CN114681438A - Application of metformin and other guanidine-containing compounds in reversing Gal-10 crystallization tendency and relieving related diseases - Google Patents
Application of metformin and other guanidine-containing compounds in reversing Gal-10 crystallization tendency and relieving related diseases Download PDFInfo
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- CN114681438A CN114681438A CN202210534642.4A CN202210534642A CN114681438A CN 114681438 A CN114681438 A CN 114681438A CN 202210534642 A CN202210534642 A CN 202210534642A CN 114681438 A CN114681438 A CN 114681438A
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- A61K31/13—Amines
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Abstract
The invention belongs to the field of biological medicine, and particularly relates to application of metformin and other guanidine-containing compounds in reversing Gal-10 crystallization tendency and relieving related diseases. The Gal-10 crystal related diseases comprise infectious diseases and inflammatory diseases.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of a guanidine-containing compound in reversing Gal-10 crystallization tendency and relieving related diseases.
Background
Various functions in the human body often require different proteins to perform. These proteins, either dissolved in body fluids or bound to membrane structures such as cell membranes, rarely form crystals. Abnormal protein crystals produced in human cells are often of a pathological nature.
In 1853, Charcot and Robin found a morphologically diverse deposit of crystals in the blood and spleen of a leukemia patient, followed by 1872, Leyden found the same crystals in the sputum of an asthma patient, also known as Charcot-Leyden crystals (CLCs, Charcot-Leyden crystals). The Xiacao-Leyten crystal is a rhombic colorless transparent compass-like crystal, has long tips at two ends, different sizes and strong refractivity, and is formed by fusing eosinophilic granules after eosinophilic granulocyte is broken. Initially, CLCs were considered inorganic crystals and were not confirmed to consist of protein crystals until 1950. Studies have shown that the major component in CLCs is galectin-10 (Gal-10). Gal-10 is a protein that is very abundant in eosinophils and basophils, and its formation is closely associated with the release of eosinophil extracellular traps, as well as with a variety of diseases including, but not limited to, infectious diseases: suppurative lymphadenitis, eosinophilic cystitis, liver abscess, etc., inflammatory diseases: asthma, allergic rhinitis, eosinophilic colitis, etc. The treatment of these diseases usually has different treatment methods aiming at different focuses, and there are few therapeutic drugs targeting the marker CLCs/Gal-10 protein.
At present, the targeted regulatory molecule aiming at Gal-10 only has one artificial monoclonal antibody, and the monoclonal antibody of the crystal can dissolve the crystal in vitro and can inhibit the natural lung immune response of mice caused by CLCs. However, the monoclonal antibody drugs are difficult to solve due to high cost, poor stability, difficult transportation of subsequent drugs, harsh storage conditions and the like, and the subsequent development of the drugs is difficult.
Compared with monoclonal antibody medicines, the organic micromolecule medicines have the advantages of simple structure, good medicine forming property and easiness in industrial production. It is crucial to find small molecules that regulate Gal-10 assembly and inhibit its pathological role.
Disclosure of Invention
The invention aims to screen a small-molecule inhibitor targeting CLCs, and further develop a potential drug capable of regulating and controlling CLCs assembly and inhibiting related inflammation.
In a first aspect, the present invention provides the use of a guanidine-containing compound for solubilizing crystals of Gal-10 and for the manufacture of a medicament for a disease associated with crystals of Gal-10.
Preferably, the guanidine-containing compound is represented by the following general formula:
wherein R is1、R2、R3、R4、R5、R6Each independently may be a hydrogen atom or a substituent.
Preferably, the substituents include, but are not limited to, the following substituents and heteroatom-containing substituents that are common: alkyl, guanidino, diazo, carboxyl, sulfonic acid group, hydrocarbyloxycarbonyl, formyl, haloformyl, oxo, carbamoyl, cyano, phenolic alkyl, phenolic hydroxyl, alcoholic hydroxyl, amino, hydrocarbyloxy, nitro, nitroso, mercapto, amino, nitro, acyl, silicon, acyloxy, oxyacylA group, a borono group, a hydroxyamino group, a nitroso group, a silyl group; the substituents may also include C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Cycloalkyl, aryl, heteroaryl, heterocyclyl- (CH2) n-, aryl-C1-6Alkyl-, heteroaryl-C1-6Alkyl-, aryl- (CH)2) n-O-, heteroaryl- (CH)2)n-O-、C3-8cycloalkyl-C (O) -, heterocyclyl-C (O) -, aryl-C (O) -, or heteroaryl-C (O) -, wherein C1-6Alkyl radical, C1-6Alkoxy radical, C3-8Cycloalkyl, aryl, heterocyclyl- (CH)2) n-, aryl-C1-6Alkyl-, heteroaryl-C1-6Alkyl-, aryl- (CH)2) n-O-, heteroaryl- (CH)2)n-O-、C3-8cycloalkyl-C (O) -, heterocyclyl-C (O) -, aryl-C (O) -.
Preferably, the substituent contains a carbon atom, and the number of carbon atoms thereof is not particularly limited;
preferably, the substituent contains 1 to 20 carbon atoms; specifically, the carbon atoms include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 carbon atoms.
