CN112494494B

CN112494494B - Novel pharmaceutical application of BML-28-4

Info

Publication number: CN112494494B
Application number: CN202011516755.9A
Authority: CN
Inventors: 卜晔; 杜建勇; 郑丽霞; 吴青; 朱小君; 熊敬维
Original assignee: Xinyoukang Pharmaceutical Technology Nanjing Co ltd
Current assignee: Xinyoukang Pharmaceutical Technology Nanjing Co ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2022-04-08
Anticipated expiration: 2040-12-21
Also published as: CN112494494A

Abstract

The invention discloses a pharmaceutical application of BML-284. The application is the application of BML-284 or pharmaceutically acceptable salts thereof in preparing a medicament for promoting myocardial cell proliferation. Experiments prove that the BML-284 can promote the rat myocardial cell proliferation. Therefore, BML-284 can be used for preparing medicaments for promoting the proliferation of myocardial cells, medicaments for treating or preventing heart diseases and other related fields, and provides a new medicament and a new treatment idea for treating or preventing heart diseases such as myocardial infarction.

Description

Novel pharmaceutical application of BML-28-4

Technical Field

The invention belongs to the field of medicines, and particularly relates to a new application of a BML-28-4 medicine.

Background

Cardiovascular diseases have become the first killer threatening human health currently, and 4000 million heart failure patients worldwide have become the main cause of human death. It has been found that in mammals, cardiomyocytes gradually lose proliferative capacity after adult life, and once myocardial infarction occurs, the loss of cardiomyocytes cannot be reversed. About 20-40 million myocardial cells exist in adults, about 25% of myocardial cells are lost within hours after the occurrence of myocardial infarction, the proliferation capacity of the remaining myocardial cells is very limited and is not enough to recover the systolic function of the heart, and finally, the heart failure of patients dies. To solve this problem, besides surgery, basic transformation studies mainly include finding cardiac stem cells or precursor cells, transplanting the cells to the myocardial infarction area by induced differentiation, but lack of sufficient molecular markers and low transplantation efficiency limit the use of this technology; the fibroblasts are transdifferentiated into cardiomyocytes using reprogramming techniques, or supplemented to the missing myocardium by means of promoting endogenous cardiomyocyte proliferation. The existing research shows that the heart of lower vertebrates such as zebra fish, salamander and the like has strong regeneration capacity, and newborn suckling mice also have certain regeneration capacity, and the regeneration capacity is mainly realized through myocardial cell proliferation induced by injury, and the capacity disappears after the adult. Adult hearts have not been considered to regenerate in the past, but extensive evidence suggests that there is a slow renewal of mammalian cardiomyocytes, and studies have found that neonatal cardiomyocytes are derived from existing myocardium. Therefore, an increasing number of scientists are interested in a strategy that induces the proliferation of endogenous cardiomyocytes. Therefore, the search for drugs capable of inducing the proliferation of endogenous cardiac muscle cells is of great significance for the treatment of cardiovascular diseases of human beings.

BML-284 is a potent, selective activator of Wnt signaling pathway. BML-284 enhances beta-catenin and increases the transcription and protein levels of p-gp. The Wnt agonist BML-284 also induced catenin in HEK-293T cells and apoptosis-dependent transcriptional activity of T cells (EC50 ═ 700 nM). BML-284 significantly increased the migration and invasion capacity of MNK45 and AGS cells, partially restoring the migration and invasion capacity of cells inhibited by pizotifen. However, no reports on the induction of endogenous cardiomyocyte proliferation of BML-284 exist.

Disclosure of Invention

The invention aims to provide a new application of BML-284 or a pharmaceutically acceptable salt thereof.

The BML-284 and CAS No.853220-52-7 have a structural formula shown in formula I:

the new application of BML-284 or the pharmaceutically acceptable salt thereof provided by the invention is the application of the BML-284 or the pharmaceutically acceptable salt thereof in preparing products for promoting the proliferation of myocardial cells.

The cardiomyocytes can be human or mammalian cardiomyocytes. The product may be a pharmaceutical product.

The invention also aims to provide application of BML-284 or pharmaceutically acceptable salts thereof in preparing products for preventing and/or treating cardiovascular diseases. The product may be a pharmaceutical product.

