CN112603914B - New application of phenylephrine hydrochloride medicament - Google Patents

Abstract

The invention discloses a medical application of phenylephrine. The application is the application of phenylephrine or pharmaceutically acceptable salts and esters thereof in preparing medicines for promoting myocardial cell proliferation. Experiments prove that the phenylephrine can promote the rat myocardial cell proliferation. Therefore, the phenylephrine 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

New application of phenylephrine hydrochloride medicament

Technical Field

The invention belongs to the field of medicines, and particularly relates to a new application of phenylephrine hydrochloride.

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 endogenous cardiomyocyte proliferation is of great significance for the treatment of cardiovascular diseases in humans.

Phenylephrine hydrochloride (Phenylephrine HCl) is a selective alpha 1-adrenergic receptor agonist. It can be used for preventing and treating hypotension caused by spinal anesthesia, general anesthesia, chlorpromazine, etc., and also can be used for supraventricular tachycardia and mydriasis examination, etc. But no report about the induction of endogenous cardiomyocyte proliferation by phenylephrine hydrochloride is available.

Disclosure of Invention

The invention aims to provide a new application of Phenylephrine (phenyleephrine) or pharmaceutically acceptable salts and esters thereof.

The phenylephrine CAS No.61-76-7 has a structural formula shown in formula I:

the pharmaceutically acceptable salt can be specifically a hydrochloride.

The new application of Phenylephrine (Phenylephrine) or pharmaceutically acceptable salts and esters thereof provided by the invention is the application of Phenylephrine (Phenylephrine) or pharmaceutically acceptable salts and esters thereof in the preparation of products for promoting the proliferation of myocardial cells.

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

The invention also aims to provide application of Phenylephrine (Phenylephrine) or pharmaceutically acceptable salts and esters 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 cardiomyopathy resulting from loss, injury, or death of other cardiomyocytes, and the like.

Products prepared from Phenylephrine (Phenylephrine) or pharmaceutically acceptable salts and esters thereof as active ingredients 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 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 Phenylephrine (Phenylephrine) or pharmaceutically acceptable salts and esters thereof into a culture medium containing the myocardial cells.

The final concentration of the Phenylephrine (Phenylephrine) or the pharmaceutically acceptable salt and ester thereof in the culture medium is 0.5-2 mu mol/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 Phenylephrine (phenyleephrine) or pharmaceutically acceptable salts and esters thereof.

The concentration of Phenylephrine (Phenylephrine) or pharmaceutically acceptable salts and esters thereof in the treatment system is 0.5-2 mu mol/L. The cardiomyocytes can be human or mammalian cardiomyocytes.

Experiments prove that the Phenylephrine (Phenylephrine) can promote the rat myocardial cell proliferation. Therefore, the Phenylephrine (Phenylephrine) can be used for preparing medicaments for promoting the proliferation of the myocardial cells and medicaments for treating or preventing heart diseases and the like, and provides a new medicament and a new treatment idea for treating or preventing the heart diseases such as myocardial infarction.

Drawings

FIG. 1 is a graph showing the effect of Phenylephrine (Phenylephrine) on the proliferation of cardiomyocytes in newborn rats.

FIG. 2 is a graph showing the effect of Phenylephrine (Phenylephrine) in promoting the entry of myocardial cells of newborn rats into the cell cycle (PH 3 is taken as a cell cycle activity marker);

FIG. 3 is a graph showing the effect of Phenylephrine (Phenylephrine) in promoting the entry of myocardial cells of newborn rats 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.

Phenylephrine hydrochloride (Phenylephrine) used in the examples below is a DMSO solution of Phenylephrine hydrochloride (Phenylephrine). Phenylephrine Hcl, manufacturer TargetMol, cat # T0453.

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.

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.

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

Anti-Ki67 used in the examples described below is a primary antibody manufactured by abcam, Inc. under the designation ab 15580.

Example 1 in vitro test for the promotion of proliferation of rat cardiomyocytes by Phenylephrine hydrochloride (Phenylephrine HCl)

(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: phenylephrine hydrochloride 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 is added for treatment.

(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, the mAG-hGeminin (1/110) positive cardiomyocytes increased 4.3-fold after phenylephrine hydrochloride treatment compared to the blank control (0.5% FBS + DMSO). Mean ± SEM; p < 0.001.

Example 2 in vitro test of phenylephrine hydrochloride to promote entry of neonatal rat cardiomyocytes into the cell cycle

Rat cardiomyocytes from 3 days of birth were isolated, supplemented with phenylephrine hydrochloride (final concentration in culture medium of 2 μmol/l), with DMEM + DMSO as control (control), washed three times with PBS after 48H, fixed 4% PFA (paraformaldehyde) for 15min at room temperature, washed three times with PBS, blocked 1H at room temperature with 1% BSA/PBS/0.1% Triton, and 1H followed by the addition of a primary antibody Anti-ti- α -actin (1:300, sigma, mouse, a7811), phosho-Histone H3(Ser10) antibody (1:300, CST, rabbit,9701S), Anti-Ki67(1:300, abcam, rabbit, 15580) overnight at 4 ℃, washed 4 times with 1% BSA/PBS/0.02% Tween, and 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. 2 and 3. As can be seen from fig. 2 and 3, PH 3-positive cardiomyocytes and Ki 67-positive cardiomyocytes in the phenylephrine hydrochloride-treated newborn rats were significantly increased compared to the control group. Mean ± SEM, × P < 0.005.

SEQUENCE LISTING

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

New application of phenylephrine hydrochloride medicament

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 836

<212> DNA

<213> Artificial sequence

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Claims (4)

1. A method of promoting cardiomyocyte proliferation in vitro comprising treating the cardiomyocytes with phenylephrine or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein: the concentration of the phenylephrine or the pharmaceutically acceptable salt thereof in the treatment system is 0.5-2 mmol/L.

3. The method according to claim 1 or 2, characterized in that: the cardiac muscle cell is a human cardiac muscle cell.

4. The method according to claim 1 or 2, characterized in that: the cardiac muscle cell is a cardiac muscle cell of a mammal.

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