Preferably, the hydrocarbyl group includes, but is not limited to, methyl (-CH)3) Ethyl (-C)2H5) Propyl (-C)3H7) Butyl (-C)4H9) Pentyl group (-C)5H11)。
Preferably, the guanidine-containing compound comprises metformin, 1-methylguanidine, 1-dimethylguanidine, 1,3, 3-tetramethylguanidine, 1-ethylguanidine, 1-phenylbiguanide, 1- (o-tolyl) biguanide, streptomycin, cimetidine.
Preferably, the guanidine-containing compound is metformin, which has the following structural formula:
preferably, the guanidine-containing compound is 1-methylguanidine, the structural formula of the 1-methylguanidine is as follows:
preferably, the guanidine-containing compound is 1, 1-dimethylguanidine, and the structural formula of the 1, 1-dimethylguanidine is as follows:
preferably, the guanidine-containing compound is 1,1,3, 3-tetramethylguanidine, the structure of which is 1,1,3, 3-tetramethylguanidine
The formula is as follows:
preferably, the guanidine-containing compound is 1-ethylguanidine, the structural formula of the 1-ethylguanidine is as follows:
preferably, the guanidine-containing compound is 1-phenyl biguanide, the structural formula of the 1-phenyl biguanide being as follows:
preferably, the guanidine-containing compound is 1- (o-tolyl) biguanide, the structural formula of which is as follows:
preferably, the guanidine-containing compound is streptomycin, which has the following structural formula:
preferably, the guanidine-containing compound is cimetidine, which has the formula:
the terms "Gal-10 crystals", "galectin-10", "Charcot-leyden crystals", "CLCs", "CLC crystals" as used herein are used interchangeably and refer to crystals formed from galectin-10 (Gal-10). The crystals formed from galectin-10 are generally biconical hexagonal crystals, approximately 20-40 μm in length and approximately 2-4 μm in width. The term "Gal-10 crystals" is broad enough to cover both human proteins and homologues of any species.
The "dissolution of Gal-10 crystals" in the invention comprises reversing the crystallization tendency of Gal-10, reducing the crystallization speed of Gal-10 and increasing the dissolution rate of Gal-10.
Preferably, the Gal-10 crystal related diseases comprise infectious diseases and inflammatory diseases.
Preferably, the etiological agent of the infectious disease includes bacteria, mycoplasma, chlamydia, mycobacteria, fungi, viruses, parasites, and the like. Illustratively, the infectious diseases include suppurative lymphadenitis, eosinophilic cystitis, liver abscess, etc.
Preferably, the inflammation comprises any inflammation well known in the art, as demonstrated by the specific examples of the present invention, including inflammation that causes an increase in IL-1 β, IL-6, TNF- α, CCL-2 at the gene level upon stimulation with CLCs (Gal-10 crystals), in particular, such as asthma, rhinitis, colitis, and the like.
Preferably, the inflammation may be caused by allergy.
Preferably, the colitis is eosinophilic colitis.
In another aspect, the present invention provides a pharmaceutical composition comprising a guanidine-containing compound as described above.
More specifically, the guanidine-containing compounds are useful as active ingredients in pharmaceutical compositions.
The pharmaceutical composition of the present invention may be administered by any of the following means: oral, aerosol inhalation, rectal, nasal, buccal, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intravenous administration.
Preferably, the pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, lozenges, syrups, liquids (solutions), emulsions, suspensions, controlled release preparations, aerosols, films, injections, intravenous drip, transdermal preparations, ointments, lotions, adhesive preparations, suppositories, pellets, nasal preparations, pulmonary preparations, eye drops and the like, oral or parenteral preparations.
As described in the embodiments of the present invention, the pharmaceutical composition may be a liquid, the solvent is PBS (phosphate buffer saline, also referred to as PBS in the embodiments), and the guanidine-containing compound may be configured into various concentrations of solutions according to its solubility, specifically, the concentrations are expressed in molar concentration. The liquid may also comprise any pharmaceutically acceptable solvent, and the liquid may also contain other pharmaceutically common ingredients.
Pbs is phosphate buffered saline solution, usually as solvent, acting as dissolution protection agent, and is the most widely used buffer in biochemical research, with Na as the main component2HPO4、KH2PO4NaCl and KCl due to Na2HPO4And KH2PO4They have secondary dissociation, and the pH value range of buffering is wide; while NaCl and KCl mainly act to increase the salt ion concentration. The buffered pH of the PBS was wide ranging, as the pH of the PBS was 7.4, as used herein.
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
Preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the national food and drug administration for use in humans or livestock.
The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.
In another aspect, the invention provides a method of treating a Gal-10 crystal-related disease, the method comprising administering to a subject a guanidine-containing compound of the invention.
Preferably, the Gal-10 crystal related diseases comprise infectious diseases and inflammatory diseases.
Preferably, the etiological agent of the infectious disease includes bacteria, mycoplasma, chlamydia, mycobacteria, fungi, viruses, parasites, and the like. Illustratively, the infectious diseases include suppurative lymphadenitis, eosinophilic cystitis, liver abscess, etc.
Preferably, the inflammation includes asthma, rhinitis, colitis, and the like.
Preferably, the inflammation may be caused by allergy.
Preferably, the colitis is eosinophilic colitis.
Preferably, the subject of the present invention includes a human or non-human animal, exemplary of which include: mouse, pig, cow, horse, sheep, monkey, rabbit, etc.
