Use of ethyl 4-hydroxy-2-quinolinecarboxylate in preparing drugs or reagents for promoting hepatocyte proliferation

Abstract

The present invention discloses the use of ethyl 4-hydroxy-2-quinolinecarboxylate in the preparation of a drug or reagent for promoting hepatocyte proliferation. The present invention confirms through in vitro cell experiments that ethyl 4-hydroxy-2-quinolinecarboxylate has a significant effect on promoting hepatocyte proliferation and is non-toxic to cells in a concentration range of 1 to 500 μM. Ethyl 4-hydroxy-2-quinolinecarboxylate can be used to promote hepatocyte proliferation in humans, rats or other animals, including the process of hepatocyte proliferation and the process of hepatocyte regeneration after partial liver resection.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a new medical application of ethyl 4-hydroxy-2-quinolinecarboxylate, and in particular to an application of ethyl 4-hydroxy-2-quinolinecarboxylate in preparing a drug or reagent for promoting hepatocyte proliferation. Background Art

[0002] For liver tumors or other serious liver diseases, strategies such as partial hepatectomy, liver transplantation or hepatitis treatment all require liver regeneration mechanisms to repair the liver and its functions. However, the mechanism of liver regeneration is very complex, which results in relatively slow development of related basic research and treatment methods. Therefore, the continuous development of new research tools for studying liver regeneration mechanisms and new safe and effective methods to promote liver regeneration are important issues in the current field of liver regeneration. Therefore, screening substances that have the effect of promoting hepatocyte proliferation has the potential to be developed into research tools for liver regeneration mechanisms, and is also an important development direction for hepatocyte biology research. At the same time, studying the effects of compounds on the survival rate of normal hepatocytes (especially evaluating the possible toxic effects of compounds on hepatocytes) has also been one of the important research tasks in the development of compound bioactivity. In addition, substances that promote hepatocyte proliferation are also considered to have development potential and can be used to develop tools to promote liver regeneration methods.

[0003] At present, some people have modified the chemical structure of ethyl 4-hydroxy-2-quinolinecarboxylate to study the possible biological activities of its analogs. For example, some people have modified the chemical structure of the target compound and synthesized two analogs respectively, and studied the electrophysiological effects of these two analogs on neural tissue.

[0004] However, there are relatively few studies directly on the biological activity of ethyl 4-hydroxy-2-quinolinecarboxylate, and it is unclear what effect ethyl 4-hydroxy-2-quinolinecarboxylate may have on hepatocytes, especially the survival rate of hepatocytes. Summary of the invention

[0005] The purpose of the present invention is to provide application of 4-hydroxy-2-quinolinecarboxylic acid ethyl ester in preparing a medicine or reagent for promoting liver cell proliferation.

[0006] Chinese name: 4-Hydroxy-2-quinolinecarboxylic acid ethyl ester

[0007] English name: Ethyl 4-hydroxy-2-quinolinecarboxylate

[0008] Alias: ①2-Quinolinecarboxylic acid,4-hydroxy-,ethyl ester;

[0009] ②4-Hydroxy-quinoline-2-carboxylic acid ethyl ester

[0010] Relative molecular weight: 217.22

[0011] Structural formula:

[0012]

[0013] To achieve the above object, the technical solution of the present invention is as follows:

[0014] One aspect of the present invention provides the use of ethyl 4-hydroxy-2-quinolinecarboxylate or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof for preparing a drug for promoting hepatocyte proliferation.

[0015] Preferably, the concentration of ethyl 4-hydroxy-2-quinolinecarboxylate is 1.0 to 500 μM.

[0016] The pharmaceutically acceptable salts include conventional pharmaceutically acceptable salts formed by reacting the compounds of the present invention with inorganic acids or organic acids. For example, conventional pharmaceutically acceptable salts can be prepared by reacting the compounds of the present invention with inorganic acids or organic acids, wherein the inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and the organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid, or sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts formed by the compounds of the present invention with inorganic bases, or methylamine salts, ethylamine salts or ethanolamine salts formed by the compounds of the present invention with organic bases.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0018] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compound of general formula I of the present invention, its pharmaceutically acceptable salt or solvate thereof, and the mixture thereof, without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0019] The drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or can be mixed or wrapped with other substances and introduced into the body.

[0020] When necessary, one or more pharmaceutically acceptable carriers may be added to the above-mentioned drugs, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0021] 4-hydroxy-2-quinolinecarboxylic acid ethyl ester or its pharmaceutically acceptable salts, esters, solvates, stereoisomers, tautomers, prodrugs are used as active ingredients, used alone or in combination or formulated with other drugs, excipients, etc. into various dosage forms, including but not limited to tablets, powders, pills, injections, capsules, films, suppositories, ointments, granules, etc. The drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0022] Another aspect of the present invention provides the use of ethyl 4-hydroxy-2-quinolinecarboxylate in preparing a reagent for promoting hepatocyte proliferation.

