CN115531386A

CN115531386A - Application of OLB-01 hydrochloride in preparation of medicine for treating duchenne muscular dystrophy

Info

Publication number: CN115531386A
Application number: CN202211211472.2A
Authority: CN
Inventors: 杨细飞; 谢永美; 李书鹏; 杨超; 谷鑫
Original assignee: Shenzhen Olive Biomedical Technology Co ltd
Current assignee: Shenzhen Olive Biomedical Technology Co ltd
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2022-12-30

Abstract

The invention discloses an application of OLB-01 hydrochloride in preparation of a medicine for treating duchenne muscular dystrophy, and belongs to the technical field of medicines. The invention provides a theoretical basis for selection of the medicament for clinically treating duchenne muscular dystrophy, and has wide application prospect and medical value.

Description

Application of OLB-01 hydrochloride in preparation of medicine for treating duchenne muscular dystrophy

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of OLB-01 hydrochloride in preparation of a medicine for treating duchenne muscular dystrophy.

Background

Duchenne Muscular Dystrophy (DMD) is an X-linked genetic neuromuscular disease, most women are carriers of pathogenic genes, almost only men, and the influence on the livelihood male infants up to 1/3600 globally has become one of the most common rare pediatric diseases threatening children globally. DMD genetic mutations result in loss of dystrophin expression, resulting in severe muscle wasting, heart failure and respiratory failure in DMD patients, and death in most patients before 30 years of age.

Because research and development difficulty of rare drugs is high, cost is high, audiences are few, and few medical enterprises are willing to invest in research, patients with rare diseases face the dilemma of 'no medical treatment for diseases' and 'no medical treatment for medicines' at birth. For a long time, there are no effective clinical intervention measures or drugs for DMD at home and abroad. The FDA approved a new drug, etoxindys 51 (Eteplirsen), in an expedited approval mode (only 12 clinical trials were conducted), however there is not enough evidence to demonstrate the therapeutic effect of Eteplirsen injections on DMD.

OLB-01 is 4-hydroxyphenylethyl ((3,5,6-trimethylpyrazin-2-yl) methyl) carbonate and no report on treatment of DMD by OLB-01 was found.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of OLB-01 hydrochloride in the preparation of a drug for treating duchenne muscular dystrophy.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an application of OLB-01 hydrochloride in preparing a medicine for treating duchenne muscular dystrophy.

Preferably, the OLB-01 hydrochloride is capable of ameliorating symptoms in duchenne muscular dystrophy, including one or more of skeletal muscle hypertrophy, behavioral dysfunction, muscle weakness, muscle injury, inflammation, muscle fibrosis, collagen fibrosis.

Preferably, the formula of OLB-01 is as follows:

preferably, the medicament comprises OLB-01 hydrochloride and pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicament comprises tablets, capsules, granules, pills, solutions, sprays or creams.

Preferably, the inflammation comprises gastrocnemius inflammation.

Preferably, the behavioral functions include exercise, gait and limb coordination abilities.

Preferably, the OLB-01 hydrochloride content in the medicament is 1-99%.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses application of OLB-01 hydrochloride in preparation of a drug for treating duchenne muscular dystrophy for the first time. The OLB-01 hydrochloride can effectively improve skeletal muscle hypertrophy symptoms and reduce weight of a DMD mouse, can also obviously improve motor function, muscle strength, muscle injury, gait and limb coordination capacity of the DMD mouse, and can also reduce gastrocnemius inflammation, muscle fibrosis and collagen fiber degree of the DMD mouse, so that duchenne muscular dystrophy can be effectively treated. The invention provides a theoretical basis for selection of the medicament for clinically treating duchenne muscular dystrophy, and has wide application prospect and medical value.

Drawings

FIG. 1 is a graph showing the comparison of the body weights of different treatment groups, and B is a graph showing the comparison of the heart weights of different treatment groups; c is a graph comparing the weights of gastrocnemius muscles in different treatment groups; d is a comparison graph of triceps muscle weights of different treatment groups;

FIG. 2A shows the comparative results of the grip tests of different treatment groups; b is a climbing pole test comparison result of different processing groups;

in fig. 3, A1 is the comparison result of the stride distance of the left forelimb of different treatment groups; a2 is the comparison result of the stride distances of the right front limbs of different treatment groups; a3 is the comparison result of the stride distances of the left hind limbs of different treatment groups; a4, comparison results of gait of the right hind limbs of different treatment groups are obtained; b1 is the comparison result of the swing speeds of the left forelimbs of different treatment groups; b2 is the comparison result of the swing speeds of the right front limbs of different treatment groups; b3 is the comparison result of the swing speeds of the left hind limbs of different treatment groups; b4, comparing the swing speeds of the right hind limbs of different treatment groups; c1 is the comparison result of the walking speeds of the left forelimbs of different treatment groups; c2 is the comparison result of the walking speeds of the right forelimb of different treatment groups; c3 is the comparison result of the walking speeds of the left hind limbs of different treatment groups; c4 is the comparison result of the walking speeds of the right hind limbs of different treatment groups;

