Application of allicin or precursor or derivative of allicin in preparation of medicine for treating CFTR-mediated diseases

Abstract

The invention relates to the field of medicines, in particular to application of allicin or a precursor or a derivative thereof to preparation of a medicine for treating CFTR-mediated diseases. The invention further discloses application of the allicin or the precursor or the derivative of the allicin to preparation of the CFTR activating agent or the CFTR correcting agent. According to the present invention, the first research results show that allicin can activate the CFTR channel function in a dose-dependent manner: opening the CFTR channel; 2, correction of CFTR channel dysfunction: the positioning efficiency of a CFTR subunit membrane can be improved by protecting the completeness of a CFTR subunit, so that allicin or a precursor or a derivative thereof can be used for preparing a CFTR activator or a CFTR corrector, the insulin secretion level can be improved through a CFTR channel, and the allicin or the precursor or the derivative thereof can be used for treating pancreatic insufficiency.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to use of allicin or its precursor or derivatives in preparing medicines for treating CFTR-mediated diseases. Background Art

[0002] CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride ion channel that is widely expressed on the apical membrane of epithelial cells in the airways, prostate, ovaries, gastrointestinal tract, pancreas, liver, lungs, testicles, and sweat glands (see Figure 2 , Zielenski J, 2000), involved in the regulation of multiple ion transporters, including sodium channels, chloride / bicarbonate buffer, nephron exchanger (Na + / H + ) and water channels. CFTR plays a key role in maintaining the homeostasis of ions, pH and water in secretory epithelial mucus, and is also involved in processes such as cell proliferation, migration, and regulation of ion concentration. Therefore, abnormalities in the activity of this channel are associated with the pathogenesis of a variety of diseases. Loss of function or reduced activity of the CFTR chloride channel can lead to cystic fibrosis, chronic pancreatitis, male infertility, constipation, and dry eye disease. Therefore, the discovery of efficient and specific regulators of the CFTR chloride channel is of great value for studying the pathological mechanisms and treatments of these diseases.

[0003] Mutations in the CFTR gene can cause loss of function or reduced activity of its ion channels, leading to cystic fibrosis, a disease that affects the physiological functions of multiple organs. The most common of these is the ΔF508-CFTR deletion mutation, which is present in at least one allele in 90% of cystic fibrosis patients. The deletion of phenylalanine at position 508 affects the function of CFTR in two ways and ultimately causes the loss of CFTR's chloride ion transport function. On the one hand, the protein cannot reach the plasma membrane normally, and on the other hand, even if it can reach the plasma membrane, the chloride ion transport activity of the channel is significantly reduced. Although some small molecule compounds that can correct the functional defects of ΔF508-CFTR have been discovered internationally, little progress has been made in the research of natural products. At present, there are reports internationally showing that a certain dose of curcumin can partially correct the upper membrane disorder of ΔF508-CFTR and thus rescue its ion channel function. In China, only the research group of Professor Zhou Wenliang of Sun Yat-sen University found that naringenin has a dose-dependent activation effect on CFTR of respiratory epithelial cells; Professor Yang Hong of Liaoning Normal University led a team to screen 500 traditional Chinese herbal medicines mainly based on high-throughput screening technology, and found that naringenin has an activating effect on CFTR channels, suggesting that naringenin may develop into a lead drug for the treatment of CFTR-related diseases including bronchiectasis.

[0004] Garlic is the bulb of Allium sativum L., a plant of the genus Allium in the Liliaceae family. It is a plant with a long history of being both a medicine and food. Garlic has antibacterial, antioxidant, lipid-lowering, blood pressure-lowering, liver-protecting, anti-tumor and cardiovascular and cerebrovascular disease prevention and treatment effects, and has become a hot topic of research at home and abroad. At present, it is internationally recognized that the main active ingredients of garlic are alliin and a series of sulfur-containing compounds such as allicin (IUPAC name: 3-prop-2-enylsulfinylsulfanylprop-1-ene) produced by the reaction of alliin and alliinase. There is no allicin in whole garlic. Only when the garlic is cut, the alliinase in different subcellular locations of garlic cells contacts and reacts with alliin to produce allicin.

[0005]

[0006] As the main active ingredient of garlic, allicin has biological activities such as anti-tumor, anti-atherosclerosis, lung protection, liver protection, and killing pathogens. However, there are no reports that allicin can fully regulate CFTR channel function and affect insulin secretion levels. Summary of the invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide the use of allicin or its precursor or derivatives in the preparation of drugs for treating CFTR-mediated diseases.

[0008] To this end, the present invention provides the following technical solutions:

[0009] Use of allicin or its precursor or derivative in preparing CFTR activator or CFTR corrector.

[0010] Use of allicin or its precursor or derivative in preparing medicine for treating CFTR-mediated diseases.

