CN113967207B - Use of 4-isothioureidobutyronitrile hydrochloride for treating mycobacterial infections - Google Patents

Use of 4-isothioureidobutyronitrile hydrochloride for treating mycobacterial infections Download PDF

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CN113967207B
CN113967207B CN202111534092.8A CN202111534092A CN113967207B CN 113967207 B CN113967207 B CN 113967207B CN 202111534092 A CN202111534092 A CN 202111534092A CN 113967207 B CN113967207 B CN 113967207B
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hydrochloride
isothioureidobutyronitrile
mycobacterium
tuberculosis
drug
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CN113967207A (en
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徐蕾
李安龙
包嘉佳
赖晓霏
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Chongqing Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of 4-isothioureidobutyronitrile hydrochloride in treating mycobacterial infection. The invention finds that the compound 4-isothioureidobutyronitrile hydrochloride has new application, can inhibit the survival of mycobacteria by being used alone or being used together with first-line antitubercular drugs, and effectively treats the mycobacterial infection, namely tuberculosis, thereby solving the problem of drug resistance of the tuberculosis to the existing first-line antitubercular drugs and providing a new means for treating the tuberculosis.

Description

Use of 4-isothioureidobutyronitrile hydrochloride for treating mycobacterial infections
Technical Field
The invention relates to the technical field of medicines, in particular to application of 4-isothioureidobutyronitrile hydrochloride in treating mycobacterial infection.
Background
Tuberculosis is an infectious disease caused by infection with Mycobacterium tuberculosis (Mtb). Estimated according to the latest World Health Organization, about 900 million people suffer from tuberculosis, and about 150 million people die of tuberculosis every year. Despite global efforts to control mycobacterium tuberculosis infection, tuberculosis (tuberculosis) remains a major public health risk and threatens the health of people worldwide.
Rifampicin is a first-line antitubercular drug used clinically, is a broad-spectrum antibiotic drug belonging to a rifamycin family, has a strong antibacterial effect on tubercle bacillus, and also has a curative effect on gram-positive or gram-negative bacteria, viruses and the like. However, in recent years, with the emergence of drug-resistant bacteria, the treatment of rifampicin has become increasingly fatiguing. The rifampicin and other antituberculosis drugs are used together to play a synergistic role, and the generation of drug-resistant strains can be delayed. In addition to rifampin, the first-line anti-tuberculosis drugs (first-line anti-tuberculosis drugs) commonly used in clinical practice include isoniazid, ethambutol, pyrazinamide, streptomycin, rifabutin, rifapentine. However, with the increasingly prominent drug resistance problem of tuberculosis, new tuberculosis treatment drugs are searched, and the research and development of new drug treatment modes are urgent.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide the use of 4-isothioureidobutyronitrile hydrochloride for the treatment of mycobacterial infections, which can treat mycobacterial infections by using 4-isothioureidobutyronitrile hydrochloride alone or by combining 4-isothioureidobutyronitrile hydrochloride with a first-line antitubercular drug, and which solves the problem of resistance of tuberculosis to first-line antitubercular drugs in the prior art.
To achieve the above objects and other related objects, the present invention provides, in a first aspect, the use of 4-isothioureidobutyronitrile hydrochloride in the manufacture of a medicament for the treatment of a mycobacterial infection.
4-isothioureidobutyronitrile hydrochloride (4-isothiuridine butyronitrile hydrochloride, kevetrin hydrochloride) with molecular formula C 5 H 10 ClN 3 S, the molecular weight is 179.67, the action target point is MDM-2/p53, and the molecular structural formula is as follows:
Figure BDA0003411942970000021
further, the mycobacterium is selected from at least one of Mycobacterium smegmatis, attenuated Mycobacterium bovis, mycobacterium tuberculosis, mycobacterium bovis, mycobacterium marinum, mycobacterium leprae, M.
In a second aspect, the invention provides the use of 4-isothioureidobutyronitrile hydrochloride in combination with a first-line antitubercular drug for the manufacture of a medicament for the treatment of mycobacterial infections.
