Application of promethazine hydrochloride in preparation of medicine for inhibiting Mycobacterium kansasii

Abstract

The promethazine hydrochloride disclosed by the invention has a very good antibacterial effect on the Mycobacterium kansasii, the MIC of the promethazine hydrochloride on the standard strain of the Mycobacterium kansasii is 1.6 mu g / ml, and the MIC of the promethazine hydrochloride on the clinical rifampicin drug-resistant bacteria is 2 mu g / ml. Promethazine hydrochloride has low cytotoxicity and good safety, and can be used as a candidate drug to be applied to the development of anti-Mycobacterium kansasii infection drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of a small molecule compound promethazine hydrochloride in the preparation of a drug for inhibiting Mycobacterium kansasii. Background Art

[0002] Nontuberculous mycobacteria (NTM) refer to mycobacteria other than Mycobacterium tuberculosis (MTB) complex and Mycobacterium leprae. Currently, more than 200 species of NTM have been discovered, and some species can cause opportunistic infections in humans (Johnston JC, Chiang L, Elwood K. Mycobacterium kansasii[J]. Microbiol Spectr, 2017, 5(1). doi:10.1128 / microbiolspec.TNMI7-0011-2016.). The incidence of NTM is gradually increasing worldwide, and in some developed countries it even exceeds that of MTB (Baldwin SL, Larsen SE, Ordway D, et al. The complexities and challenges of preventing and treating nontuberculous mycobacterial diseases [J]. PLoS Neglected Trop Dis, 2019, 13(2): e0007083.). Mycobacterium kansasii is currently one of the most common pathogenic NTM (Falkinham JO. Epidemiology of infection by nontuberculous mycobacteria [J]. Clin Microbiol Rev, 1996, 9(2): 177-215.). Mycobacterium kansasii can cause lesions in multiple parts of the body, including the lungs, lymph nodes, skin, and skeletal muscles, as well as systemic disseminated diseases, with pulmonary lesions being the most common (Chinese Society of Tuberculosis Medicine. Guidelines for the diagnosis and treatment of nontuberculous mycobacterial diseases (2020 edition) [J]. Chinese Journal of Tuberculosis and Respiratory Diseases, 2020, 43(11): 918-46). Its infection is most similar to MTB infection among all NTM infections (Matveychuk A, Fuks L, Priess R, et al. Clinical and radiological features of Mycobacterium kansasii and other NTM infections [J]. Respir Med, 2012, 106(10): 1472-7.). Chronic cavitation in the upper lobes of the lungs is the most common manifestation of its lung infection, and patients may initially be misdiagnosed as pulmonary tuberculosis (Akram SM, Rawla P. Mycobacterium kansasii infection [M]. Treasure Island (FL): Stat Pearls). Publishing,2023.), and the treatment plans for the two are different.Although the treatment success rate of Mycobacterium kansasii is as high as 89.9%, the all-cause mortality rate of patients within 8 years is as high as 59.2%. Mycobacterium kansasii faces multiple difficulties such as high incidence of drug resistance, long treatment cycle, and few available therapeutic drugs. It is urgent to develop new drugs, especially for rifampicin-resistant strains.

[0003] Promethazine hydrochloride is a multi-purpose drug that has been widely used in clinical practice. Its pharmacological effects involve multiple aspects. First, as a competitive H1 receptor antagonist, it can compete with histamine released by tissues for H1 receptors, effectively antagonizing the contraction and contracture of gastrointestinal tract, trachea, bronchus or bronchiolar smooth muscle caused by histamine, thereby relieving the spasm and congestion of bronchial smooth muscle. Secondly, promethazine hydrochloride can inhibit the emetic chemoreceptor area of ​​the medulla oblongata to achieve an antiemetic effect. In addition, it acts on the vestibular and vomiting centers and midbrain medullary receptors through its central anticholinergic properties, blocking the excitement of vagal impulses of cholinergic synapses in the vestibular nucleus, and exerting an anti-motion sickness effect. Promethazine hydrochloride also has a mild sedative effect, which can weaken the activity of nerve cells in the subcortical limbic system, cause drowsiness and sleep, and can enhance the central inhibitory effects of hypnotic drugs, sedatives and anesthetics. Currently, there is no relevant research on the use of promethazine hydrochloride in the field of Mycobacterium kansasii disease, especially no reports on its in vitro inhibitory activity against Mycobacterium kansasii. Summary of the invention

[0004] The present invention finds that promethazine hydrochloride has good antibacterial activity against Mycobacterium kansasii in vitro, has low cytotoxicity and good safety. Based on this, the present invention is completed.