Preferably, the subject of the invention is a human.
As used herein, a method of "treating" a disease or disorder refers to curing the disease or disorder and/or alleviating or eradicating the symptoms associated with the disease or disorder, thereby alleviating the patient's suffering.
In another aspect, the present invention provides a method for solubilizing crystals of Gal-10, which comprises contacting crystals of Gal-10 with the guanidine-containing compound of the present invention.
Preferably, the method is performed in vitro.
Preferably, the method is non-therapeutic.
More specifically, the dissolution of Gal-10 crystals according to the present invention can also be referred to as accelerating the dissolution of Gal-10 crystals.
Drawings
FIG. 1 is a graph of the results of measurements of the morphological changes of CLC crystals in different salt solutions under a light microscope.
FIG. 2 is a graph of the statistical results of the relative area of CLC crystals in different salt solutions over time, A: (CH)3)4NCl，B：KCl，C：NaCl，D：GdmCl。
FIG. 3 is a graph of the statistical results of the intrinsic onset rates of dissolution of CLC crystals in different salt solutions.
FIG. 4 is a graph showing the results of the detection of the morphological changes of CLC crystals in metformin solutions of different concentrations under a light mirror.
Figure 5 is a graph of the statistical results of the relative area of CLC crystals as a function of time in different concentrations of cimetidine.
FIG. 6 is a graph of the statistical results of the relative area of CLC crystals over time in different concentrations of streptomycin.
Figure 7 is a graph of the statistical results of the relative area of CLC crystals in different concentrations of metformin as a function of time.
FIG. 8 is a graph of the statistical results of the intrinsic onset rates of dissolution of CLC crystals in different solutions containing guanidine compounds. (this cannot be called "salt")
FIG. 9 is a graph showing the results of IL-1. beta., IL-6, TNF-. alpha., CCL-2qPCR in mice treated with tracheal injection, A: IL-1. beta., B: IL-6, C: TNF- α, D: CCL-2.
FIG. 10 is a graph showing the results of IL-1. beta., IL-6, TNF-. alpha., CCL-2qPCR in mice treated with oral administration, A: IL-1. beta., B: IL-6, C: TNF- α, D: CCL-2.
FIG. 11 is a graph showing the results of ELISA assay of IL-1. beta. IL-6, TNF-. alpha.and CCL-2 expression levels in mice treated with tracheal injection, A: IL-1. beta., B: IL-6, C: TNF- α, D: CCL-2.
FIG. 12 is a graph showing the results of measuring the expression levels of IL-1. beta., IL-6, TNF-. alpha., and CCL-2 in mice treated with oral administration by ELISA, A: IL-1. beta., B: IL-6, C: TNF- α, D: CCL-2.
Fig. 13 is the staining results of pathological samples of lung tissue, a: 6 hours, B: for 12 hours.
FIG. 14 is a graph of the statistical results of the relative area of CLC crystals as a function of time in different guanidine compound-containing solutions at different concentrations, A: 1-ethylguanidine, B: 1, 1-dimethylguanidine, C: 1-methylguanidine, D: 1,1,3, 3-tetramethylguanidine, E: 1-phenyl biguanide, F: 1- (o-tolyl) biguanide.
FIG. 15 is a graph of the statistical results of the intrinsic onset rates of dissolution of CLC crystals in different solutions containing guanidine compounds.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 preparation of CLCs and in vitro characterization experiment of salt-solubilized CLCs
1. Experimental Material
1.1 plasmid: after humanization codon optimization of the Gal-10 protein sequence, its N-terminus was added (MASTTHHHHHDTDIPTTGGGSRPDDDDDKENLYFQGHM) and cloned into pET-28a vector plasmid via NcoI/XhoI double cleavage sites to form pET-28a-6His-TEV-Gal 10.
1.2 reagent materials and instrumentation
1.2.1 chemical reagents:
kanamycin (Kanamycin) and Ampicillin (Ampicillin) were purchased from Tiangen biotechnology limited; Isopropyl-beta-D-thiogalactoside (IPTG) and E.coli BL21(DE3) were purchased from Beijing Quanjin; SDS, Trizol and imidazole were purchased from Sigma; TEV enzyme was purchased from Beijing Yiqiao Shenzhou, Inc.; coomassie brilliant blue dye liquor (self-made); cDNA reverse transcription reagents were purchased from Takara; the real-time fluorescent quantitative PCR reagent is purchased from Shanghai Roche GmbH; elisa kit from R&D Systems, Inc.; NaCl was purchased from Dalochi chemical reagent works, Tianjin; (CH)3)4NCl, KCl, GdmCl, Kac, KBr, streptomycin, cimetidine, dexamethasone, 1-methylguanidine, 1,3, 3-tetramethylguanidine, 1-dimethylguanidine, 1-ethylguanidine, 1-phenylbiguanide and 1- (o-tolyl) biguanide are all available from Shanghai Michelin Biochemical technology Ltd; DEPC water, ELISA stop solution, TMB single-component color developing solution from Solebao.