Further, the first-line antituberculosis drug is at least one selected from rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, rifabutin, rifapentine, preferably rifampicin.
Further, the mycobacterium is selected from at least one of Mycobacterium smegmatis, attenuated Mycobacterium bovis, mycobacterium tuberculosis, mycobacterium bovis, mycobacterium marinum, mycobacterium leprae, M.
In a third aspect, the invention provides a medicament for the treatment of mycobacterial infections comprising a safe and effective amount of 4-isothioureidobutyronitrile hydrochloride.
Further, the drug is used in combination with a first line antitubercular drug.
Further, the first-line antituberculosis drug is at least one selected from rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, rifabutin and rifapentine, preferably rifampicin.
Further, the mycobacterium is selected from at least one of Mycobacterium smegmatis, attenuated Mycobacterium bovis, mycobacterium tuberculosis, mycobacterium bovis, mycobacterium marinum, mycobacterium leprae, M.avium, and M.cheloni.
Further, in the medicine, the dosage of the 4-isothioureidobutyronitrile hydrochloride is 0-40 umol/L (excluding 0), preferably 10-40 umol/L, and more preferably 20-40 umol/L.
In a fourth aspect, the present invention provides a combination comprising a safe and effective amount of 4-isothioureidobutyronitrile hydrochloride and at least one first line antitubercular agent for use in the treatment of a mycobacterial infection.
Further, the first-line antituberculosis drug is at least one selected from rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, rifabutin, rifapentine, preferably rifampicin.
Further, the mycobacterium is selected from at least one of Mycobacterium smegmatis, attenuated Mycobacterium bovis, mycobacterium tuberculosis, mycobacterium bovis, mycobacterium marinum, mycobacterium leprae, M.avium, and M.cheloni.
Further, in the combined medicine, the dosage of the 4-isothioureidobutyronitrile hydrochloride is 0-40 umol/L (excluding 0), preferably 10-40 umol/L, and more preferably 20-40 umol/L.
In the present invention, the form of the pharmaceutical preparation is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, and the like. The administration forms can be rich, and can be gastrointestinal administration or parenteral administration; known routes of administration for each drug are generally recommended. The subject may be a mammal or a mammalian macrophage; the mammal is preferably a rodentia, artiodactyla, perissodactyla, lagomorpha, primate, or the like; the primate is preferably a monkey, ape or human; the macrophage can be an ex vivo macrophage.
As described above, the use of 4-isothioureidobutyronitrile hydrochloride according to the invention for the treatment of mycobacterial infections has the following beneficial effects:
the invention provides a new application of a compound 4-isothioureidobutyronitrile hydrochloride, and through further research and test on the combined treatment of mycobacterium in macrophages by the 4-isothioureidobutyronitrile hydrochloride and rifampicin, the invention discovers that the 4-isothioureidobutyronitrile hydrochloride can effectively inhibit the survival of the mycobacterium, and the 4-isothioureidobutyronitrile hydrochloride can be combined with a first-line antitubercular drug (such as rifampicin) to effectively treat the infection of the mycobacterium. Therefore, the invention provides a method for independently using the compound 4-isothioureidobutyronitrile hydrochloride and combining with a first-line antituberculosis drug to inhibit the survival of mycobacteria and treat mycobacterial infection, namely tuberculosis, thereby solving the problem of drug resistance of the tuberculosis to the existing first-line antituberculosis drug and providing a new means for treating the tuberculosis.
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FIG. 1 is a graph showing the results of activity measurement of cells treated with 4-isothioureidobutyronitrile hydrochloride for 48 hours in example 1 of the present invention.
FIG. 2 is a graph showing the colony count of the plated plate after 4-isothioureidobutyronitrile hydrochloride treated M.smegmatis macrophage for 48 hours in example 2 of the present invention.