[0011] Optionally, the CFTR-mediated diseases include cystic fibrosis, CF-related diabetes, pancreatic insufficiency, insulin deficiency, emphysema, chronic bronchitis, chronic obstructive pulmonary disease, chronic sinusitis, hereditary hemochromatosis, protein C deficiency, alcoholic chronic pancreatitis, idiopathic chronic pancreatitis, hereditary pancreatitis, dry eye disease, habitual constipation, secretory diarrhea, polycystic kidney disease, localized liver fibrosis, biliary cirrhosis, gallstones, hereditary angioedema type 1, familial hypercholesterolemia, type 1 chylomicronemia, lipoprotein deficiency, fucosidase deficiency, mucopolysaccharidosis, Crigler-Najjar syndrome, congenital bilateral absence of the vas deferens, congenital absence of the uterus and vagina, malignant tumor proliferation, IPEX syndrome, diabetes, Iar on dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, congenital deglycosylation disorder, hereditary emphysema, congenital hyperthyroidism, brittle bone disease, diabetes insipidus, hereditary dysfibrinogenemia, ACT deficiency, nephrogenic diabetes insipidus, Charcot-Marie-Tooth disease, Pelvic floor disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy (Steele-Rchardson-Olszewski syndrome), Pick's dementia, Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentate-rubral-pallidular Lewy body atrophy, myotonic dystrophy, hereditary Creutzfeldt-Jakob disease, Fabry disease, Steffen-Strauss syndrome, Sjögren's disease, etc.

[0012] Optionally, the malignant neoplastic growth comprises gastrointestinal cancer, prostate cancer or breast cancer.

[0013] Use of allicin or its precursor or derivative in preparing a drug for regulating CFTR in cells.

[0014] A method for activating cell CFTR, comprising contacting allicin or its precursor or derivative with the cell.

[0015] Optionally, the concentration of the allicin or its precursor or its derivative is 0.1-15 mg / ml.

[0016] A method for correcting CFTR channel dysfunction in a cell, comprising contacting the cell with allicin or its precursor or derivative.

[0017] Optionally, the concentration of the allicin or its precursor or its derivative is 0.1-15 μg / ml.

[0018] A pharmaceutical composition for treating CFTR-mediated diseases comprises allicin or its precursor or derivative and pharmaceutically acceptable excipients.

[0019] Optionally, the dosage form of the pharmaceutical composition includes a liquid dosage form and a solid dosage form;

[0020] Optionally, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, injections or ointments;

[0021] Optionally, the auxiliary materials include any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0022] The technical solution of the present invention has the following advantages:

[0023] 1. Use of allicin or its precursor or derivatives in the preparation of CFTR activators or CFTR correctors. The present invention first studies and finds that allicin has: 1. Dose-dependent activation of CFTR channel function: opening CFTR channels; 2. Correction of CFTR channel dysfunction: possibly by protecting the integrity of CFTR subunits and improving the membrane localization efficiency of CFTR subunits. Therefore, allicin or its precursor or derivatives can be used to prepare CFTR activators or CFTR correctors, and increase the level of insulin secretion through CFTR channels, and can be used to treat pancreatic insufficiency.

[0024] Furthermore, the use of allicin or its precursor or its derivative in the preparation of a drug for treating CFTR-mediated diseases. The present invention has found that allicin or its precursor or its derivative can be used to treat CFTR-mediated insulin deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is a schematic diagram of the structure of the CFTR ion channel;

[0027] Figure 2 is the distribution of CFTR ion channels in the human body;

[0028] Figure 3 It is a cell model for fluorescence detection of CFTR ion channels;

[0029] Figure 4 Lentiviral transfection CFPAC-1 identification laser confocal results;

[0030] Figure 5 Lentiviral transfection CFPAC-1 identified flow cytometry results;

[0031] Figure 6 The fluorescence quenching experiment results of different compounds affecting the iodide ion influx of CFPAC-1 / wt-hCFTR / EYFP (H408Q) cells in Example 1;

[0032] Figure 7 The fluorescence quenching experiment results of the blank control group and the inhibitor group on the iodide ion influx of CFPAC-1 / wt-hCFTR / EYFP (H408Q) cells in Example 1;

[0033] Figure 8 The MQAE experimental results of the effects of different compounds on chloride ion efflux in CFPAC-1 / wt-hCFTR / EYFP (H408Q) cells in Example 1;

[0034] Fig. 9 The fluorescence quenching experiment results of different compounds affecting the iodide ion influx of CFPAC-1 / EYFP (H408Q) cells in Example 2;

[0035] Fig.10 The MQAE experimental results of the effects of different compounds on chloride ion efflux in CFPAC-1 / EYFP (H408Q) cells in Example 2;

[0036] Fig.11 is a bar graph of CFTR gene expression levels in cells of each treatment group in Example 2;

[0037] Fig.12 It is a mosaic of the bands showing the expression level of the 105KDa band in the CFTR channel of each treatment group in Example 3;

[0038] Fig.13 yes Fig.12 Quantitative result diagram of

[0039] Fig.14 It is a statistical analysis of the effects of each treatment group on the intracellular cAMP level of CFPAC-1 cells. DETAILED DESCRIPTION

[0040] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0041] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0042] As used herein, the CFTR ion channel is a symmetrical, multidomain protein structure consisting of five functional domains (see Figure 1 ): two hydrophobic transmembrane domains (MSDs), each of which includes six transmembrane helices, which determine the formation of chloride ion selective pores; two hydrophilic nucleotide binding domains (NBDs), which control the opening and closing of the channel by binding-hydrolyzing ATP cycle reactions; a regulatory domain (R Domain), located in the middle, these parts are integrated together to form the CFTR channel. (Xu Lina, 2008).