FIG. 4 is a comparison of the serum CK activity of mice from different treatment groups;

FIG. 5 is a comparison of the effects of different treatment groups on gastrocnemius inflammation in mice;

FIG. 6 is a comparison of nuclear deposition areas caused by gastrocnemius inflammation in mice for different treatment groups;

FIG. 7 is a comparison of the effects of different treatment groups on the fibrotic lesions of the muscle of DMD mice;

FIG. 8 is a comparison of the degree of muscle fibrosis in DMD mice between different treatment groups;

FIG. 9 is a comparison of the effect of different treatment groups on DMD mouse collagen fibers;

FIG. 10 is a graph showing the comparison of the degree of collagen fibrosis in DMD mice between different treatment groups;

FIG. 11 shows the structure of OLB-01.

Detailed Description

In the present invention, the OLB-01 is 4-hydroxyphenylethyl ((3,5,6-trimethylpyrazin-2-yl) methyl) carbonate with the molecular formula C ₁₇ H ₂₀ N ₂ O ₄ Molecular weight 316.36, formula shown below:

in the present invention, the OLB-01 hydrochloride is capable of ameliorating the symptoms of duchenne muscular dystrophy, preferably including one or more of skeletal muscle hypertrophy, behavioral dysfunction, muscle weakness, muscle injury, inflammation, muscle fibrosis, collagen fibrosis. The inflammation refers to infiltration of large inflammatory cells, and preferably includes gastrocnemius inflammation. The behavioral functions preferably include motion, gait and limb coordination abilities.

In the invention, the medicine comprises OLB-01 hydrochloride and pharmaceutically acceptable auxiliary materials, and the content of the OLB-01 hydrochloride in the medicine is preferably 1-99%. The medicament may be prepared according to methods well known in the art. For this purpose, the medicaments according to the invention can, if desired, be mixed with one or more adjuvants and brought into a suitable administration form or dosage form which can be used as medicaments.

The auxiliary materials comprise a binding agent, a lubricant, a flavoring agent, a filling agent, a disintegrating agent and the like, wherein the binding agent comprises one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, arabic gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like. Such as one or more of talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The correctant comprises one or more of aspartame, sucrose, simple syrup, pericarpium Citri Junoris syrup, fructus Citri syrup, fructus Pruni Pseudocerasi syrup, stevioside, sorbitol, mannitol, acacia, gelatin, sodium alginate, and tartaric acid. The filler comprises one or more of starch, microcrystalline cellulose, sucrose, dextrin and lactose. The disintegrant comprises one or more of crospolyvinylpyrrolidone, croscarmellose sodium and sodium carboxymethyl starch. The dosage form of the medicament preferably comprises tablets, capsules, granules, pills, solutions, sprays or creams.

The medicament of the invention can be administered in unit dosage form, and the administration route can be intestinal tract or parenteral tract, such as oral administration, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum, etc., preferably oral administration. The route of administration of the drug of the present invention may be injection, including intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and the like.

The dose of the drug of the present invention to be administered depends on many factors such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, character and individual response of the patient or animal, the administration route, the number of administrations, the therapeutic purpose, and thus the therapeutic dose of the present invention can be widely varied. Generally, the dosages of the drugs of the present invention are well known to those skilled in the art. The amount of the drug contained in the drug preparation of the invention can be properly adjusted according to the actual amount of the drug, so as to meet the requirement of the effective treatment amount and fulfill the aim of treating Duchenne muscular dystrophy. The daily dose of the drug of the present invention is 0.001 to 150mg/kg body weight, preferably 0.01 to 100mg/kg body weight, and more preferably 0.01 to 60mg/kg body weight.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the embodiment of the invention, an mdx mouse is a model mouse which is generally accepted to be used for researching Duchenne muscular dystrophy due to DMD gene defect and causes muscular dystrophy protein deficiency, and an experimental animal mdx type male mouse used in the invention is a model mouse with Dmd gene frameshift mutation, and the product is B10-DMD-KO and is purchased from Jiangsu Jiejicaokang biotechnology GmbH.

