CN114642662A

CN114642662A - Novel application of MK-5046 medicine

Info

Publication number: CN114642662A
Application number: CN202011516817.6A
Authority: CN
Inventors: 卜晔; 杜建勇; 郑丽霞; 吴青; 朱小君; 熊敬维
Original assignee: Nanjing Jingruikang Molecular Medicine Technology Co ltd; Xinyoukang Pharmaceutical Technology Nanjing Co ltd
Current assignee: Nanjing Jingruikang Molecular Medicine Technology Co ltd; Xinyoukang Pharmaceutical Technology Nanjing Co ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2022-06-21
Anticipated expiration: 2040-12-21
Also published as: CN114642662B

Abstract

The invention discloses a pharmaceutical application of MK-5046. The application is the application of MK-5046 or pharmaceutically acceptable salts thereof in preparation of medicines for promoting myocardial cell proliferation. Experiments prove that MK-5046 can promote rat myocardial cell proliferation. Therefore, MK-5046 can be used for preparing medicaments for promoting myocardial cell proliferation, 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 application of MK-5046 medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to a new application of an MK-5046 medicine.

Background

Cardiovascular diseases have become the first killer threatening human health at present, and 4000 million heart failure patients are 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.

MK-5046 is an orally active, potent, selective agonist of the G protein-coupled receptor Bombesin subtype-3(BRS-3), whose therapeutic efficacy in treating obesity is currently being evaluated. However, no reports are reported on MK-5046 in inducing endogenous cardiomyocyte proliferation.

Disclosure of Invention

The invention aims to provide a new application of MK-5046 or a pharmaceutically acceptable salt thereof.

The structural formula of MK-5046 and CAS No.1022152-70-0 is shown in formula I:

the novel application of MK-5046 or a pharmaceutically acceptable salt thereof provided by the invention is application of the MK-5046 or the pharmaceutically acceptable salt thereof in preparation of a product for promoting proliferation of myocardial cells.

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

Another object of the present invention is to provide MK-5046 or a pharmaceutically acceptable salt thereof for use in the preparation of a product 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.

Products prepared by using MK-5046 or a pharmaceutically acceptable salt thereof as an active ingredient and used for promoting myocardial cell proliferation and products prepared for preventing and/or treating cardiovascular diseases 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 above drugs in various dosage forms can be prepared according to conventional methods 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 MK-5046 or pharmaceutically acceptable salt thereof into a culture medium containing the myocardial cells.

The final concentration of MK-5046 or a pharmaceutically acceptable salt thereof in the 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 MK-5046 or a pharmaceutically acceptable salt thereof.

The concentration of MK-5046 or a pharmaceutically acceptable salt thereof in the treatment system is 0.5-2 μmol/L. The cardiomyocytes can be human or mammalian cardiomyocytes.

Experiments prove that MK-5046 can promote rat myocardial cell proliferation. Therefore, MK-5046 can be used for preparing medicaments for promoting myocardial cell proliferation, 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 MK-5046 on the proliferation of cardiomyocytes in neonatal rats.

Detailed Description

The present invention will be further illustrated with reference to the following 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.

MK-5046, used in the examples below, is a DMSO solution of MK-5046. MK-5046 manufacturer MedChemExpress (MCE), cat # HY-14342.

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 recombinant plasmid into cells by using a calcium phosphate coprecipitation method, removing a culture solution containing coprecipitation particles the next day after transfection, washing the cells by using a PBS buffer solution, subpackaging the cells into a 6-pore plate (adding 3ml of DMEM culture solution containing 5% fetal calf serum into each pore), and standing for 6 hours to make the cells 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.

Example 1 in vitro test for MK-5046 promotion of rat cardiomyocyte proliferation

(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

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

a. experimental groups: MK-5046 was added (final concentration in medium 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 above 2 groups were treated for 24 hours, nuclei were stained with hoechst fluorescent dye, and then photographed with a high content living cell analyzer (Molecular Device), and mAG-hGeminin (1/110) positive cardiomyocytes and total cell numbers were 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 1.7-fold after MK-5046 treatment compared to the blank control (0.5% FBS + DMSO). Mean ± SEM; p < 0.005.

SEQUENCE LISTING

<110> Xin Youkang pharmaceutical technology (Nanjing) Limited Nanjing Jingruikang molecular pharmaceutical technology Limited

New application of <120> MK-5046 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 application of MK-5046 or a pharmaceutically acceptable salt thereof in preparing a product for promoting the proliferation of myocardial cells;

MK-5046, CAS No. 1022152-70-0.
2. Use according to claim 1, characterized in that: the cardiac muscle cell is human or mammalian cardiac muscle cell.
The use of MK-5046 or a pharmaceutically acceptable salt thereof in the manufacture of a product for the prevention and/or treatment of cardiovascular disease; MK-5046, CAS No. 1022152-70-0.
4. Use according to claim 3, characterized in that: the cardiovascular disease is heart disease caused by myocardial cell loss, damage or death due to various reasons.
5. Use according to any one of claims 1 to 4, characterized in that: the product is a medicine.
6. A method for culturing cardiomyocytes in vitro, comprising adding MK-5046 or a pharmaceutically acceptable salt thereof to a culture medium comprising cardiomyocytes.
7. The method of claim 6, wherein: the final concentration of MK-5046 or a pharmaceutically acceptable salt thereof in the medium is 0.5 to 2 μmol/L.
8. A method for promoting cardiomyocyte proliferation in vitro comprising treating cardiomyocytes with MK-5046 or a pharmaceutically acceptable salt thereof.
9. The method of claim 8, wherein: the concentration of MK-5046 or a pharmaceutically acceptable salt thereof in the treatment system is 0.5-2 μmol/L.
10. The method according to any one of claims 7-9, wherein: the cardiac muscle cell is human or mammalian cardiac muscle cell.