FIG. 3 is a graph showing the colony count of the plate coated after 48 hours of the co-action of 4-isothioureidobutyronitrile hydrochloride with rifampicin on M.smegmatis macrophage in example 2 of the present invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
4-isothioureidobutyronitrile hydrochloride (4-isothioureido nitrile hydrochloride, kevetrin hydrochloride) is a specific agonist of tumor suppressor protein p53, and can effectively inhibit the occurrence and development of tumors. The 4-isothioureidobutyronitrile hydrochloride can promote the specific combination of p53 and DNA, and the activity of the hydrochloride is regulated and controlled by post-translational modification such as phosphorylation, acetylation, methylation, ubiquitination and the like. The wild type inhibits the survival of cancer cells, while the mutant type enhances the survival of cancer cells in the body. Thus, 4-isothioureidobutyronitrile hydrochloride exerts important biological functions by participating in apoptosis.
Through further research and test on 4-isothioureidobutyronitrile hydrochloride and the combination treatment of mycobacterium in macrophage with rifampicin, the invention discovers the new application of the compound 4-isothioureidobutyronitrile hydrochloride: 4-isothioureidobutyronitrile hydrochloride can inhibit the survival of mycobacteria; 4-Isothioureidobutyronitrile hydrochloride is used in combination with rifampin to treat mycobacterial infections.
It should be noted that the mycobacterium specifically used in the examples of the present invention is mycobacterium smegmatis, which is an ideal model bacterium for studying tuberculosis. Of course, other types of mycobacteria such as Mycobacterium tuberculosis, mycobacterium bovis, attenuated Mycobacterium bovis, mycobacterium marinum, mycobacterium leprae, M.avium, M.cheloniae, etc. may be used with the same technical effect.
Example 1
In this example, the damage of 4-isothioureidobutyronitrile hydrochloride to cells is determined by in vitro experiments to define a safe administration range, and the specific operations are as follows:
1. experimental Material
Cell: RAW264.7 mouse mononuclear phagocytic leukemia cells;
reagent: dimethyl sulfoxide (DMSO), a DMEM high-glucose cell culture medium, fetal calf serum, a phosphate buffer solution and 4-isothioureidobutyronitrile hydrochloride powder.
2. Experimental procedure
Fetal bovine serum was added to cell culture media and RAW264.7 cells were seeded into 96-well cell culture plates at 1 × 10 per well 4 (ii) individual cells; after the cells adhere to the wall, 4-isothioureidobutyronitrile hydrochloride solution (prepared by DMSO) is added, five experimental groups are set in the experiment, wherein the five experimental groups respectively comprise groups with the concentration of 4-isothioureidobutyronitrile hydrochloride of 10, 20, 30, 40 and 50umol/L and a control group without the addition of 4-isothioureidobutyronitrile hydrochloride.
After the addition of the drug, the samples were incubated in a 37 ℃ thermostatted cell incubator for 48 hours.
After 48 hours of treatment, the cell supernatant was discarded, 100uL of a cell culture medium containing fetal bovine serum and 10uL of CCK-8 reagent were newly added to each well of the 96-well plate, and the 96-well plate was incubated at 37 ℃ for 1 hour.
After 1 hour, the OD was measured at a wavelength of 450 nm.
3. Results of the experiment
The activity of the cells after 48h treatment with 4-isothioureidobutyronitrile hydrochloride was determined and the results are shown in FIG. 1. From figure 1, it is drawn that the safe application range of 4-isothioureidobutyronitrile hydrochloride in cell experiments is 0-40 umol/L.
Example 2
In this example, in vitro experiments prove that 4-isothioureidobutyronitrile hydrochloride and its combination with rifampicin can effectively inhibit survival of mycobacteria in macrophages, the specific operations are as follows:
1. experimental Material
Cell: RAW264.7 mouse mononuclear phagocytic leukemia cells;
bacteria: mycobacterium smegmatis;
reagent: dimethyl sulfoxide (DMSO), DMEM (DMEM) high-glucose cell culture medium, fetal calf serum, phosphate buffer (used for washing before cell digestion and preparing cell lysate), triton X-100 (used for preparing 1% cell lysate), middlebrook7H9 broth base, agar powder, 4-isothioureidobutyronitrile hydrochloride powder and rifampicin powder.