[0043] As used herein, the terms "activate CFTR channels" and "open CFTR channels" can be used interchangeably and refer to causing the normally structured CFTR channels to function as chloride ion channels, releasing chloride ions to the luminal surface of epithelial cells, mediating fluid transport and maintaining the balance of electrolytes and potentials inside and outside the cells by increasing the concentration of chloride ions in the tubular cavity. The iodide ion influx test and the chloride ion efflux test can verify the principle of allicin activating CFTR channels: the experiment used a CFPAC-1 cell model co-transfected with a wild-type CFTR channel and halogen ion-sensitive EYFP (enhanced yellow fluorescence protein). Based on the characteristic that fluorescent proteins are sensitive to halogen ions, 137 mM isotonic solution containing iodide ions was injected into the extracellular space. If the channel is open, a fluorescence quenching signal can be detected. This change in fluorescence intensity can indirectly reflect the channel's ion transport, that is, the activity of the channel. See the cell model for fluorescence detection of CFTR ion channels in the iodide ion influx test. Figure 3 .

[0044] As used herein, the terms "correcting CFTR channels" and "protecting CFTR integrity" mainly refer to overcoming CFTR subunit transport barriers, preventing endoplasmic reticulum degradation of misfolded protein subunits, protecting the integrity of channel proteins, and ultimately enabling the expression of partially active mutant channels on the cell surface. Among them, ΔF508-CFTR is the main form of this type of mutation, and it is also the most important of all mutation forms, accounting for about 90%. Since the function of ΔF508 mutation is still impaired after being placed on the membrane, its channel activity must be corrected. The iodide ion influx test and the chloride ion efflux test can verify the principle of allicin protecting CFTR integrity and / or correcting CFTR channels: the CFPAC-1 cell model transfected with halogen ion-sensitive EYFP (enhanced yellow fluorescence protein) was used in the experiment. After 24 hours of induction treatment with the test compound, according to the characteristics of fluorescent protein being sensitive to halogen ions, 137mM isotonic solution containing iodine ions was injected into the cell extracellularly. If the channel is open, a fluorescence quenching signal can be detected. The change in this fluorescence intensity can indirectly reflect the channel's ion transport, that is, the activity of the channel.

[0045] The cell platform involved in the following embodiments: CFPAC-1 cells (human pancreatic cancer cells; the cells are derived from the liver metastasis area of ​​pancreatic ductal carcinoma of a cystic fibrosis patient, and express carcinoembryonic antigen, pancreatic carcinoma fetal antigen, and pancreatic cancer-associated antigen. The cells exhibit ion transport activity consistent with cystic fibrosis, express the cystic fibrosis transmembrane regulator, i.e., CFTR, and carry the most common CFTR mutation: ΔF508.

[0046] Cell model: CFPAC-1 / wt-hCFTR / EYFP (H408Q): used to test the activation effect of allicin or its precursors or derivatives on CFTR channels. Construction method: The cell model was constructed using lentiviral transfection technology, and all steps were carried out according to the instructions for lentiviral transfection of GeneCare.

[0047] CFPAC-1 / EYFP (H408Q): used to test the effect of allicin or its precursor or derivative on CFTR (ΔF508), and the effect of allicin or its precursor or derivative on correcting the function of CFTR protein channel. Construction method: The cell model was constructed using lentiviral transfection technology, and all steps were carried out according to the instructions for lentiviral transfection of GeneCare.

[0048] The cell model constructed above was identified by laser confocal microscopy and flow cytometry. The results of laser confocal microscopy identification of EYFP expression were as follows: Figure 4As shown in Figure 1, laser confocal imaging showed that EYFP was successfully transfected into CFPAC-1 cells and expressed normally (the white area was originally green fluorescence). At the same time, the cell model constructed above was identified by flow cytometry, and the identification results were as follows: Figure 5 As shown, flow cytometry results showed that EYFP was successfully transfected into CFPAC-1 cells and expressed normally. The above results indicate that both cell models were successfully constructed.

[0049] Example 1 Allicin activates or opens CFTR channels

[0050] 1. Cell model: CFPAC-1 / wt-hCFTR / EYFP(H408Q).

[0051] 2. Test system: forskolin + test compound; the test compounds and their concentrations in the test system are: alliinase group (10-15 mg / ml), alliin group (0.1-15 mg / ml), allicin group (0.1-15 mg / ml), CFTR inhibitor: CFTRinh-172 or CFTRinh-101.