[0005] In a first aspect, the present invention provides the use of promethazine hydrochloride in the preparation of a product for inhibiting Mycobacterium kansasii, wherein the product contains promethazine hydrochloride, and the chemical formula of the promethazine hydrochloride is as follows:

[0006]

[0007] Furthermore, the Mycobacterium kansasii includes a clinical isolate of Mycobacterium kansasii, a standard strain of Mycobacterium kansasii, or Mycobacterium kansasii carried by a person infected with Mycobacterium kansasii.

[0008] Furthermore, the product for inhibiting Mycobacterium kansasii is an experimental reagent or an antibacterial agent.

[0009] Furthermore, the antibacterial agent can be a cleaning agent, including using promethazine hydrochloride and its pharmaceutically acceptable substance form to inhibit / eliminate Mycobacterium kansasii.

[0010] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the product.

[0011] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0012] Furthermore, the water-soluble carrier material includes, but is not limited to, one or more of polyethylene glycol, polyvinyl pyrrolidone and / or organic acid.

[0013] Furthermore, the poorly soluble carrier material includes but is not limited to one or more of ethyl cellulose and / or cholesterol stearate.

[0014] Furthermore, the enteric carrier material includes but is not limited to one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0015] Furthermore, the product can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0016] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0017] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0018] Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and / or aluminum silicate.

[0019] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0020] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0021] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0022] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0023] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0024] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0025] Furthermore, the pills may be prepared using a wide variety of carriers known in the art, including diluents and absorbents, binders and / or disintegrants.

[0026] Furthermore, the diluent and absorbent include, but are not limited to, one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin and / or talc.

[0027] Furthermore, the adhesive includes, but is not limited to, one or more of gum arabic, gum tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste and / or batter.

[0028] Furthermore, the disintegrant includes, but is not limited to, one or more of agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0029] Furthermore, the suppository may be made of a wide variety of carriers known in the art, including but not limited to one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and / or semi-synthetic glycerides.

[0030] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0031] Furthermore, the injectable preparation can use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0032] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0033] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0034] In a second aspect, the present invention provides the use of promethazine hydrochloride in the preparation of a drug for preventing and / or treating a disease caused by Mycobacterium kansasii infection, wherein the drug contains promethazine hydrochloride, and the chemical formula of the promethazine hydrochloride is as follows:

[0035]

[0036] Furthermore, the Mycobacterium kansasii includes a clinical isolate of Mycobacterium kansasii, a standard strain of Mycobacterium kansasii, or Mycobacterium kansasii carried by a person infected with Mycobacterium kansasii.

[0037] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

[0038] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0039] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, lozenges, suppositories and / or lyophilized powder injections.

[0040] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0041] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0042] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

[0043] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0044] Beneficial Effects

[0045] The promethazine hydrochloride in the present invention has a good antibacterial effect on Mycobacterium kansasii, and the MIC of promethazine hydrochloride to the standard strain of Mycobacterium kansasii is 1.6 μg / ml, and the MIC to clinical rifampicin-resistant bacteria is 2 μg / ml. Promethazine hydrochloride has low cytotoxicity and good safety, and can be used as a candidate drug for the development of drugs against Mycobacterium kansasii infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the effect of promethazine hydrochloride on the survival rate of A549 and THP-1 cells. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0049] The "CCK-8 method" described in this article is a cell proliferation and cytotoxicity detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt), which can be reduced to a water-soluble orange-yellow formazan dye by dehydrogenase in the mitochondria of living cells under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate, and its absorbance is measured at a wavelength of 450nm using an enzyme reader.

[0050] Example 1 Drug sensitivity test

[0051] In the present invention, the source of the standard strain of Mycobacterium kansasii is ATCC 12478. The 96-well plate microbroth dilution method was used to determine the in vitro activity of promethazine hydrochloride against the standard strain of Mycobacterium kansasii ATCC12478, and the activity of promethazine hydrochloride against rifampicin-resistant strains.