1.2.2 consumables and instrumentation:
Ni-NTA affinity chromatography column from GE; 10kDa concentration tubes were purchased from Millipore; an electric heating constant temperature incubator (XMTD HH.B 11-600); vertical pressure steamSterilizing pots (Shanghai Bo Xue, China); constant temperature bacteria incubator (shanghai bo news, china); PCR instrument (Biometra tgradent); 384-well real-time fluorescent quantitative PCR instrument (Roche 480 II); desktop centrifuges (eppendorf, Centrifuge 5415D); an electric constant temperature water tank (SHH W21600); a micro ultraviolet spectrophotometer (NanoDrop 2000); electronic balance (Sartorius 2000S); an optical inverted microscope (XDS-1B); micropipettes (eppendorf Research plus); ultra low temperature refrigerator at-80 deg.C (SANYO, MDF-382E); high speed refrigerated centrifuge (Beckman, germany); a pH meter (Thermo Orion 868); magnetic stirrers (IKA RH-KT/C); an ultrasonic crusher;
pure 25 protein purification system (superdex75, GE Healthcare); SDS-PAGE electrophoresis apparatus (Bio-Rad); gel imager (Tanon); 1mL single use sterile syringe (available from Bidi medical devices, Inc.).
2. Experimental methods
2.1 expression, purification and purification of Gal10 recombinant proteinsCLCPreparation of crystals
2.1.1 transformation:
1) placing BL21(DE3) competent cells and pET-28a-6His-TEV-gal10 recombinant plasmid on ice for thawing;
2) adding 0.5. mu.L (0.2. mu.g/. mu.L) of recombinant plasmid into BL21(DE3) competent cells, and incubating on ice for 15-20 min;
3) carrying out water bath heat shock at 42 ℃ for 90 s;
4) rapidly placing on ice, and ice-cooling for 5 min;
5) adding 500 μ L of nonresistant LB, and culturing at 37 deg.C for 40-50 min;
6) mu.L of the bacterial suspension was inoculated on LB solid medium containing Kanamycin (25. mu.g/mL) and cultured overnight in a 37 ℃ incubator.
2.1.2 bacterial culture:
with Kanamycin (25. mu.g/mL) as a selection marker, the positive monoclonal BL21(DE3)/pET-28a-gal10-TEV-6His on the solid medium in the above step was picked up, inoculated into 20mL LB liquid medium containing Kanamycin resistance, and shake-cultured at 37 ℃ and 210r/min for 8 h. The bacterial liquid is inoculated to 1L LB liquid culture medium containing Kanamycin resistance according to the proportion of 1:100, and cultured under the condition of 37 ℃ and 210r/min oscillation.
2.13 inducible expression and acquisition of a protein of interest:
1) when the optical density (OD600) of Escherichia coli at 600nm is 0.6-0.8, adding IPTG with final concentration of 1mM, culturing at 28 deg.C under oscillation condition of 210r/min for 12-16h, and inducing target protein expression;
2) the overnight expressed bacterial culture was enriched, centrifuged at 6000g at 4 ℃ for 20min and the supernatant discarded. Using Buffer Lysis Buffer (50mM NaH)2PO4(ii) a 300mM NaCl pH7.4), concentrating the suspension in a beaker with a volume of about 60-80mL, and adding PMSF (1: 100) (final concentration of 1mM) to prevent target protein degradation;
3) ultrasonic bacteria disruption: placing the beaker on ice for ultrasonic treatment, wherein the power is 25%, the working time is 25min, the ultrasonic on time is 3s, and the ultrasonic off time is 9 s;
4) collecting protein: the cell debris was removed by centrifugation (4 ℃, 12000g, 30min), the nucleic acid released after cell disruption was disrupted and the supernatant was collected, the soluble protein of interest being present in the supernatant.
2.1.4 purification of Gal-10 protein:
2.1.4.1Ni-NTA affinity column chromatography:
1) balancing Ni columns: the elution of the protein can be started by penetrating the lysine Buffer out of the Ni column by the capacity of about 2-3 column volumes;
2) centrifuging to obtain a supernatant sample, and passing through the Ni column for 2-3 times;
3) washing the column with Lysis Buffer containing 20mM imidazole and 0.1% Empigen detergent, in order to elute the hetero-proteins;
4) washing the column with Lysis Buffer containing 500mM imidazole to remove the target protein from the nickel column;
5) concentration: and (3) putting the target protein eluted and collected in the step (4) into a 10kDa concentration tube, centrifuging at 4 ℃ and 3000 Xg for 10min, and adding a small amount of lysine Buffer or PBS to dilute imidazole in the concentration process so as to prevent the target protein from aggregating and precipitating.
2.1.4.2
pure 25 protein purification:
1) cleaning a chromatographic column: the chromatography column was washed with about 8mL of sterile water. Placing the pump head in a PBS solution, repeating the steps and performing to wash about 36 mL;
2) parameters are as follows: system flow: 0.4 mL/min; column position: 2; alarm delta column compressed enabled: 3.0; alarm pre column compressed enabled: 5.0;
3) loading: washing the sample loading ring with PBS for 2-3 times, then injecting the concentrated sample of about 1.5mL into the sample loading ring, and finally slightly sucking the PBS and injecting the sample into the sample loading ring to avoid sample residue. Put the collection tube, select inject valid: inject;
4) collecting: when the UV280 marking line representing the protein content rises, collecting the target protein by using an automatic sample collector, and setting the collection amount of each tube to be 0.3 mL;
5) cleaning a chromatographic column: after the sample is collected, the pump head is placed in PBS, the pump head is continuously operated until the sample passes through 20mL, the pump head is replaced by sterile water for cleaning, the operation is carried out for 10mL, and then the pump head is replaced by 20% ethanol solution for operating for 20mL, and then the machine can be shut down;
6) protein treatment: measuring the concentration of each tube of the collected protein, marking, quickly freezing in liquid nitrogen, and freezing and storing in a refrigerator at-80 deg.C. The experimental antechamber is statically placed at a room temperature and slowly melts.
