA of the knockout FKBP5 gene is shown in SEQ ID NO. 1 and/or SEQ ID NO. 2.
Use of fkbp5 inhibitor SAFit2 in the manufacture of a medicament for the prevention of post-traumatic stress disorder.
8. The use of claim 7, wherein SAFit2 further comprises a derivative in salt form.
9. The use according to claim 1 or 7, wherein the dosage form of the medicament comprises at least one of a tablet, a capsule, a drop, an injectable powder and an injectable solution.
10. The use according to any one of claims 1 to 6, wherein the mass percentage of SAFit% 2 in the medicament is 5% to 90%.