[0023] Preferably, the reagent comprises a biochemical reagent having the effect of promoting hepatocyte proliferation as one of the experimental objectives.

[0024] Preferably, the reagent is a chemical or composition required for cell experiments or animal experiments with promoting hepatocyte proliferation as one of the experimental objectives.

[0025] The present invention discloses the following technical effects:

[0026] 1.4-Hydroxy-2-quinolinecarboxylic acid ethyl ester has the effect of promoting hepatocyte proliferation.

[0027] 2.4-Hydroxy-2-quinolinecarboxylic acid ethyl ester can promote the cell proliferation of normal human liver cells or normal rat liver cells.

[0028] 3. Regarding the cell viability of normal human hepatocytes or normal rat hepatocytes, ethyl 4-hydroxy-2-quinolinecarboxylate had no obvious cytotoxicity within the concentration range of 1-500 μM.

[0029] 4. It is expected that ethyl 4-hydroxy-2-quinolinecarboxylate can be used to promote the proliferation of hepatocytes in humans, rats or other animals, including the process of hepatocyte proliferation and the process of hepatocyte regeneration after partial liver resection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. It is obvious that the following drawings are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 Figure 4 is the effect of ethyl 4-hydroxy-2-quinolinecarboxylate on the proliferation of LO2 cells and BRL-3A cells.

[0032] Figure 1 a is the effect of ethyl 4-hydroxy-2-quinolinecarboxylate on the proliferation of LO2 cells;

[0033] Figure 1 b is the effect of 4-hydroxy-2-quinolinecarboxylic acid ethyl ester on the proliferation of BRL-3A cells.

[0034] LO2 cells are normal human hepatocytes, and BRL-3A cells are normal rat hepatocytes.

[0035] “*” indicates p<0.05; “**” indicates p<0.01

[0036] Figure 2 The figure shows the effect of L-ascorbic acid on the proliferation of LO2 cells and BRL-3A cells.

[0037] Figure 2 a is the effect of L-ascorbic acid on LO2 cell proliferation;

[0038] Figure 2 b is the effect of L-ascorbic acid on the proliferation of BRL-3A cells.

[0039] LO2 cells are normal human hepatocytes, and BRL-3A cells are normal rat hepatocytes.

[0040] “*” indicates p<0.05; “**” indicates p<0.01 DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually performed under normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0042] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0043] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Due to space limitations, they will not be described one by one here.

[0044] Example 1

[0045] LO2 cells and BRL-3A cells were treated with ethyl 4-hydroxy-2-quinolinecarboxylate, and L-ascorbic acid was selected as a comparison object. The cell survival rate was detected by tetramethylthiazolyl blue (MTT) method to determine whether ethyl 4-hydroxy-2-quinolinecarboxylate has the effect of promoting cell proliferation.

[0046] The cells used in the experiment are shown in Table 1, the main reagents used in the experiment are shown in Table 2, and the instruments used are shown in Table 3.

[0047] Table 1 Cell sources

[0048] Cell Type How to get it BRL-3A Wuhan Prosai Biotechnology Co., Ltd., Wuhan, China LO2 Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

[0049] Table 2 Main reagents

[0050]

[0051]

[0052] Table 3 Main instruments

[0053] Experimental instruments model Manufacturer <![CDATA[CO2 Incubator with Constant Temperature and Humidity]]> Forma3111 ThermoFisher, USA Multifunctional ELISA reader SynergyHIMF BioTek, USA Microplate oscillator MM-2 Shanghai, Precision Instruments

[0054] Experimental methods

[0055] S1. Cell culture

[0056] LO2 was cultured using DMEM high-glucose medium containing 1% penicillin-streptomycin and 10% fetal bovine serum, and placed in a constant temperature and humidity cell culture incubator at 37°C and 5% CO2; BRL-3A culture medium contained 1% penicillin-streptomycin, 10% fetal bovine serum, and 89% MEM-EBSS culture medium, and was cultured in a constant temperature and humidity cell culture incubator at 37°C and 5% CO2.

[0057] 2. MTT method

[0058] Cells were inoculated in a 96-well plate, with 20,000 cells in each well, and cultured in an incubator for 14-16 hours. Drugs were added, and after 24 hours of culture, 0.5 mg / mL of MTT culture medium was added for 4 hours, 150 μL of DMSO was added, and the plate was shaken for 10 minutes. The absorbance was measured at 490 nm using a multifunctional microplate reader. DMSO was used as the solvent control. 1% DMSO was used as the blank solvent group.