2.1.4.3SDS-PAGE identification of expression products:
1) sample preparation: a whole bacteria sample, a supernatant sample after centrifugation, a protein supernatant sample after passing through a chromatographic column, a 20mM imidazole sample, a 500mM imidazole sample, and protein samples collected at three positions of a peak head, a peak tip and a peak tail in a molecular sieve;
2) preparing glue: 5% concentrated gum; selecting 15% of separation gel according to the size of the protein;
3) preparing a sample: mixing the 2X Loading Buffer with the sample at a ratio of 1: 1;
4) loading: 5 mu L of whole bacteria sample, 3 mu L of marker and 10 mu L of other samples;
5) glue running: running concentrated glue at constant pressure of 80V; constant pressure 120V running separation glue;
6) dyeing: placing the protein gel in Coomassie brilliant blue dye solution, and heating with microwave for 2 min;
7) and (3) decoloring: placing the dyed protein glue in tap water, and heating with microwave for 20-40 min.
2.1.5CLC Crystal Generation and concentration determination:
1) mixing TEV enzyme and pET-28a-gal10-TEV-6His recombinant protein according to the mass ratio of 1:10, and carrying out enzyme digestion incubation on a shaker at 4 ℃ overnight;
2) centrifuging for 10min at the temperature of 4 ℃ at the temperature of 600g, sucking out the supernatant after the centrifugation is finished, adding a proper amount of PBS (phosphate buffer solution) for heavy suspension, and repeating for three times to obtain pure CLCs;
3) the CLCs were disassembled by 6M guanidine hydrochloride, and the concentration of CLCs was measured using a spectrophotometer One-Drop.
2.2 in vitro characterization experiments of salt-solubilized CLCs
2.2.1 dissolving the CLCs in PBS (pH7.4), adjusting the concentration to make the concentration of the CLCs be 0.6 mg/ml;
2.2.2 selection of (CH) with consideration of solubility and safety3)4Preparing salt solution by using six salts of NCl, KCl, NaCl, GdmCl, KAc and KBr, wherein the molar concentration of the salt solution is 4M, and the pH value is 7.4;
2.2.3 mixing the CLCs and the saline solution in proportion, diluting them to working concentrations:
1) diluting CLCs with working concentration of 0.15 mg/ml;
2) dilution (CH)3)4NCl, molarity gradient 1.0M, 1.5M, 2.0M, 3.0M;
3) KCl is diluted, and the molar concentration gradient is 1.0M, 1.5M, 2.0M and 3.0M;
4) diluting NaCl with molar concentration gradient of 1.0M, 1.5M, 2.0M and 3.0M;
5) GdmCl is diluted, and the molar concentration gradient is 0.2M, 0.4M, 0.5M and 0.6M;
2.2.4 dropping the mixed liquid on a crystal plate to observe by an optical microscope, and taking pictures at intervals (the detection result of the morphological change of the CLC crystal in different salt solutions under the optical microscope is shown in figure 1);
2.2.5 processing the obtained image by using Photoshop, intercepting the same area, calculating the intercepted area by using ImageJ, and normalizing the data obtained at the first time point for 10min (the statistical result of the change of the relative area of the CLC crystal in salt solutions with different concentrations along with the time is shown in a figure 2);
2.2.6 according to R (t) ═ k.A (t) · [1-exp (. beta. DELTA.. mu. (c))]Determining the initial reaction rate Rint(statistics of the intrinsic onset rates of CLC crystal dissolution in different zwitterion solutions are shown in figure 3).
+The above experimental results demonstrate that Gal-10 crystals have a selective response to the above selected salt ions, in which Gdm shows
The Gal-10 crystals are dissolved, and the dissolution of Gal-10 crystals is time-dependent.
2.3 in vitro characterization experiment for dissolving CLCs with guanidine Compound
2.3.1 dissolving CLCs in PBS (pH7.4), adjusting the concentration to make the concentration of CLCs 0.3 mg/ml;
2.3.2 dissolving metformin in PBS buffer in a molarity gradient of 0.2mM, 2.0mM, 10.0mM, 20.0mM, 100.0mM, 200.0mM, 300.0mM, 400mM, 800 mM;
the structural formula of the metformin is as follows:
2.3.3 dissolving streptomycin in PBS buffer with a molarity gradient of 0.2mM, 2.0mM, 10.0mM, 20.0 mM;
the structural formula of streptomycin is as follows:
2.3.4 dissolution of cimetidine in PBS buffer with a molarity gradient of 0.2mM, 2.0mM, 10.0mM, 20.0 mM;
the structural formula of cimetidine is as follows:
2.3.5 mixing the salt solution with the CLCs in a volume ratio of 1:1, dropping on a crystal plate, observing with an optical microscope, and taking pictures at intervals; (the detection results of the morphological changes of the CLC crystals in different solutions containing guanidine compounds under a light mirror are shown in FIG. 4)
2.3.6 processing the obtained image with Photoshop, intercepting the same area, calculating the intercepted area with ImageJ, and normalizing the first time point for 10 min; (statistics of the relative area of CLC crystals in different solutions containing guanidine Compound over time are shown in FIGS. 5-7)
2.3.7 according to R (t) k. A (t). [1-exp (. beta.. DELTA.. mu. (c))]Determining the initial reaction rate Rint. (statistics of the intrinsic onset rates of CLC crystal dissolution in different solutions containing guanidine Compound in FIG. 8)
The above results show that of the three small molecule drugs, metformin has better solubility in dissolving Gal-10 crystals.