[0052] 3. Experimental methods and results:

[0053] (1) Iodide ion influx test and results

[0054] The method for determining the activator is as follows: CFPAC-1 / wt-hCFTR / EYFP (H408Q) cells are plated on a black-walled, transparent-bottomed 96-well plate (Corning-Costar 3603, NK, USA) at a density of 20,000 cells per well. The culture medium is DMEM complete culture medium (containing 10% fetal bovine serum, 2mM glutamine, and 500U / ml penicillin / streptomycin). The cells are cultured in a carbon dioxide incubator (37°C, 90% humidity, 5% CO2 concentration, and 95% air concentration). After normal culture at 37°C until a monolayer cell membrane is formed, the test can be performed. 50 μl of a mixture of a PBS solution containing the test compound (concentration is as shown in the test system) and 10 μM forskolin is added to each well. After incubation at 37°C for 10 minutes, the kinetics of the quenching of intracellular EYFP fluorescence by the influx of iodide ions through the CFTR chloride ion channel is measured using a fluorescence detector. At the same time, a control group is set up as follows:

[0055] Blank control group: no test compound was added.

[0056] DMSO group: 50 μl DMSO was added (final concentration: 0.5%).

[0057] Allicin group: 50 μl of a mixture of allicin (final concentrations of 0.5, 2, 4, and 8 mg / ml, respectively) and forskolin (final concentration of 10 μM) in PBS was added to each well.

[0058] Alliinase group: 50 μl of a mixture of alliinase (final concentration of 5 mg / ml) and forskolin (final concentration of 10 μM) in PBS solution was added to each well.

[0059] Alliin group: 50 μl of a mixture of alliin (final concentrations of 2.5 and 5 mg / ml, respectively) and forskolin (final concentration of 10 μM) in PBS solution was added to each well.

[0060] Inhibitor 172 experimental group: 50 μl of a mixture of PBS solution containing allicin (concentration of 2 mg / ml), CFTRinh-172 (concentration of 20 μM) and forskolin (concentration of 10 μM) was added to each well.

[0061] Inhibitor 101 experimental group: 50 μl of a mixture of PBS solution containing allicin (concentration of 2 mg / ml), CFTRinh-101 (concentration of 20 μM) and forskolin (concentration of 10 μM) was added to each well.

[0062] Each well was one replicate, and each group corresponded to 3-6 replicates.

[0063] The fluorescence analysis method is: use Varioskan Flash full wavelength scanning reader to record the fluorescence change value: 5 consecutive complete readings are used as the baseline, then 120μl PBS containing 137mM NaI (instead of NaCl) is quickly injected into the cell culture well, and 15-25 readings are continued. The activity of CFTR channel on the cell membrane for iodide ion transport is directly reflected by the fluorescence value and time change curve.

[0064] The results are as follows Figure 6-Figure 7 As shown, the following results can be obtained:

[0065] a. Figure 6 In the figure, the larger the fluorescence quenching difference, the stronger the activation effect of the compound on the CFTR channel. It can be seen that a certain concentration of allicin can activate the CFTR channel function in a dose-dependent manner; alliin also has the ability to activate the CFTR channel, but not as significantly as allicin; alliinase has no activation effect.

[0066] b. Figure 7It can be seen that there is almost no fluorescence quenching. This activation effect is independent of the reaction system and the use of solvents such as DMSO, and can be inhibited by CFTR-specific inhibitors. It is thus determined that the fluorescence quenching caused by the influx of I ions is indeed caused by the activation of CFTR channels by garlic sulfur-containing compounds or their precursors or derivatives.

[0067] c. Since the allicin solution itself is a light yellow solution, the fluorescence baseline continues to increase with the increase in concentration, and the CFTR inhibitor can only inhibit the inhibition of 2mg / ml allicin. Allicin treatment above 2mg / ml may greatly increase cell membrane permeability or activate other ion channel functions, so no in-depth screening was performed. Therefore, in all subsequent inhibitor verification experiments, the highest concentration of allicin was 2mg / ml.

[0068] (2) Chloride ion efflux test and results - MQAE test

[0069] MQAE is the most widely used new type of chloride ion fluorescent probe, which can be used to detect the intracellular chloride ion concentration. Its fluorescence intensity decreases proportionally with the increase of chloride ions in the cell.

[0070] The determination method is as follows: 1.5 ml of CFPAC-1 / wt-hCFTR / EYFP (H408Q) cell solution is inoculated in a laser confocal dish, and DMEM complete culture medium (containing 10% fetal bovine serum, 2 mM glutamine, 500 U / ml penicillin / streptomycin) is used, and the cells are normally cultured in a carbon dioxide incubator (37°C, humidity of 90%, CO2 concentration of 5%, air concentration of 95%) until a monolayer cell membrane is formed, and then the CFPAC-1 / wt-hCFTR / EYFP (H408Q) cells are treated with a gradient concentration (final concentration of 0.1-15 mg / ml) of allicin solution for 10 minutes, and then MQAE is used to develop intracellular chloride ions and the results are recorded by laser confocal. At the same time, a blank control group is set up, and an equal volume of complete culture medium is added to the blank control group. At the same time, a CFTRinh-172+allicin group was set up, and a mixed solution containing allicin (final concentration of 0.1-15 mg / ml) + CFTRinh-172 (final concentration of 20 μM) was added. At the same time, a CFTRinh-101+allicin group was set up, and a mixed solution containing allicin (final concentration of 0.1-15 mg / ml) + CFTRinh-101 (final concentration of 20 μM) was added. Each concentration of the test compound corresponds to 3 replicates.