Further, in some embodiments of the invention, the cardiovascular disease may be a heart disease resulting from loss, injury or death of cardiomyocytes.

Further, in some embodiments of the present invention, the above-mentioned heart diseases include, but are not limited to, myocardial infarction, heart failure, and other cardiomyopathies resulting from loss, injury, or death of cardiomyocytes, and the like.

The product for promoting the proliferation of the myocardial cells and the product for preventing and/or treating cardiovascular diseases, which are prepared by using BML-284 or pharmaceutically acceptable salts and esters thereof as an active ingredient, also belong to the protection scope of the invention.

When necessary, one or more pharmaceutically acceptable carriers can be added into the medicine; the carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The above medicine can be made into various forms such as injection, tablet, powder, granule, capsule, oral liquid, paste, cream, etc.; the medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The above drugs can be introduced into body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

It is still another object of the present invention to provide a method for culturing cardiomyocytes in vitro.

The method for culturing the myocardial cells in vitro comprises the step of adding BML-284 or pharmaceutically acceptable salt thereof into a culture medium containing the myocardial cells.

The final concentration of BML-284 or a pharmaceutically acceptable salt thereof in the culture medium is 0.5-2 μmol/L. The cardiomyocytes can be human or mammalian cardiomyocytes.

The invention also provides a method for promoting the proliferation of the myocardial cells in vitro.

The method for promoting the proliferation of the myocardial cells in vitro comprises the step of treating the myocardial cells with BML-284.

The concentration of the BML-284 or the pharmaceutically acceptable salt thereof in the treatment system is 0.5-2 mu mol/L. The cardiomyocytes can be human or mammalian cardiomyocytes.

Experiments prove that BML-284 can promote the proliferation of rat cardiac muscle cells and can also promote the proliferation of human induced pluripotent stem cell-induced cardiac muscle cells (hipSC-derived cardiac muscle cells). Therefore, BML-284 can be used for preparing medicaments for promoting the proliferation of myocardial cells, medicaments for treating or preventing heart diseases and other related fields, and provides a new medicament and a new treatment idea for treating or preventing heart diseases such as myocardial infarction.

Drawings

FIG. 1 is a graph showing the effect of BML-284 on the proliferation of cardiomyocytes in newborn rats.

FIG. 2 is a graph showing the effect of BML-284 on the proliferation of human induced pluripotent stem cell-induced cardiomyocytes (hipSC-derived cardiomyocytes).

FIG. 3 is a graph showing the effect of BML-284 in promoting entry of neonatal rat cardiomyocytes into the cell cycle (PH 3 is a cell cycle activity marker);

FIG. 4 is a graph showing the effect of BML-284 in promoting entry of neonatal rat cardiomyocytes into the cell cycle (Ki 67 is used as a cell cycle activity marker).

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. BML-284 used in the examples below was a DMSO solution of BML-284. BML-284 manufacturer MedChemExpress (MCE), cat # 19987.

In the following examples "FBS" is fetal bovine serum.

The cTnT-mAG-hGeminin (1/110) virus used in the following examples was constructed as follows:

the cTnT myocardial specific promoter (shown as a sequence 1 in a sequence table) and mAG-hGeminin (1/110) (GenBank: NM-015895) are assembled and cloned on a pShuttle vector (Addgene 16402) by pEASY-Uni Seamless Cloning and Assembly Kit (all-type gold CU101-01), and then are subjected to enzyme digestion by a restriction enzyme PmeI to collect a linear plasmid; the linearized plasmid was co-transformed with pAdEasy1 DNA (Addgene 16400) to BJ5183 competent, recombinant plasmid was selected and amplified, and 293A cells were transfected with the recombinant plasmid (7.5X 10 cells were transfected the day before transfection)⁵293A cells were plated in a 60mm dish and cultured in DMEM medium containing 5% fetal bovine serum until the cell count reached 1.0-1.5X 10⁶Then, transfecting the cells with the recombinant plasmid by using a calcium phosphate coprecipitation method, removing a culture solution containing coprecipitation particles the next day after transfection, washing with a PBS buffer solution, subpackaging the cells into a 6-hole plate (3 ml of DMEM culture solution containing 5% fetal calf serum is added into each hole), and standing for 6 hours to allow the cells to adhere to the wall; covering the agarose after 6 hours for the formation of viral plaques (plaques should form within 10-21 days, adding agarose/DMEM mixture every 4-5 days or when the medium turns yellow); after obtaining the initial virus, the adenovirus is purified by 2-3 rounds of amplification and cesium chloride density gradient centrifugation.