Example 1

(1) Laboratory animal

10 weeks old mdx type male mice (Dmd gene frameshift mutated mouse model, strain B10-DMD-KO), animals were housed in a temperature (20 + -2 deg.C) and humidity (55 + -5%) stable 12-hour light and dark cycle room with free access to water.

(2) Experimental protocol

The mdx type male mice were randomly divided into 3 groups of normal control group, model group, OLB-01 hydrochloride group, 6 mdx type male mice per group, orally administered daily for 56 days. Specific groups and doses of administration are shown in table 1:

TABLE 1 specific grouping and dosing of experimental mice

(3) Effect of OLB-01 hydrochloride on skeletal muscle hypertrophy and body weight of DMD mice

The experimental method comprises the following steps: the weights of the mice in each group were weighed once before administration every Monday morning after the start of the experiment, and after 8 weeks of administration, the heart, the left and right gastrocnemius muscles, and the left and right brachial triceps muscles were weighed during the material drawing.

The results in figure 1 show that mdx mice are heavier than normal wild-type mice, the body weight of the mdx mice can be reduced to a certain extent through administration treatment, and OLB-01 hydrochloride can obviously reduce skeletal muscle hypertrophy lesion of the mdx mice, which indicates that OLB-01 hydrochloride can improve skeletal muscle hypertrophy symptoms and reduce body weight of DMD mice. Data are presented as mean ± SEM. * WT compared to mdx group, # mdx compared to dosing group, # p <0.05, # p <0.01, # p <0.001, # p # and <0.0001. Each group n =6.

(4) Effect of OLB-01 hydrochloride on DMD mouse behaviourology

At 10 weeks of age of mdx mice, dosing was started according to the protocol described in step (2) for a total of 8 weeks, and then motor function was examined for each group.

The method for testing the holding power comprises the following steps: the strength of the muscle of the limbs of the mice is directly evaluated by measuring the grasping power of the limbs of the mice. The mouse is lightly placed on the central platform of the grip plate, the tail of the mouse is lightly pulled to enable the mouse to grasp the grip plate, the force is timely applied when the mouse grabs the grip plate with force, the mouse is pulled backwards, the small claw is loosened, and the maximum grasping force of the small plate is recorded daily at the moment. After the formal experiment is started, each visual test is performed once, each test is repeated for three more, and the maximum numerical value in the results obtained in three times is taken as an evaluation value.

The pole climbing test method comprises the following steps: the pole climbing test was used to assess the mobility and coordination of the limbs of the mice. A long rod with the length of about 50cm and the diameter of about 1cm is manufactured, and gauze is wound on the rod to increase friction. The wooden pole is vertically placed on a horizontal desktop, the head of the mouse is downwards lightly placed on the top of the dry pole, the mouse autonomously crawls downwards under the driving of no external force, and the time (pole climbing time) for the mouse to climb to the bottom platform from the top of the post is recorded. The mice with disqualified status were removed by continuous training for 3 days, 3 times a day, before administration. After the beginning of the official experiment, the test is carried out once a month, the test result takes 15 seconds as a boundary value, and the test result is recorded by 15 seconds after exceeding 15 seconds. This experimental procedure was repeated three times each time, and the average pole-climbing time value of the results of three times per mouse was calculated as an evaluation value.

The results of figure 2 show that the motion capability of the mdx mice is reduced, the pole climbing time of the mdx mice can be reduced by OLB-01 hydrochloride, and the forelimb grip strength test result shows that the drug can remarkably improve the grip strength level of the mice. Data are presented as mean ± SEM. * WT compared to mdx group, # mdx compared to dosing group, # p <0.05, # p <0.01, # p <0.001, # p # and <0.0001. Each group n =6.

(5) Effect of OLB-01 hydrochloride on DMD mouse gait

At 10 weeks of age of mdx mice, dosing was started according to the protocol described in step (2) for a total of 8 weeks, and then the gait of each group of mice was examined.