[0071] The results are as follows Figure 8As shown in the figure, with the increase of allicin concentration, the intracellular chloride ion concentration decreased significantly, and the chloride ion efflux induced by 2 mg / ml allicin could be completely blocked by pretreatment with CFTR-specific inhibitor, proving that allicin mediates the reduction of intracellular chloride ion levels by activating CFTR channels (the white area was originally blue fluorescence, and the higher the fluorescence intensity, the lower the intracellular chloride ion concentration).

[0072] Example 2 Allicin corrects ΔF508-CFTR channel function

[0073] 1. Cell model: CFPAC-1 / EYFP (H408Q)

[0074] 2. Preparation of cell induction model: CFPAC-1 / EYFP (H408Q) cell model was used in the experiment. After 24 hours of induction treatment with 0.1-15 μg / ml (final concentration) of allicin, based on the characteristics of fluorescent protein being sensitive to halogen ions, recording the changes in fluorescence intensity can indirectly reflect the transport of ions by the channel.

[0075] 3. Test system: forskolin + test compound; test compound and concentration: allicin group (2 mg / ml), standard CFTR inhibitors: CFTRinh-172 and CFTRinh-101.

[0076] IV. Experimental Methods

[0077] 1) Iodide ion influx test and results

[0078] The method is the same as "(1) Iodide ion influx test and results" in Example 1. The method is as follows: CFPAC-1 / EYFP (H408Q) cells (induced cell model or uninduced cell model, 5 μg / ml allicin induction) are plated on a 96-well plate with a black wall and a transparent bottom (Corning-Costar 3603, NK, USA) at a density of 20,000 cells per well. The culture medium is DMEM complete culture medium (containing 10% fetal bovine serum, 2 mM glutamine, 500 U / ml penicillin / streptomycin). The cells are cultured in a carbon dioxide incubator (37°C, 90% humidity, 5% CO2 concentration, 95% air concentration). After normal culture at 37°C until about 80% confluence is formed, 0.1-15 μg / ml of the test compound is added for 24 hours of induction treatment before detection. Add 50 μl of a mixture of the test compound (concentration as shown in the test system) and 10 μM forskolin in PBS to each well, incubate at 37°C for 10 minutes, and use a fluorescence detector to measure the kinetics of iodide ion influx through the CFTR chloride ion channel on the quenching of intracellular EYFP fluorescence. At the same time, set up a control group as follows:

[0079] Blank control group: no test compound was added.

[0080] Allicin group: 50 μl of a mixture of allicin (final concentration of 2 mg / ml) and forskolin (final concentration of 10 μM) in PBS solution was added to each well.

[0081] Inhibitor 172 experimental group: 50 μl of a mixture of PBS solution containing allicin (concentration of 2 mg / ml), CFTRinh-172 (concentration of 20 μM) and forskolin (concentration of 10 μM) was added to each well.

[0082] Inhibitor 101 experimental group: 50 μl of a mixture of PBS solution containing allicin (concentration of 2 mg / ml), CFTRinh-101 (concentration of 20 μM) and forskolin (concentration of 10 μM) was added to each well.

[0083] Each well was one replicate, and each group corresponded to 3-6 replicates.

[0084] The fluorescence analysis method is: use Varioskan Flash full wavelength scanning reader to record the fluorescence change value: 5 consecutive complete readings are used as the baseline, then 120μl PBS containing 137mM NaI (instead of NaCl) is quickly injected into the cell culture well, and 15-25 readings are continued. The activity of CFTR channel on the cell membrane for iodide ion transport is directly reflected by the fluorescence value and time change curve.

[0085] The results are as follows Fig. 9 As shown, only the CFTR function of the induced group was activated by 2mg / ml allicin and fluorescence quenching occurred. Incubation with 5ug / ml allicin solution restored the function of some CFTR channels in CFPAC-1 cells.

[0086] (2) Chloride ion efflux test and results - MQAE test

[0087] The method is the same as "(2), chloride ion efflux test and results - MQAE test" in Example 1, specifically: 1.5 ml of CFPAC-1 / EYFP (H408Q) cell fluid (induced or uninduced) was inoculated in a laser confocal dish, and DMEM complete culture medium (containing 10% fetal bovine serum, 2mM glutamine, 500U / ml penicillin / streptomycin) was used. After normal culture in a carbon dioxide incubator (37°C, humidity of 90%, CO2 concentration of 5%, air concentration of 95%) until 80% confluence, 0.1-15μg / ml of the test compound was added for 24 hours of induction treatment before detection. After treating CFPAC-1 / EYFP (H408Q) cells with a gradient concentration (final concentration of 0.1-15mg / ml) of allicin solution for 10 minutes, MQAE was used to develop intracellular chloride ions and the results were recorded using laser confocal. At the same time, a blank control group was set up, and an equal volume of complete culture medium was added to the blank control group. The CFTRinh-172+allicin group was added with a mixed solution containing allicin (final concentrations of 1 and 2 mg / ml, respectively) + CFTRinh-172 (final concentration of 20 μM). At the same time, the CFTRinh-101+allicin group was set up, and a mixed solution containing allicin (final concentrations of 1 and 2 mg / ml, respectively) + CFTRinh-101 (final concentration of 20 μM) was added. Each well was a replicate, and each concentration of the test compound corresponded to 3 replicates.