Human induced pluripotent stem cell-induced cardiomyocytes (hiPSC-derived cardiomyocytes) used in the following examples were prepared according to the methods described in the following references: cell Res.2017Aug; 27(8) 1002-1019.doi 10.1038/cr.2017.84.Epub 2017Jun 16.

The Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody used in the examples described below was a Secondary Antibody manufactured by Invitrogen having the trade name of A32731, available under the trade name Goat anti-Rabbit IgG (H + L) high hly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488.

The Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody used in the examples described below was a Secondary Antibody manufactured by Life technologies under the trade designation A21424 under the trade name Goat anti-Mouse IgG (H + L) high hly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555.

The Anti-Cardiac Troponin T used in the examples described below is a primary antibody manufactured by abcam, Inc. under the designation ab8295

Anti- α -Actinin used in the following examples is a first antibody manufactured by Sigma under the trade name A7811

The Phospho-Histone H3(Ser10) used in the examples described below was a primary antibody manufactured by CST Inc. under the designation 9701S

The Anti-Ki67 antibody used in the following examples is a first antibody manufactured by abcam, Inc. under the designation ab15580

Example 1 in vitro assay of BML-284 for promoting proliferation of rat cardiomyocytes

(1) Culture of cardiac muscle cells of SD rat

Cardiomyocytes from 3-day-old SD rats were isolated and cultured in DMEM high-sugar medium (Hyclone) + 5% horse serum (GIBCO) in a 37-degree, 5% carbon dioxide incubator.

(2) Experimental grouping and processing

The myocardial cells of SD rats were isolated, and 5% horse serum (GIBCO) + DMEM high-sugar medium culture (Hyclone) was added, cytarabine (final concentration 20umol/L) was added to suppress the growth of non-myocardial cells, cells were infected with cTnT-mAG-hGeminin (1/110) virus (MOI value of virus infection: 100) after 48 hours of attachment, and then changed to DMEM containing 0.5% FBS after 24 hours, and the cells were subjected to the divided-dose treatment, as follows:

a. experimental groups: BML-284 was added (final concentration in the medium was 2. mu. mol/L) for 24 hours.

b. Blank control group: the same amount of DMSO as in the a experiment group was added.

(3) Test method

After the 2 groups are respectively treated for 24 hours, the cell nucleus is stained by hoechst fluorescent dye, then a high content living cell analyzer (molecular sieve) is used for photographing, and the positive myocardial cells of mAG-hGeminin (1/110) and the total cell number are analyzed.

(4) Results

The test results are shown in FIG. 1. As can be seen in FIG. 1, mAG-hGeminin (1/110) -positive cardiomyocytes increased 2.1-fold after BML-284 treatment compared to the blank control (0.5% FBS + DMSO). Mean ± SEM; p < 0.01.

Example 2 in vitro test of BML-284 for promoting proliferation of cardiomyocytes derived from human iPSC

Obtaining human induced pluripotent stem cell induced cardiomyocytes (hiPSC-derived cardiac cytocytes) and treating with BML-284 for 48h, using RPMI1640+ DMSO as a control (control), washing three times with PBS after 48h, fixing 4% PFA (paraformaldehyde) for 15min at room temperature, washing three times with PBS, blocking 1% BSA/PBS/0.1% Triton for 1h at room temperature, adding primary antibody after 1 h: Anti-Cardiac Troponin T (1:300, abcam, mouse, ab8295), Phospho-Histone H3(Ser10) antibody (1:300, CST, rabbit,9701S), overnight at 4 deg.C, washed 4 times with 1% BSA/PBS/0.02% Tween, plus secondary antibody: goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (1:500, Invitrogen, A32731), goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (1:500, Life technologies, A21424), left at room temperature for 1H, washed 3 times with PBS/0.02% Tween, stained nuclei with DAPI, photographed with a high content living cell analyzer (Molecular Device), and analyzed for the number of cardiomyocytes and total number of cells positive to PH 3.

The test results are shown in FIG. 2. As can be seen from fig. 2, the BML-284 treated group had 18.5-fold increase in the PH 3-positive human iPSC-derived cardiomyocytes compared to the control group. Mean ± SEM; p < 0.0001.

