CN111214649A

CN111214649A - Application of BM23 peptide in preparing medicine for treating ischemic cerebrovascular disease

Info

Publication number: CN111214649A
Application number: CN202010126310.3A
Authority: CN
Inventors: 许元生; 卢圆; 汤宇晴
Original assignee: Dr August Wolff GmbH and Co KG Arzneimittel
Current assignee: Dr August Wolff GmbH and Co KG Arzneimittel
Priority date: 2020-02-27
Filing date: 2020-02-27
Publication date: 2020-06-02
Anticipated expiration: 2040-02-27
Also published as: CN111214649B

Abstract

The invention discloses an application of BM23 peptide in preparing a medicament for treating ischemic cerebrovascular diseases, wherein the amino acid sequence of BM23 peptide is shown as SEQ ID NO:1, preferably, the 1 st amino acid of the BM23 peptide is D-glutamine, the 7 th amino acid is homoarginine, and the 23 rd amino acid is amidated leucine. The inventor of the application discovers that the BM23 peptide has the application of treating ischemic stroke for the first time, provides a new targeted drug for clinical prevention and treatment of stroke, and has important medical significance and great economic value.

Description

Application of BM23 peptide in preparing medicine for treating ischemic cerebrovascular disease

Technical Field

The invention belongs to the field of medicines, and relates to application of BM23 peptide in preparation of a medicine for treating ischemic cerebrovascular diseases.

Background

The brain is one of the vital organs of human being, and its weight is only 2% of the body weight, but its oxygen consumption is 20% of the total oxygen consumption, and its required blood supply is 15% of the cardiac output, so the demand for oxygen and blood is particularly high, and it is very vulnerable to ischemic damage, among which cerebral ischemia caused by middle cerebral artery infarction is common. Cerebrovascular disease ranks second in 2003 among the causes of death from urban and rural disease, second only to malignant tumors.

Stroke (also known as stroke) and cerebrovascular accident (CVA) is an acute cerebrovascular disease; is a group of diseases that cause damage to brain tissue due to sudden rupture of cerebral blood vessels or failure of blood flow into the brain due to vascular occlusion. Cerebrovascular disorders include ischemic and hemorrhagic stroke. The incidence rate of ischemic stroke is higher than hemorrhagic stroke, and accounts for 60-70% of the total stroke.

The ischemic stroke has no real specific medicine. At present, there are three main types of drugs, the first is vasodilator, the second is drug for improving microcirculation and expanding blood volume, and the third is drug for dissolving thrombus. In recent years, vasodilating drugs have been gradually advocated for use because they may cause blood at the diseased site to flow back to healthy brain tissue; the drugs for improving microcirculation and expanding blood volume are easy to cause heart failure of heart patients and have certain limitations; the thrombolytic drug has relatively good effect, but also has certain side effect, and has time limitation, and has certain difficulty in specific application. Therefore, there is a need to develop a new drug for treating ischemic stroke.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide a novel drug useful for treating ischemic cerebrovascular diseases.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides application of BM23 peptide in preparing a medicament for treating ischemic cerebrovascular diseases.

BM23 peptide is a multifunctional growth factor belonging to TGF- β superfamily, which has been found to be used in the process of repairing cartilage and/or treating osteoarthritis.

Further, the amino acid sequence of the BM23 peptide is as shown in SEQ ID NO:1 is shown.

In a preferred embodiment of the present invention, the BM23 peptide has D-glutamine as the amino acid at position 1, homoarginine as the amino acid at position 7, and amidated leucine as the amino acid at position 23.

The modified BM23 peptide overcomes the defects of instability and short half-life of BMP-2 medicines, and has longer in-vivo efficacy duration.

In a preferred embodiment of the present invention, the ischemic cerebrovascular disease is stroke.

The invention has the beneficial effects that: through the research of the inventor, the BM23 peptide is found to have the application of treating ischemic stroke for the first time, and the invention provides a new targeted drug for the clinical prevention and treatment of stroke, and has important medical significance and great economic value.

Drawings

FIG. 1 shows the synthetic detection of BM23 peptide.

FIG. 2 shows the death of the rats in each group after 24h of treatment.

Figure 3 is the neurological score of each group of rats 24h after treatment.

Figure 4 is the cerebral infarct size of each group of rats 24h after treatment.

FIG. 5 shows the brain staining of rats in each group 24h after treatment, wherein A is a model control group, B is a BM23 peptide low dose group, and C is a BM23 peptide high dose group.