[0088] The results are as follows Figure 10-11 As shown, from Fig.10 It can be seen that only the CFTR function of the induced group was activated by 1-2 mg / ml allicin, causing chloride ion efflux and inhibited by the CFTR specific inhibitor, indicating that 24 hours of incubation with 1-2 mg / ml allicin can correct the ΔF508-CFTR dysfunction (the white area was originally blue fluorescence, and the higher the fluorescence intensity, the lower the intracellular chloride ion concentration). Fig.11 It can be seen that the 24-hour induction of concentration gradient allicin has no significant effect on the relative expression level of CFTR channel mRNA (P>0.05). The above shows that 24-hour induction of 0.1-15μg / ml allicin solution can correct the ΔF508-CFTR channel function of CFPAC-1 cells.

[0089] Example 3 Allicin protects CFTR protein integrity and corrects CFTR channel function This example investigates the effect of concentration gradient allicin (final concentration 0-15ug / ml) on CFTR protein transcription level and translation level / post-translational stability after inducing CFPAC-1 (same cell culture conditions as in Example 1 and Example 2) for 24 hours, using β-actin as the internal reference gene. The specific method is: 3-5ml CFPAC-1 cell fluid is inoculated in a 25ml screw-top cell culture bottle, and the cell confluence reaches about 80% normally. After adding concentration gradient allicin (final concentration 0-15ug / ml) and inducing at 37°C for 24 hours, CFTR protein subunits (105Kda) are detected by fluorescence quantitative PCR (using Merck SYBR Green I kit to accurately analyze the number of starting templates of CFTR) and Western blotting (WB).

[0090] 1) The results of fluorescence quantitative PCR are shown in the following table. Concentrations 1-5 in the table correspond to 2.5, 5, 10, 20, and 40 ug / ml, respectively.

[0091] Table 1. Analysis of CFTR gene expression levels in cells of each group ( n=9)

[0092]

[0093] Note: There is no significant difference among the groups.

[0094] 2) WB results are as follows Figure 12-13 and the following table.

[0095] Table 2. Analysis of protein expression levels in human pancreatic cancer cells ( n=3)

[0096]

[0097] Note: △Compared with the blank group, P<0.05; Compared with concentration 1, P<0.05.

[0098] From the above results, it can be seen that the 24-hour induction of low-concentration allicin solution did not increase the transcriptional expression level of CFTR, but increased the stability of the 105KDa protein subunit of the CFTR channel and maintained the integrity of the CFTR channel. It is reasonable to speculate that allicin therefore produces the effect of correcting the ΔF508-CFTR channel function in Example 3.

[0099] Example 4 Effect of allicin on cAMP levels in CFPAC-1 cells

[0100] This example investigates the effect of a 0-15 mg / ml allicin solution on the intracellular cAMP level of CFPAC-1 cells. The method is as follows: CFPAC-1 cells are plated on a 6-well cell culture plate at a density of 200,000 cells per well, and the culture medium is DMEM complete culture medium (containing 10% fetal bovine serum, 2 mM glutamine, 500 U / ml penicillin / streptomycin), and cultured in a carbon dioxide incubator (37°C, 90% humidity, 5% CO2 concentration, 95% air concentration) until film formation, and then 1.5 ml of the test system is added and incubated for 10-20 min according to the following experimental groups, and the specific groups are as follows:

[0101] Blank control group: no test compound was added.

[0102] Forskolin group: PBS solution containing forskolin (FSK) at a final concentration of 10 μM.

[0103] Allicin group: PBS solution containing allicin (AC) at a final concentration of 2 mg / ml.

[0104] Forskolin + allicin group: containing a final concentration of 10 μ M forskolin and a mixture of allicin with a final concentration of 2 mg / ml in PBS.

[0105] Forskolin + phosphodiesterase inhibitor group: a PBS solution containing a mixture of forskolin with a final concentration of 10 μM and a phosphodiesterase inhibitor (IBMX) at a working concentration.

[0106] Then the cells were treated with the above groups, and the intracellular cAMP level of CFPAC-1 cells was detected using the R&D Systems kit KGE012B.

[0107] The results are as follows Fig.14 As shown, the results of intracellular cAMP levels in CFPAC-1 cells of each group showed that 2 mg / ml allicin treatment significantly increased the intracellular cAMP level in CFPAC-1 cells and mediated the efflux of chloride ions or the influx of iodide ions in the cells.