[0059] Calculation formula:

[0060] Cell survival rate (%) = [(absorbance of sample group - background absorbance) / (average absorbance of blank solvent group - background absorbance)] * 100%

[0061] Table 4 Cytotoxicity determination table

[0062] project Cell viability Cytotoxicity determination 1 >90％ No cytotoxicity 2 60％–90％ Slight cytotoxicity 3 30％–59％ Moderate cytotoxicity 4 <30％ Severe cytotoxicity

[0063] S3. Statistical methods

[0064] The data in this article were statistically analyzed using one-way ANOVA, and the Least Significant Difference (LSD) was used for post hoc test to calculate the differences between the groups. When α=0.05, P<0.05 level, it was marked with "*"; when P<0.01 level, it was marked with "**".

[0065] The experimental results are as follows Figure 1 (1a, 1b), Figure 2 As shown in (2a, 2b),

[0066] 4-Hydroxy-2-quinolinecarboxylic acid ethyl ester has a proliferative effect on LO2 cells at concentrations of 1, 10, 100, 200, and 500 μM, and the mean survival rate of LO2 is between 110% and 120%, which shows that 4-hydroxy-2-quinolinecarboxylic acid ethyl ester has a very significant proliferative effect on LO2 cells; BRL-3A cells were treated with 4-hydroxy-2-quinolinecarboxylic acid ethyl ester at concentrations of 1, 10, 100, 200, and 500 μM for 24 hours, and the mean survival rate of BRL-3A cells was between 110% and 120%, indicating that 4-hydroxy-2-quinolinecarboxylic acid ethyl ester has a proliferative effect on BRL-3A cells and is extremely significant. In summary, 4-hydroxy-2-quinolinecarboxylic acid ethyl ester is non-toxic in the concentration range of 1 to 500 μM and has the effect of promoting cell proliferation.

[0067] LO2 cells were treated with L-ascorbic acid at concentrations of 1, 10, 100, 200, and 500 μM for 24 hours, and the average survival rate of LO2 cells was between 95% and 110%. At 500 μM, L-ascorbic acid has a proliferation-promoting effect, and the cell survival rate is about 110%. BRL-3A cells were treated with L-ascorbic acid at concentrations of 1, 10, 100, 200, and 500 μM for 24 hours, and the average survival rate of BRL-3A cells was between 90% and 100%, indicating that L-ascorbic acid is non-toxic within the above concentration range. In summary, L-ascorbic acid has no obvious toxicity at concentrations of 1, 10, 100, 200, and 500 μM, but cell proliferation is not obvious.

[0068] L-ascorbic acid is a well-known common antioxidant with high biological safety. Figure 2 a, b show that L-ascorbic acid has no cytotoxicity.

[0069] This study selected L-ascorbic acid (vitamin C, vitamin C, VC), which has been widely studied and known to have antioxidant effects, as a comparison object. L-ascorbic acid is a compound that is often used in biomedical research and other fields, and is known to have antioxidant and several other biological activities. L-ascorbic acid only causes cell proliferation in LO2 (normal human liver cells) at a high concentration of 500μM ( Figure 2 a). However, as the concentration increases, L-ascorbic acid still does not increase the survival rate of BRL-3A (rat normal liver cells) cells ( Figure 2 b).

[0070] This study proves that L-ascorbic acid does not promote the cell proliferation of hepatocytes, and highlights the effect of 4-hydroxy-2-quinolinecarboxylic acid ethyl ester on promoting the proliferation of hepatocytes. This study uses two normal hepatocyte cell lines to prove the effect of 4-hydroxy-2-quinolinecarboxylic acid ethyl ester on promoting the proliferation of hepatocytes and its dose-effect relationship. This new use of 4-hydroxy-2-quinolinecarboxylic acid ethyl ester is not easily inferred by existing known methods.

[0071] In summary, ethyl 4-hydroxy-2-quinolinecarboxylate is non-toxic in the concentration range of 1-500 μM, and has a very significant effect on promoting the proliferation of hepatocytes (P<0.01).

[0072] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. Use of ethyl 4-hydroxy-2-quinolinecarboxylate in the preparation of a reagent for promoting normal hepatocyte proliferation, wherein the reagent is a biochemical reagent with the promotion of hepatocyte proliferation as one of the experimental objectives, and the biochemical reagent is a chemical or composition required in a cell experiment with the promotion of LO2 normal human hepatocytes or BRL-3A normal rat hepatocytes proliferation as one of the experimental objectives.

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