Example 2 preparation of CLCs and salt solubilizationCLCsIn vitro characterization experiment of
Experimental method
1 administration by tracheal injection
1.1 Experimental animals
Mice of strain C57BL/6 were purchased from the institute for laboratory animal resources, national institute for food and drug testing. Mice (wild type, male, 6 week old, C57BL/6 strain mice, weight 20 ± 2g) were randomly divided into five groups of 6 mice each; and the following groups of infusion drugs were prepared:
1) PBS (negative control),
2)CLCs(1.5mg/ml)，
3) metformin (0.45mg/kg),
4) CLCs plus metformin (CLCs: 1.5mg/ml, metformin: 0.45mg/kg),
5) CLCs plus dexamethasone (0.5mg/kg) (positive control);
1.2 administration phase:
1) injecting tribromoethanol anesthetic into abdominal cavity of mouse by using sterile injector, the anesthetic dosage is as follows: the weight (g) of the mouse is multiplied by 15 mu L/mouse, and the mouse is fixed after the observation of the mouse is fully anesthetized (the foot squeezing reflex is negative);
2) keeping the airway of the mouse vertical, exposing a longitudinal incision with the length of 0.5-0.8cm at the position of 1.5cm of the lower jaw in the right middle of the ventral skin of the neck by using an ophthalmic scissors, and separating hypodermis, connective tissue and neck muscle along the midline in a blunt manner by using an ophthalmic curved forceps until the trachea is fully exposed;
3) inclining the mouse fixing device at about 30 degrees, sucking 50 mu L of air by using an injector, sucking 70 mu L of prepared medicine, inserting a needle below the middle thyroid cartilage of the trachea by using the injector in parallel to the longitudinal axis of the trachea, wherein the depth of the needle is about 1cm, and keeping the position to quickly drip the air and the perfusion medicine into the trachea of the mouse;
4) after the operation is finished, the respiratory frequency of the mouse can be observed to be suddenly increased, and the mouse fixing device is placed for about 1min vertical to the operation table to help the distribution of the medicine; the mouse fixing device resets, uses the curved tweezers of ophthalmology to clamp the neck tissue of the mouse layer by layer according to the dissection layering, relieves the fixation of the mouse, puts the mouse in the lateral position and observes the state of the mouse until the mouse returns to the autonomous movement.
2 oral administration
2.1 Experimental animals
Mice (wild type, male, 6 week old, C57BL/6 strain mice, weight 20 ± 2g) were randomly divided into 6 groups of 3 mice each; and the following groups of infusion drugs were prepared:
1) PBS (negative control),
2)CLCs(1.5mg/ml)，
3) metformin (0.45mg/kg),
4) CLCs plus metformin 1 × (CLCs: 1.5mg/ml, metformin: 0.45mg/kg),
5) CLCs plus metformin 3 × (CLCs: 1.5mg/ml, metformin: 1.35mg/kg),
6) CLCs plus metformin 10 × (CLCs: 1.5mg/ml, metformin: 4.5 mg/kg).
2.2 administration phase:
the mouse was held with its head, neck and body in line. The injector with the gastric perfusion needle is used, the needle enters from the mouth corner of the mouse, presses the tongue, props against the palate, slightly pushes inwards, enters the esophagus and slightly penetrates deeply and injects the liquid medicine.
3, a sample collection stage:
the sampling was started 6h and 12h after the administration by tracheal injection, and the sampling was started 6h after the oral administration group.
1) Injecting tribromoethanol anesthetic into the abdominal cavity of a mouse by using a sterile syringe, wherein the anesthetic dosage is as follows: the weight (g) of the mouse is multiplied by 15 mu L/mouse, the mouse is observed to be fully anesthetized (the foot squeezing reflex is negative), and then the mouse is sacrificed and fixed;
2) exposing the mouse trachea again along the trachea instillation incision, cutting an inverted V-shaped incision below the thyroid cartilage, and carefully and gently operating to avoid breaking the mouse trachea;
3) sucking 600 mu L of sterile PBS, then extending a liquid shifter in a cut parallel to the longitudinal axis of the trachea until the sterile PBS is completely attached to the inner wall of the mouse trachea, injecting the PBS into the mouse bronchus through the mouse trachea at a constant speed, then sucking back at a constant speed, finding a large number of bubbles in the process of sucking back, which is a sign of successful operation, repeating the injection-sucking back process for 4-5 times, then putting the recovery liquid into a 1.5mL centrifuge tube, and blowing, beating and uniformly mixing;
4) repeating the step (3), putting the recovery liquid into the same 1.5mL sterile EP tube, and storing at-80 ℃;
5) the mice were opened in the chest, the sternum was cut, the heart was extracted to cut off connective tissue and fascia between the lung tissue and the chest, the lung tissue was removed and rinsed with sterile PBS, lysed by adding 1ml Trizol and stored at-80 ℃.