[0108] In summary, we can draw the following conclusions:

[0109] 1. High concentration of allicin for a short time (10-30 min) can activate CFTR channel function in a dose-dependent manner:

[0110] ≤2mg / ml activates CFTR channels by increasing intracellular cAMP concentration and mediates intracellular chloride ion efflux.

[0111] ≥2mg / ml may increase cell membrane permeability / open other ion channels.

[0112] 2. Low concentration of allicin (5ug / ml) for 24 hours can correct CFTR channel dysfunction: it may protect the integrity of CFTR subunits and improve the efficiency of CFTR subunit membrane localization.

[0113] 3. Allicin is the first compound that exhibits dual functions of "activator" and "corrector".

[0114] Example 5 Allicin increases insulin secretion level in Min6 cells

[0115] 1. Cell model: Min6 cell line (pancreatic β cells). Min6 cell line responds well to external stimulation and glucose-stimulated insulin secretion, and its secretion curve is similar to that of the human body. Therefore, it is often used for experiments on glucose-stimulated insulin secretion and detection of pancreatic β cell function and pharmacodynamics.

[0116] 2. Test system: 2.0-25.0 mM glucose + test compound; the test compound and concentration in the test system are: allicin group (0.5-2 mg / ml), standard CFTR inhibitor: CFTRinh-172 (20 μM).

[0117] 3. Experimental methods and results: The conventional glucose-stimulated insulin secretion (GSIS) experimental method was used. The specific operation was as follows: Min6 cells grown to the logarithmic growth phase were digested with 0.25% trypsin, and then high-glucose DMEM medium containing 15% fetal bovine serum was added to terminate the digestion. Then, single-cell suspension was made by blowing and inoculating in a 24-well cell culture plate at a density of 20,000 per well, and cultured in a carbon dioxide incubator (37°C, 90% humidity, 5% CO2 concentration, and 95% air concentration). After the cells adhered to the wall, they were switched to serum-free culture medium for starvation culture for 2 hours, and then each group of test systems was added and incubated for 10 to 30 minutes. The specific groups are as follows:

[0118] Blank control group: Min6 cells were not added with any test compound.

[0119] High-glucose injury model group: Min6 cells were cultured in 35 mM glucose solution for 24 hours to induce injury.

[0120] Blank control-allicin group: 1.0 ml of PBS solution containing allicin (final concentration of 1 mg / ml) was added to each well of Min6 cells.

[0121] High glucose injury model-allicin group: After Min6 cells were cultured in 35 mM glucose solution for 24 hours to induce injury, 1.0 ml of PBS solution containing allicin (final concentration of 1 mg / ml) was added to each well.

[0122] High glucose injury model - allicin-inhibitor group: After Min6 cells were cultured in 35 mM glucose solution for 24 hours to induce injury, 1.0 ml of a mixture of allicin (concentration of 1 mg / ml) and CFTRinh-172 (concentration of 20 μM) PBS solution was added to each well.

[0123] Each well was a replicate, and each group corresponded to 3 replicates.

[0124] After the incubation, the culture medium containing the test compound was discarded, and the cells were washed three times with 0.5 ml Kreb's solution (135 mM NaCl, 3.6 mM KCl, 5 mM NaH2PO4, 0.5 mM MgCl2, 2.5 mM CaCl2, 2 mM NaH2CO3, 10 mM HEPES (pH 7.4), 0.1% BSA), and then low-glucose Kreb's solution (2.0 mM glucose, 0.1% BSA) was added and incubated at 37°C for 1 hour. After the incubation, the cell supernatant was collected with a pipette, and then Kreb's solution containing 25.0 mM glucose was added and incubated for 30 minutes. After the incubation, the cell supernatant was collected, and the insulin level was detected using an ELISA kit from Mercodia.

[0125] Experimental results:

[0126] (1) Under 2.0mM glucose, the insulin secretion of the blank control group, high glucose injury model group and high glucose injury model-allicin group (final concentration of 1mg / ml) were: control group 7.31±0.55ug / l, high glucose injury model group 5.34±0.61ug / l, high glucose injury model-allicin group 6.84±0.99ug / l. After treatment with 20uM inhibitor, the insulin level of the high glucose injury model-allicin-inhibitor group decreased to 6.21±1.05ug / l. It can be seen that under low glucose, the total insulin of the high glucose injury model-allicin group was significantly higher than that of the high glucose injury model group (P<0.05), close to the blank control group, and related to the CFTR channel. At the same time, allicin treatment did not affect the insulin secretion of normal Min6 cells under low glucose treatment (7.26±0.47ug / l), all P<0.05, and the difference was statistically significant; see the table below for details:

[0127] Table 3. Insulin secretion of each group under 2.0 mM glucose

[0128] Group Insulin secretion ug / l Blank control group 7.31±0.55ug / l* High glucose injury model group 5.34±0.61ug / l High glucose injury model- allicin group 6.84±0.99ug / l* High glucose injury model-allicin-inhibitor group 6.21±1.05ug / l

[0129] *Indicates that compared with the high-glucose injury model group, P<0.05, the difference is statistically significant.