2. Experimental procedure
Fetal bovine serum was added to cell culture medium, and RAW264.7 cells were seeded into cell culture well plates at 1 × 10 per well 6 (ii) individual cells; after the cells were attached, M.smegmatis infected cells were added at MOI (multiplicity of infection) 0.1.
Adding bacteria for 1 hour, and adding 4-isothioureidobutyronitrile hydrochloride and rifampicin; six experimental groups are set in the experiment, wherein the concentration of 4-isothioureidobutyronitrile hydrochloride is 20, 30 and 40umol/L, the concentration of the 4-isothioureidobutyronitrile hydrochloride added is 20, 30 and 40 umol/L; two control groups were set, one of which was not treated with drugs after infecting bacteria, and the other was treated with rifampicin at a drug concentration of 10umol/L after infecting bacteria.
After the addition of the drug, the samples were incubated in a 37 ℃ thermostatted cell incubator for 48 hours.
After 48 hours of treatment, 1mL of cell lysis solution (1% Triton X-100) was added to release the cells from the bacteria, the supernatant was discarded after centrifugation, and 1mL of PBS was added to resuspend the cell debris and the bacterial mixture. The bacterial suspension is diluted in gradient with the dilution times of 10 respectively 2 、10 3 、10 4 Three gradient dilution suspensions, 10uL each, were plated on 7H 9M M.solid medium.
Placing the culture medium into a 37 deg.C incubator, culturing for 3-7 days, and counting (CFU should be 10 when counting) 6 number/mL, the factor is large, so the unified unit is 10 7 one/mL).
3. Results of the experiment
4-Isothioureidobutyronitrile hydrochloride (Kevetrin in the figures and tables) acts on macrophage attenuationSpread plate colony counts 48h after toxigenic M.bovis (CFU 10. Multidot 7 pieces/mL) are shown in fig. 2 and table 1.
TABLE 1
Figure BDA0003411942970000061
4-Isothioureidobutyronitrile hydrochloride with rifampicin in macrophage attenuated M.bovis 48h later plated colonies were counted (CFU 10. Multidot 7 pieces/mL) are shown in fig. 3 and table 2.
TABLE 2
Figure BDA0003411942970000062
From the above experimental results, it can be seen that 4-isothioureidobutyronitrile hydrochloride in combination with rifampicin can effectively inhibit the survival of mycobacteria within macrophages, and the ability to inhibit the survival of mycobacteria is superior to that of rifampicin alone.
In conclusion, in vitro experiments prove that the 4-isothioureidobutyronitrile hydrochloride can effectively inhibit the survival of mycobacteria in macrophages, and the 4-isothioureidobutyronitrile hydrochloride can be used for effectively treating the infection caused by the mycobacteria by being used alone or combined with a first-line antituberculosis drug (such as rifampicin).
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims (4)

  1. The application of 4-isothioureidobutyronitrile hydrochloride in preparing medicine for treating mycobacterial infection is characterized by comprising the following steps: the mycobacterium is Mycobacterium smegmatis.
  2. The application of 4-isothioureidobutyronitrile hydrochloride and a first-line antituberculosis drug in preparing a drug for treating mycobacterial infection is characterized in that: the first-line antituberculosis drug is rifampicin, and the mycobacterium is Mycobacterium smegmatis.
  3. 3. A combination for use in the treatment of mycobacterial infections, characterized by: contains 4-isothioureidobutyronitrile hydrochloride and a first-line antitubercular drug with safe and effective dosage, wherein the first-line antitubercular drug is rifampicin, and the mycobacterium is mycobacterium smegmatis.
  4. 4. The combination according to claim 3, wherein: in the combined medicine, the dosage of the 4-isothioureidobutyronitrile hydrochloride is 0-40 umol/L, and 0 is not included.
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