[0052] 1. Experimental Materials

[0053] 1.1 Test bacteria: standard strain of Mycobacterium kansasii ATCC12478, 3 strains of clinical rifampicin-sensitive bacteria (No. 1, 2, and 3), and 2 strains of rifampicin-resistant bacteria (No. 4 and 5).

[0054] 1.2 Antibacterial drugs: promethazine hydrochloride.

[0055] 1.3 Culture medium: Cation-Adjusted Mueller-Hinton Broth (CAMHB).

[0056] 1.4 Color indicator: Resazurin.

[0057] 2. Experimental methods

[0058] 2.1 MIC detection of standard strains of Mycobacterium kansasii

[0059] Promethazine hydrochloride was diluted with CAMHB medium containing 5% OADC, then added to a 96-well plate containing 100 μl 5% OADC CAMHB broth and serially diluted, and 100 μl of medium without drug was added to the negative control wells and the positive control wells, as shown in Table 1.

[0060] Table 1 Distribution of promethazine hydrochloride concentrations in 96-well plates of standard strains of Mycobacterium kansasii

[0061] 1 2 3 4 5 6 7 8 9 10 11 12 PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS 20μM 10μM 10μM 2.5μM 1.25μM 0.625μM 0.312μM 0.156μM PBS PBS PBS PBS 20μM 10μM 5μM 2.5μM 1.25μM 0.625μM 0.312μM 0.156μM PBS PBS PBS PBS Positive Positive Positive Positive Positive Positive Positive Positive PBS PBS PBS PBS Negative Negative Negative Negative Negative Negative Negative Negative PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

[0062] Note: Drug concentration is in μM

[0063] Pick a colony of the standard strain of Mycobacterium kansasii ATCC12478 in the logarithmic growth phase, grind the bacteria and measure the turbidity to 0.5 McFarland units, then dilute 100 times, and pipette 100 μl of bacterial solution into the drug well and positive control well of the aforementioned 96-well plate. The bacterial inoculation amount is about 5×10^5 CFU / mL, and the final concentration range of promethazine hydrochloride is 20 μM to 0.156 μM. Add 100 ul of bacterial solution to the positive control well and 100 ul of culture medium to the negative control well. Seal the plate with a sealing film and incubate.

[0064] 2.2 MIC detection of rifampicin-sensitive and rifampicin-resistant clinical strains of Mycobacterium kanssa

[0065] Promethazine hydrochloride (final concentration range 4 μg / ml to 0.125 μg / ml) was serially diluted using CAMHB broth containing 5% OADC according to the above method, and 100 ul of medium without drug was added to the negative control wells and the positive control wells, as shown in Table 2.

[0066] Table 2 Distribution of various concentrations of promethazine hydrochloride in 96-well plates of clinical strains of Mycobacterium kansasii

[0067] 1 2 3 4 5 6 7 8 9 10 11 12 A PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS B PBS Negative Positive 4 4 PBS PBS PBS PBS PBS PBS PBS C PBS Negative Positive 2 2 PBS PBS PBS PBS PBS PBS PBS D PBS Negative Positive 1 1 PBS PBS PBS PBS PBS PBS PBS E PBS Negative Positive 0.5 0.5 PBS PBS PBS PBS PBS PBS PBS F PBS Negative Positive 0.25 0.25 PBS PBS PBS PBS PBS PBS PBS G PBS Negative Positive 0.125 0.125 PBS PBS PBS PBS PBS PBS PBS H PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

[0068] Note: Drug concentration is in μg / ml

[0069] Select 5 well-growing clinical strains of Mycobacterium kansasii (No. 1, 2, and 3 are rifampicin-sensitive strains, and No. 4 and 5 are rifampicin-resistant strains) and colonies of the standard strain ATCC12478, prepare a bacterial suspension concentration of 0.5 McFarland units, and then dilute it 100 times. Pipette 100 μl of bacterial solution into the aforementioned 96-well plate to ensure that the bacterial inoculation concentration is about 5×10^5 CFU / mL, add 100 ul of bacterial solution to the negative control well, and add 100 ul of culture medium to the negative control well. Seal the plate with a sealing film and incubate.