Example 3 in vitro assay of BML-284 to promote entry of neonatal rat cardiomyocytes into the cell cycle

Rat cardiomyocytes from 3 days of birth were isolated, BML-284 (final concentration in culture medium of 2. mu. mol/l) was added, DMEM + DMSO was used as a control (control), PBS was used three times after 48H, 4% PFA (paraformaldehyde) was fixed at room temperature for 15min, PBS was used three times, 1% BSA/PBS/0.1% Triton was blocked at room temperature for 1H, and primary antibodies Anti- α -Actinin (1:300, sigma, mouse, A7811), Phospho-Histone H3(Ser10) antibody (1:300, CST, rabbit,9701S), Anti-Ki67(1:300, abcam, bbrait, 15580) were used overnight at 4 ℃, PBS was used 1% BSA/0.02% Tween 4 times, secondary antibodies were added: goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (1:500, Invitrogen, a32731), goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (1:500, Life technologies, a21424), left at room temperature for 1H, washed 3 times with PBS/0.02% Tween, stained nuclei with DAPI, photographed with a confocal microscope, and statistically analyzed for the proportion of cardiomyocytes positive for PH3 and positive for Ki 67.

The test results are shown in fig. 3 and 4. As can be seen from fig. 3 and 4, PH 3-positive cardiomyocytes and Ki 67-positive cardiomyocytes in the BML-284-treated neonatal rat were significantly increased compared to the control group. Mean ± SEM; p < 0.05, P < 0.0001.

SEQUENCE LISTING

<110> Xin you kang pharmaceutical technology (Nanjing) Limited

New application of BML-28-4 medicine

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 836

<212> DNA

<213> Artificial sequence

<400> 1

tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg atcctctaga 60

actatagcta gaattcgccc ttacgggccc cccctcgagg tcgggataaa agcagtctgg 120

gctttcacat gacagcatct ggggctgcgg cagagggtcg ggtccgaagc gctgccttat 180

cagcgtcccc agccctggga ggtgacagct ggctggcttg tgtcagcccc tcgggcactc 240

acgtatctcc gtccgacggg tttaaaatag caaaactctg aggccacaca atagcttggg 300

cttatatggg ctcctgtggg ggaaggggga gcacggaggg ggccggggcc gctgctgcca 360

aaatagcagc tcacaagtgt tgcattcctc tctgggcgcc gggcacattc ctgctggctc 420

tgcccgcccc ggggtgggcg ccggggggac cttaaagcct ctgcccccca aggagccctt 480

cccagacagc cgccggcacc caccgctccg tgggacgatc cccgaagctc tagagcttta 540

ttgcggtagt ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct 600

cgaacttaag ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa 660

gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt 720

tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc 780

actcccagtt caattacagc tcttaaggct agagtactta atacgactca ctatag 836

Claims

The use of BML-284 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and/or treatment of cardiovascular disease; the cardiovascular disease is heart disease caused by myocardial cell loss, damage or death due to various reasons;

the structural formula of the BML-284 is shown as a formula I:

(formula I).
2. A method of culturing cardiomyocytes in vitro, comprising adding BML-284 of claim 1 or a pharmaceutically acceptable salt thereof to a culture medium comprising cardiomyocytes.
3. The method of claim 2, wherein: the final concentration of the BML-284 or the pharmaceutically acceptable salt thereof in the culture medium is 0.5-2 mmol/L.
4. A method according to claim 2 or 3, characterized in that: the cardiomyocyte is a human cardiomyocyte.
5. A method according to claim 2 or 3, characterized in that: the cardiac muscle cell is a cardiac muscle cell of a mammal.
6. A method for promoting cardiomyocyte proliferation in vitro, comprising treating cardiomyocytes with BML-284 or a pharmaceutically acceptable salt thereof as defined in claim 1.
7. The method of claim 6, wherein: the concentration of the BML-284 or the pharmaceutically acceptable salt thereof in the treatment system is 0.5-2 mmol/L.
8. The method according to claim 6 or 7, characterized in that: the cardiomyocyte is a human cardiomyocyte.
9. The method according to claim 6 or 7, characterized in that: the cardiac muscle cell is a cardiac muscle cell of a mammal.