The gait detection method comprises the following steps: in the case of muscle damage, the gait of mice is slightly altered. By adopting a walking tracking analysis system, the natural gait of the mouse is obtained, and gait detection and analysis are carried out. The system adopts Germany The Imaging Source high-speed kilomega network port camera (150 frames/second) and a Sony CCD photosensitive principle to track The mouse in real time, adopts international advanced edge measurement, image noise reduction, binarization algorithm and other technologies, can comprehensively and intelligently identify The body size, the head, the tail, the four limbs and other parts of The mouse, and automatically classifies footprints so as to realize accurate measurement of indexes such as The area, the stride, the walking period, the movement speed, the support duration, the swing speed, the swing duration, the average intensity of The footprints and The like of each footprints of The mouse. Before the instrument is used, the width of the walking platform walkway is adjusted to be just suitable for the body type of the mouse, the phenomenon that the walkway is too wide or too narrow is avoided, and a ruler is arranged. And the camera is set, and the gain value and the white balance value are properly adjusted, so that the recording and shooting effects are optimal. The whole gait test is carried out in a quiet and dark environment. Before formal test, the mouse is subjected to walking training, and the mouse repeatedly walks through a glass walkway of 50cm in length for many times to be familiar with the environment. During formal testing, a mouse is put into the glass walkway from the entrance of the walkway, the mouse walks along the glass walkway, walking data is collected by a high-speed gigabit net portal camera, and screening and self-analysis are performed through system software WalkAnalysator. If the mouse is afraid of or smells the phenomenon of pause or turning back during walking, the measurement needs to be carried out again. If the footprint is analyzed automatically for errors, manual analysis may be used to calibrate the footprint. After the analysis is completed, the statistics of the footprint data and the analysis of the diagram can be checked, and the data can be exported.

The results in fig. 3 show that the limb movement ability of the model group mice is impaired to some extent compared with the normal wild type mice, and the OLB-01 hydrochloride administration group can improve the limb coordination ability of the mdx mice. Data are presented as mean ± SEM. * WT compared to mdx group, # mdx compared to dosing group, # p <0.05, # p <0.01, # p <0.001, # p # and <0.0001. Each group n =6.

(6) Effect of OLB-01 hydrochloride on muscle injury in DMD mice

After the mdx mice are 10 weeks old, administration is started according to the protocol described in step (2) for 8 weeks in total, and then the CK activity in the serum of each group of mice is measured.

The CK detection method comprises the following steps: blood was collected from the inferior vena cava prior to euthanasia of each group of mice. Blood samples were centrifuged at 1500g for 10 minutes at 4 ℃ and then serum CK activity levels were measured using a HITACHI7080 automated clinical analyzer.

As shown in FIG. 4, creatine kinase is mainly present in skeletal muscle, brain and cardiac muscle, and compared with normal wild-type mice, creatine kinase is significantly increased in model mice, severe damage may occur in mouse muscle, and the degree of muscle damage in mdx mice can be improved after OLB-01 hydrochloride administration. Data are presented as mean ± SEM. * WT compared to mdx group, # mdx compared to dosing group, # p <0.05, # p <0.01, # p <0.001, # p # and <0.0001. Each group n =6.

(7) Effect of OLB-01 hydrochloride on gastrocnemius inflammation in DMD mice

At the age of 10 weeks of mdx mice, administration was started according to the protocol described in step (2) for a total of 8 weeks, and then gastrocnemius inflammatory lesions were examined in each group of mice.

And (3) an HE dyeing method: both lateral gastrocnemius muscles of each group of mice were sectioned at a thickness of 5 μm, and then H & E staining was performed. The H & E staining procedure was as follows: baking for 30min, dewaxing for twice with xylene, respectively 15min,100% ethanol, 95% ethanol, 75% ethanol, 30% ethanol, performing gradient hydration with warm water for 2min, staining with hematoxylin for 6min, returning blue with tap water for 6min, staining with eosin for 3min, washing with hyperchromic solution, and air drying. And (5) sealing the neutral resin. After staining, histological images were taken on a 10-fold objective inverted microscope. Two equal-size areas are selected from each Image, nuclear deposition caused by inflammation in the areas is manually circled through Image J, and the muscle inflammation degree is automatically identified by software.

The results in figure 5 show that compared with normal wild type mice, the gastrocnemius section of the mice in the model group has large inflammatory cell infiltration, and the OLB-01 hydrochloride administration groups can obviously reduce inflammatory lesions of the gastrocnemius of the mice.

The results in FIG. 6 show that the nucleus deposition area caused by gastrocnemius inflammation is significantly increased in the model group mice compared with the normal wild-type mice, and the nucleus deposition area caused by gastrocnemius inflammation can be significantly reduced in each of the OLB-01 hydrochloride administration groups.

(8) Effect of OLB-01 hydrochloride on muscle fibrosis lesions in DMD mice

At 10 weeks of age of mdx mice, administration was started according to the protocol described in step (2) for a total of 8 weeks, and then muscle fibrosis lesions were examined in each group of mice.