Figure 6 is the MDA concentration in each group of rats 24h after treatment.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

Example 1 Synthesis of BM23 peptide

BM23 peptide was synthesized by conventional solid phase process with purity > 98% (see FIG. 1) and 500 mg.

BM23 peptide sequence:

GlnLeuLysHisArgAsnHarHisArgIleLysThrGlySerThrAsnHisGlyLeu GlyLeu ValGlnSerLeu (SEQ ID NO: 1); wherein the amino acids at positions 1, 7 and 23 are D-glutamine, homoarginine and amidated leucine, respectively.

Example 2 therapeutic Effect of BM23 peptide on the ischemic Stroke model in MCAO rats

(I) Experimental method

Molding: 50 male SPF SD rats of 6-7 weeks old, weighing 178.3-219.6g, are inhaled and anesthetized with 2% isoflurane after fasting for 12h without water prohibition, and are fixed on an operating table in a supine position. The neck skin was cut open, the subcutaneous tissue and muscles were bluntly isolated, the Common Carotid Artery (CCA) was exposed, and the Internal Carotid Artery (ICA) and External Carotid Artery (ECA) continued to be free, with a ligature placed on each of the ICA and ECA. Cutting a small opening at 45 degrees in an oblique way by using an eye scissors (about 2mm away from the bifurcation of the common carotid Artery) on CCA, introducing a embolus line, ligating a hanging line which is reserved in the common carotid Artery to reduce bleeding, adjusting the direction and the angle of the embolus line, slightly pushing the embolus line, inserting the embolus line into the ICA, then loosening an Artery clamp on the ICA, and continuously inserting the embolus line until the embolus line reaches the starting part of the MCA (stopping when resistance occurs) and the length is about 18-20 mm to form Middle Cerebral Artery interruption (MCAO for short). And (5) tightening the fixing thread by the double knots, suturing the skin after confirming no bleeding, exposing the tail part of the fixing thread out of the skin, and fixing the thread. After 1.5h, the plug thread is softly and slowly drawn out by about 10mm from the tail part of the plug thread exposed out of the skin, blood flow reperfusion is carried out, and the residual tail end of the plug thread is cut off; and (5) returning the animals to cages for breeding.

In addition, sham-operated control SD rats were prepared in the same manner as described above except that the introduction of the tether and the related steps were not included.

Grouping: after the animals are awake, the animals are scored for nerve function, rats successfully modeled are selected and randomly divided into 3 groups according to the body weight, namely a model control group, a BM23 peptide low-dose group and a BM23 peptide high-dose group. In addition, sham control groups were included. The number of animals in each group is shown in Table 1.

Administration: after 1.5h of blood reperfusion, each group was treated with the corresponding drug as shown in table 1 by tail vein injection (the solvent for each group of drugs was normal saline).

Table 1 dosing regimen for each group of rats

Group of

Test article

Number of animals

Route of administration

Dosage to be administered

Volume of administration

Frequency of administration

Sham control group

Physiological saline

8

i.v. (intravenous injection)

/

5mL/kg

Single pass

Model control group

Physiological saline

12

i.v. (intravenous injection)

/

5mL/kg

Single pass

BM23 peptide Low dose group

BM23

12

i.v. (intravenous injection)

1mg/kg

5mL/kg

Single pass

BM23 peptide high dose group

BM23

12

i.v. (intravenous injection)

10mg/kg

5mL/kg

Single pass

General observations were: the overall status of the animal during the experiment was observed and totaled the mortality rate 24 hours after treatment.

And (3) nerve function scoring: before grouping and 24 hours after treatment, neurological grading was performed according to the motor performance of each group of animals. The scoring criteria were as follows:

0 point no symptoms of nerve damage;

1 minute of mild neurological impairment, incomplete extension of the contralateral forepaw or delayed/absent pain retraction;

2, dividing the degree of focal neurological deficit, and turning to the outside;

3, pouring the severe focal neurological deficit to the opposite side;

walking was not spontaneous in 4 minutes, and the level of consciousness decreased or lost.

Biochemical determination of serum: after 24 hours of treatment, the anesthesia test animals were injected intraperitoneally with 10% chloral hydrate at 3.5mL/kg, and the abdominal aorta was bled until death by blood loss. Centrifuging blood at 3000/r for 10min, separating serum, and storing at 4 deg.C. The MDA (malondialdehyde) kit (Shanghai Biyuntian biotechnology limited company, specification: 100T) is adopted to measure the MDA content in serum.