Detection of therapeutic effects
1. RNA extraction and cDNA reverse transcription
1.1RNA extraction:
1) placing the lysate stored in Trizol on ice for slow thawing;
2) after the RNA sample is completely thawed, the sample is taken to room temperature and placed for 10min to fully dissolve the RNA, chloroform is added according to the proportion of Trizol to chloroform of 5:1(v/v), the mixture is forcibly shaken to be fully and uniformly mixed, and the mixture is placed for 10-15min at room temperature;
3) centrifuging at 12000r/min at 4 deg.C for 15 min;
4) transferring the upper aqueous phase to a new 1.5mL centrifuge tube without RNase;
5) adding 0.6-0.8 times volume of pre-cooled isopropanol, mixing, and standing at-20 deg.C for more than 30 min;
6) centrifuging at 4 ℃ at 12000r/min for 15min, and removing the supernatant to see that RNA precipitates are at the bottom of the centrifuge tube;
7) adding 1mL of 75% ethanol prepared by DEPC-treated water or enzyme-free sterile water, and gently oscillating the centrifugal tube to resuspend the precipitate;
8) centrifuging at 4 ℃ at 12000r/min for 15min, discarding the supernatant as much as possible, and repeating the step 7-8;
9) drying at room temperature until 75% ethanol is evaporated;
10) dissolving the RNA precipitate with DEPC-treated water or enzyme-free sterile water;
11) taking 2 mu L of RNA sample, measuring the RNA concentration by using a trace ultraviolet spectrophotometer, adding a proper amount of DEPC treated water or enzyme-free sterile water to adjust the RNA solution concentration to 200 ng/mu L, and freezing and storing at-80 ℃.
1.2 reverse transcription of RNA into cDNA:
reverse Transcription of cDNA was performed according to the instruction of cDNA Reverse Transcription kit (cat. no RR036A) of Takara;
1)5×PrimeScript RT Master Mix(Perfect Real Time):2μL
1×Total RNA：0.5μg
RNase Free dH2O up to 10μL
2) after gentle and uniform mixing, carrying out reverse transcription reaction under the following conditions:
15min at 37 ℃ (reverse transcription)
5sec at 85 ℃ (inactivation reaction of reverse transcriptase)
Short-term storage at 4 ℃.
2. Real-time fluorescent quantitative PCR (RT-qPCR) detection of gene expression changes
According to
480SYBR Green I Master instructions for real-time fluorescent quantitative PCR experiments;
1) the working solution added to each reaction system was as follows:
2) adding the working solution into a 384-well plate, adding 1 mu L of cDNA obtained by the reversion of the above step into each well, flatly pasting a sealing film on the 384-well plate, and instantly leaving for 90 seconds;
3) the 384 well plate was placed on the reaction tray as specified in the instrument and set up for the following reaction protocol;
4) after the reaction is finished, use
480 software calculates a reaction dissolution curve (Melt curve), observes whether an amplification curve is a single peak to eliminate non-specific amplification, automatically calculates the relative expression quantity of the detection gene according to Ct values of a target gene and an internal reference gene, and has the algorithm as follows: relative gene expression value of 2(purpose Ct)Gene-Ct reference gene))；
5) Plotted in Graphpad Prism 8.0 software and analyzed for significant differences.
The results of tracheal injection qPCR are shown in FIG. 9, and IL-1 beta, IL-6, TNF-alpha and CCL-2 genes are caused after CLCs stimulation
The level was increased, and the metformin treated group significantly reduced the expression of inflammatory factors in lung tissue and was statistically significant.
The qPCR results of the oral administration group are shown in FIG. 10, and IL-1 beta, IL-6, TNF-alpha and CCL-2 are caused after the CLCs are stimulated
Due to the increase of the level, the figure shows that the test result of the administration for 6h shows that the oral metformin treatment group 1x can obviously reduce the inflammation in the lung tissue
The factor expression, after increasing the concentration, may be due to the toxic effect on the cells, the inflammation reduction effect is not 1x good.