[0130] (2) Under 25.0 mM glucose, the insulin secretion levels of the blank control group, high glucose injury model group and high glucose injury model-allicin group (AC concentration was 1 mg / ml) were: 12.33±0.81 ug / l for the control group, 7.97±0.94 ug / l for the high glucose injury model-allicin group, and 9.18±0.58 ug / l for the high glucose injury model-allicin group. After treatment with 20 uM inhibitor, the insulin level in the high glucose injury model-allicin-inhibitor group decreased to 8.41±1.01 ug / l. It can be seen that under high glucose conditions, the total insulin level in the high glucose injury model-allicin group was significantly higher than that in the high glucose injury model group (P<0.05), and was related to the CFTR channel. At the same time, allicin treatment did not affect the insulin secretion of normal Min6 cells under high glucose treatment (11.94±0.93 ug / l), all P<0.05, and the differences were statistically significant.

[0131] Table 4. Insulin secretion of each group under 25.0 mM glucose

[0132] Group Insulin secretion ug / l Blank control group 12.33±0.81ug / l* High glucose injury model group 7.97±0.94ug / l High glucose injury model- allicin group 9.18±0.58ug / l* High glucose injury model-allicin-inhibitor group 8.41±1.01ug / l

[0133] *Indicates that compared with the high-glucose injury model group, P<0.05, the difference is statistically significant.

[0134] (3) The total amount of insulin in the blank control group with 25.0 mM glucose was significantly higher than that in the blank control group with 2.0 mM glucose, P < 0.001, which proved that the Min6 cells used in this experiment had good function.

[0135] in conclusion:

[0136] (1) Under low-sugar and high-sugar conditions, allicin can significantly increase the insulin secretion of Min6 cells after high-sugar injury.

[0137] (2) 0.5-2 mg / ml allicin can increase the insulin secretion of Min6 cells after high glucose injury through CFTR channel in a dose-dependent manner.

[0138] It is explained that the allicin or its precursor or derivatives involved in the present invention can be used to activate insulin secretion function and treat pancreatic insufficiency and insulin deficiency.

[0139] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of allicin or its precursor or derivative in the preparation of CFTR activator or CFTR corrector.

2. Use of allicin or its precursor or derivative in the preparation of drugs for treating CFTR-mediated related diseases.

3. The use according to claim 2, characterized in that: The CFTR-mediated related diseases include cystic fibrosis, CF-related diabetes, pancreatic insufficiency, insulin deficiency, emphysema, chronic bronchitis, chronic obstructive pulmonary disease, chronic sinusitis, hereditary hemochromatosis, protein C deficiency, alcoholic chronic pancreatitis, idiopathic chronic pancreatitis, hereditary pancreatitis, dry eye disease, habitual constipation, secretory diarrhea, polycystic kidney disease, localized liver fibrosis, biliary cirrhosis, gallstones, hereditary angioedema type 1, familial hypercholesterolemia, type 1 chylomicronemia, lipoprotein deficiency, fucosidase deficiency, mucopolysaccharidosis, Crigler-Najjar syndrome, congenital bilateral absence of the vas deferens, congenital absence of the uterus and vagina, malignant tumor proliferation, IPEX syndrome, diabetes, Iar on dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, congenital deglycosylation disorder, hereditary emphysema, congenital hyperthyroidism, brittle bone disease, diabetes insipidus, hereditary dysfibrinogenemia, ACT deficiency, nephrogenic diabetes insipidus, Charcot-Marie-Tooth disease, Pelvic floor disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy (Steele-Rchardson-Olszewski syndrome), Pick's dementia, Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidum Lewy body atrophy, myotonic dystrophy, hereditary Creutzfeldt-Jakob disease, Fabry disease, Steffen-Strauss syndrome, or Sjögren's disease; Optionally, the malignant neoplastic growth comprises gastrointestinal cancer, prostate cancer or breast cancer.

4. Use of allicin or its precursor or derivative in the preparation of drugs for regulating CFTR in cells.

5. A method for activating cell CFTR, characterized in that: The allicin or its precursor or derivative according to claim 1 is contacted with cells.

6. The method for activating cell CFTR according to claim 5, characterized in that: The concentration of the allicin or its precursor or its derivative is 0.1-15 mg / ml; Optionally, allicin or its precursor or derivative comprises alliin.

7. A method for correcting cellular CFTR channel dysfunction, characterized in that: The allicin or its precursor or derivative according to claim 1 is contacted with cells.

8. The method for correcting cellular CFTR channel dysfunction according to claim 7, characterized in that: The concentration of the allicin or its precursor or its derivative is 0.1-15 μg / ml.

9. A pharmaceutical composition for treating CFTR-mediated diseases, characterized in that: The invention comprises the allicin or its precursor or derivative as claimed in claim 1 and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The dosage form of the pharmaceutical composition includes a liquid dosage form and a solid dosage form; Optionally, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, injections or ointments; Optionally, the auxiliary materials include any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

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