Cerebral infarction volume measurement: after blood is taken, 6 rats in each group are randomly taken, brains are taken out, liquid nitrogen is placed into the rats for fixation for 5-7 s, the rats are taken out and placed in a mold, 4 knives are coronal-incised after visual crossing, the 1 st knife is arranged between the anterior pole of the brain and the visual crossing connecting line, the 2 nd knife is arranged at the visual crossing position, the 3 rd knife is arranged at the funnel handle position, the 4 th knife is arranged between the funnel handle and the posterior leaf tail pole, the thickness is about 2mm, brain slices are quickly placed in phosphoric acid buffer solution of 2% triphenyltetrazolium chloride (TTC) to be incubated for 30min in a dark place at 37 ℃, and the brain slices are turned over once every 3-5 min in. The percentage of cerebral infarction was calculated by taking a photograph with a digital camera (normal tissue stained red and cerebral infarction tissue white) and analyzing with Image pro-Plus Image analysis software.

And (3) data statistics: data statistics were performed using Graphpad Prism software, and all measurements were expressed as means plus minus standard deviation (x ± s). The comparison of the means between groups is carried out by One-Way analysis of variance (One-Way ANOVA), two-by-two comparison of the means between groups, and t-test or Dunnett's T3. .

(II) results of the experiment

(1) Effect of BM23 peptide on mortality in MCAO rats

Mortality in each group after 24h of treatment is shown in figure 2. As can be seen from fig. 2, the mortality rate in the model control group was much higher than that in the treatment group, indicating that BM23 peptide significantly reduced the mortality rate in MCAO rats.

(2) Effect of BM23 peptides on the neurological function of MCAO rats

By observing the behavior of the movements of the animals in each group, it can be found that (the result is shown in fig. 3): after 24h of treatment, compared with a model control group (2.53 +/-0.58), the neurological function scores of the BM23 peptide low-dose group (1.36 +/-0.62) and the BM23 peptide high-dose group (0.85 +/-0.37) MCAO rats are reduced in a dose-dependent manner, and the difference has a remarkable statistical significance (P is less than 0.01), which indicates that the BM23 peptide treatment has a certain improvement effect on the neurological function.

(3) Effect of BM23 peptide on cerebral infarct size in MCAO rats

After modeling, compared with a sham operation group, the cerebral infarction area of the MCAO rat of the model control group is obviously increased (P is less than 0.01). After 24h of treatment, compared with a model control group (27.62 +/-3.67), the cerebral infarction areas of MCAO rats in a BM23 peptide low dose group (15.16 +/-4.13) and a BM23 peptide high dose group (8.58 +/-2.79) are reduced in a dose-dependent manner, and the differences have significant statistical significance (P is less than 0.01), shown in figures 4 and 5 (in figure 5, A is the model control group, B is the BM23 peptide low dose group, and C is the BM23 peptide high dose group), which indicates that the BM23 peptide treatment can significantly reduce the cerebral infarction areas of the MCAO rats.

(4) Effect of BM23 peptide on serum biochemistry of MCAO rats

After modeling, compared with a sham operation group, the MDA concentration of the model control group is obviously increased (P is less than 0.01). After 24h of treatment, compared with the model control group (1.2 +/-0.52), the MDA levels of the BM23 peptide low dose group (0.76 +/-0.31) and the BM23 peptide high dose group (0.29 +/-0.16) are reduced in a dose-dependent manner, and the difference is significant and statistically significant (P is less than 0.01), and the BM23 peptide treatment is suggested to reduce the MDA level in the serum of MCAO rats as shown in FIG. 6.

In conclusion, each experimental data shows that the BM23 peptide has obvious therapeutic effect on a rat MCAO model and is likely to be developed into a novel medicament for treating ischemic cerebrovascular diseases, in particular to a novel medicament for treating ischemic stroke.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Guangzhou Zhicheng medical science and technology Limited

Application of <120> BM23 peptide in preparation of medicine for treating ischemic cerebrovascular disease

<160>1

<170>PatentIn version 3.3

<210>1

<211>25

<212>PRT

<213> Artificial sequence

<400>1

Gln Leu Lys His Arg Asn His Ala Arg His Arg Ile Lys Thr Gly Ser

1 5 10 15

Thr Asn His Gly Leu Val Gln Ser Leu

20 25

Claims

Application of BM23 peptide in preparing medicine for treating ischemic cerebrovascular disease.
2. The use of claim 1, wherein the BM23 peptide has an amino acid sequence as set forth in SEQ ID NO:1 is shown.
3. The use of claim 2, wherein the amino acid at position 1 of the BM23 peptide is a D-glutamine, the amino acid at position 7 is a homoarginine, and the amino acid at position 23 is an amidated leucine.
4. The use of claim 1, wherein the ischemic cerebrovascular disease is stroke.