[0070] 2.3 Adding color indicator

[0071] The 96-well culture plate was taken out, and resazurin indicator was added to each well, and the culture was continued to be incubated.

[0072] 2.4 Record the color of each well and determine the MIC

[0073] After the incubation, the 96-well culture plate was taken out, and the color changes of each well in the 96 microplate were observed and recorded (blue indicates no strain growth, pink indicates strain growth), and the MIC (minimum inhibitory concentration is defined as the lowest drug concentration that inhibits visible bacterial growth) was determined. The MIC value is defined as the drug concentration value at which the color changes from blue to pink after adding the resazurin indicator 24-48 hours after the 96-well plate was cultured in a 37°C constant temperature incubator for 10 days, and pink indicates bacterial growth.

[0074] 3. Experimental results

[0075] The MIC of promethazine hydrochloride against the standard strain of Mycobacterium kansasii ATCC12478 was 1.6 μg / ml, and the MIC against two clinical rifampicin-resistant strains was 2 μg / ml.

[0076] Table 3 Minimum inhibitory concentration of promethazine hydrochloride against clinical strains of Mycobacterium kansasii

[0077]

[0078] The experimental results show that promethazine hydrochloride has a good inhibitory effect on Mycobacterium kansasii in vitro and is worthy of further research.

[0079] Example 2 Cytotoxicity Experiment

[0080] 1. Experimental Methods

[0081] The cytotoxicity of promethazine hydrochloride was evaluated against human non-small cell lung cancer cells (A549) and human monocytic leukemia cell line (THP-1):

[0082] (1) Dilute the A549 cell content to 2.5×10^5 cells / ml, pipette 100 μl into a 96-well plate, and culture until it adheres to the wall and grows;

[0083] (2) Dilute THP1 cells to a density of 5 × 10^5 cells / ml and add 50 nM sodium dodecyl sulfate (PMA). Take 100 μl and transfer to a 96-well plate for culture to induce differentiation into macrophages and adhere to the wall for growth.

[0084] (3) After the two cell lines were completely attached, the culture medium was replaced with DMEM or RPMI medium containing promethazine hydrochloride (20 μM, 10 μM, 5 μM). 10 μl of CCK8 was added 24 h after the addition of the drug, and the absorbance at 450 nm was measured.

[0085] 2. Experimental results

[0086] After co-culture with promethazine hydrochloride at concentrations of 5μM, 10μM, and 20μM for 24 hours, the cell viabilities of A549 were 99.43%, 99.28%, and 100.50%, respectively, and there was no statistical difference between the concentrations and the control group; after co-culture with promethazine hydrochloride at concentrations of 5μM, 10μM, and 20μM for 24 hours, the cell viabilities of THP-1 were 94.80%, 97.56%, and 103.69%, respectively, and there was no statistical difference between the cell viabilities at the concentrations and the control group.

Claims

1. The use of promethazine hydrochloride in the preparation of a product for inhibiting Mycobacterium kansasii, wherein the product contains promethazine hydrochloride, and the chemical formula of the promethazine hydrochloride is as follows:

2. The product for inhibiting Mycobacterium kansasii as claimed in claim 1 is an experimental reagent or an antibacterial agent.

3. The antibacterial agent as claimed in claim 1 can be a cleaning agent, comprising using promethazine hydrochloride and its pharmaceutically acceptable substance form to inhibit / eliminate Mycobacterium tuberculosis.

4. Use of promethazine hydrochloride in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium kansasii infection, wherein the drug contains promethazine hydrochloride, and the chemical formula of the promethazine hydrochloride is as follows:

5. The Mycobacterium kansasii according to claim 1 or 4 comprises a clinical isolate of Mycobacterium kansasii, a standard strain of Mycobacterium kansasii or Mycobacterium kansasii carried by a Mycobacterium kansasii infected person.

6. The use as claimed in claim 4, wherein one or more pharmaceutically acceptable carriers or excipients may be added to the medicine.

7. The use according to claim 6, wherein the carrier material includes but is not limited to one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

8. The use as claimed in claim 4, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, transdermal agents, suppositories and / or lyophilized powder injections.

9. The use according to claim 8, wherein the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

10. The use according to claim 4, wherein the drug can be administered by injection, cavity administration, respiratory tract administration or mucosal administration.

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