Immunohistochemistry for fibrinectin: the bilateral gastrocnemius muscles of the mice were sectioned at a thickness of 5 μm, and then immunohistochemical staining was performed. The procedure for immunohistochemical staining of fibrinectin is as follows: dewaxing and rehydrating the slices conventionally, soaking the slices in an antigen retrieval solution (1X), heating for 10 minutes by using a microwave oven with medium fire, and washing for 3 times for 3min by using PBS (PH 7.4); incubating with hydrogen peroxide blocking solution for 15min; washing with PBS on a shaker for 10min,3 times; incubation for 1h with protease blocking solution or 3% BSA (containing 0.3% Triyon-100); directly incubating for 1h with a correspondingly diluted primary antibody working solution at room temperature, and then transferring to a refrigerator at 4 ℃ for overnight; the overnight sections were taken out and equilibrated at room temperature for 1h, then washed with PBS on a shaker for 10min 4 times; taking a proper amount of secondary antibody working solution to incubate for 1h; washing with PBS for 5min on a shaking bed for 3 times; placing the treated slices in DAB working solution for dyeing for 1-10 min; washing with PBS twice; dehydrating with 75%, 85%, 95%, and 100% ethanol for 3min each time; the xylene is transparent for 2 times, 5min each time; and sealing the sheet by using neutral resin. After staining, histological images were taken on an inverted microscope using a 10-fold objective. Two equal-size areas of each Image were selected and the fibrin-stained areas in the areas were manually circled by Image J to assess the degree of fibrosis.

The results in FIG. 7 and FIG. 8 show that wild-type mouse has very little fibrin, and mdx mouse can see a great deal of fibrin deposition, which indicates that the mice in the model group have serious fibrosis lesion, and OLB-01 hydrochloride can obviously reduce muscle fibrosis lesion of mdx mouse after being administrated.

(9) Effect of OLB-01 hydrochloride on DMD mouse collagen fibers

At the age of 10 weeks of mdx mice, administration was started according to the protocol described in step (2) for a total of 8 weeks, and then the diaphragm collagen fibers of each group of mice were examined.

Masson staining method: the mouse diaphragm muscle was sectioned at a thickness of 5 μm, and then Masson staining was performed. The Masson staining procedure was as follows: the slices are dewaxed and rehydrated conventionally and stained with hematoxylin for 5-10 min. Then differentiating with acid ethanol differentiation solution, soaking in distilled water for 1min, and turning blue. And dyeing with ponceau fuschin dyeing solution for 10min. Washing with weak acid working solution for 1min; washing with phosphomolybdic acid solution for 2min; washing with weak acid working solution for 1min. Then, the fabric is dyed in aniline blue staining solution for 2min. Washing with prepared weak acid working solution for 1min. Finally, dehydrating with 95% ethanol for 10s, and dehydrating with anhydrous ethanol for 3 times, 10s each time; xylene was clear 3 times for 2min each time. And (5) sealing by using neutral gum. After staining, histological images were acquired on a digital scanning microscope imaging system (M8, precision, germany) using a 10-fold objective lens. The red-blue area was directly identified by MIPAR image analysis software and the percentage of blue area over the entire tissue area was calculated, which reflects the degree of fibrosis.

The results in FIGS. 9 and 10 show that, compared to normal wild-type mice, the deposition of large collagen fibers was observed in the model mice, and that the dose of OLB-01 hydrochloride significantly reduced the collagen fibers in the mdx mice.

In conclusion, the OLB-01 hydrochloride can effectively improve skeletal muscle hypertrophy symptoms and reduce weight of a DMD mouse, can also obviously improve motor function, muscle strength, muscle injury, gait and limb coordination capacity of the DMD mouse, and can also reduce gastrocnemius inflammation, muscle fibrosis and collagen fiber degree of the DMD mouse, so that duchenne muscular dystrophy can be effectively treated.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

Use of olb-01 hydrochloride in the manufacture of a medicament for the treatment of duchenne muscular dystrophy.
2. The use of claim 1, wherein said OLB-01 hydrochloride is capable of ameliorating symptoms in duchenne muscular dystrophy, said symptoms comprising one or more of skeletal muscle hypertrophy, behavioral dysfunction, muscle weakness, muscle injury, inflammation, muscle fibrosis, collagen fibrosis.
3. The use of claim 1, wherein OLB-01 has the formula:
4. the use of claim 1, wherein the medicament comprises OLB-01 hydrochloride and a pharmaceutically acceptable excipient.
5. The use according to claim 1, wherein the medicament is in the form of a tablet, capsule, granule, pill, solution, spray or cream.
6. The use of claim 2, wherein the inflammation comprises gastrocnemius inflammation.
7. The use of claim 2, wherein the behavioral functions include motion, gait and limb coordination abilities.
8. The use of claim 1, wherein the medicament contains OLB-01 hydrochloride in an amount of 1 to 99%.