3. Enzyme linked immunosorbent assay (ELISA) for detecting protein changes
3.1 coating of ELISA 96-well plates: diluting the capture antibody by sterile PBS according to the working concentration, adding 100 mu L/hole of the diluted capture antibody into a 96-hole enzyme label plate with high binding force, completely covering the upper surface by a sealing film, and incubating overnight at room temperature;
3.2 detection process:
1) discarding the capture antibody, adding 200 μ L of washing solution into each well, washing, discarding the washing solution, and drying the residual liquid with filter paper, repeating for 3 times;
2) add 300. mu.L of dilution (PBS containing 1% BSA) per well and incubate for at least 1h at room temperature;
3) discard the dilution and repeat step 1);
4) preparing a standard substance by using a new diluent, adding 100 mu L of the prepared standard substance or sample into each hole, repeating the holes for each sample, shaking up gently, pasting a cover plate, and incubating for 2h at 37 ℃;
5) discarding the sample and repeating step 1);
6) diluting the detection antibody to a working concentration by using a new diluent, adding 100 mu L of detection antibody working solution into each hole, laminating plates, and incubating for 2h at room temperature;
7) discarding the detection antibody and repeating step 1);
8) diluting Streptavidin-HRP with new diluent to working solution concentration according to the ratio of 1:40, adding 100 μ L into each well, laminating, and incubating at room temperature in dark for 20 min;
9) discarding Streptavidin-HRP working solution and repeating step 1);
10) adding 100 μ L of TMB single-component color developing solution into each well, pasting the plates, and incubating for 10-20min at room temperature in a dark place;
11) adding 50 mu L of chromogenic termination solution into each hole, vibrating and uniformly mixing, detecting OD values of 450nm and 570nm by using a full-function micropore plate detector, and subtracting the OD value of 570nm from the OD value of 450 nm;
12) plotted in Graphpad Prism 8.0 software and analyzed for significant differences. (FIG. 11, FIG. 12)
The results of tracheal injection ELISA are shown in FIG. 11, and IL-1 beta, IL-6, NF-alpha, CCL-2 protein is induced after CLCs stimulation
The level is increased, and the metformin treatment group can obviously reduce inflammatory factors in bronchoalveolar lavage fluid and has statistics
The meaning of learning.
The results of ELISA in the oral group are shown in FIG. 12, and IL-1 beta, IL-6, TNF-alpha and CCL-2 are caused after the CLCs are stimulated
The increase of protein level, the oral metformin treatment group 1X significantly reduced inflammatory factors in bronchoalveolar lavage fluid.
4. Lung tissue pathology sample H of mouse in tracheal injection administration group&E dyeing
4.1 tracheal injection administration group after 6h and 12h, left lung tissue (n ═ 3) of mice was carefully harvested, washed in PBS and placed in a 10mL tube containing 8mL paraformaldehyde (4%), and then left to stand at room temperature for 48-72 h;
4.2 dehydration, embedding, dewaxing, dyeing, dehydration mounting (this step is completed by Wuhan Severe Biotech Co., Ltd.)
The results are shown in FIG. 13, and the lung tissue inflammatory cell immersion after mouse trachea injection of metforminThe moistening condition is obviously relieved.
Example 3 Structure Effect relationship (other guanidine-containing Compounds)
1. Diluting the CLC crystals to a concentration of 0.3mg/ml
2. Dissolving 1-methylguanidine in PBS buffer solution, wherein the molar concentration gradient is 2 mM; 20 mM; 100 mM; 200 mM; 400 mM.
3. Dissolving 1,1,3, 3-tetramethylguanidine in PBS buffer solution, wherein the molar concentration gradient is 0.2 mM; 1.0 mM; 4.0; 10.0 mM.
4. Dissolving 1, 1-dimethylguanidine in PBS buffer solution with a molar concentration gradient of 2 mM; 20 mM; 100 mM; 200 mM; 400 mM.
5. Dissolving 1-ethylguanidine in PBS buffer solution, and performing molar concentration gradient of 2 mM; 20 mM; 100 mM; 200 mM; 400 mM.
6. Dissolving 1-phenyl biguanide in PBS buffer with a molar concentration gradient of 0.2 mM; 2.0 mM; 4.0 mM; 10.0 mM; 20.0 mM.
7. Dissolving 1- (o-tolyl) biguanide in PBS buffer with a molar gradient of 0.2 mM; 2.0 mM; 4.0 mM; 10.0 mM.
8. Mixing the salt solution and the CLC crystal in a volume ratio of 1:1, observing on a crystal plate, and taking pictures at intervals;
9. intercepting the same area by using Photoshop, calculating the intercepted area by using ImageJ, and taking the first time point for 10min to normalize;
10. the initial reaction rate Rint is calculated from r (t) k · a (t) · [1-exp (β Δ μ (c)) ].
The statistical results of the change of the relative area of CLC crystals over time in different guanidine compound-containing solutions of different concentrations are shown in the figure
FIG. 14 shows the statistical results of the intrinsic initial rates of dissolution of CLC crystals in different solutions containing guanidine compounds, as shown in FIG. 15.
The above experimental results demonstrate that the number of non-polar functional groups and the ability of the guanidine ion derivative to dissolve Gal-10 crystals
And (4) positively correlating.
Claims (10)
1. The application of the guanidine-containing compound in dissolving Gal-10 crystals and preparing medicaments for treating Gal-10 crystal related diseases;
preferably, the guanidine-containing compound is represented by the following general formula:
wherein R is1、R2、R3、R4、R5、R6Each independently is a hydrogen atom or a substituent;
preferably, the substituents include, but are not limited to, the following: a hydrocarbon group, a guanidino group, a phenyl group, an o-tolyl group, a diazo group, a carboxyl group, a sulfonic group, a hydrocarbyloxycarbonyl group, a formyl group, a haloformyl group, an oxo group, a carbamoyl group, a cyano group, a phenolic hydrocarbon group, a phenolic hydroxyl group, an alcoholic hydroxyl group, an amino group, a hydrocarbyloxy group, a nitro group, a nitroso group, a mercapto group, an amino group, a nitro group, an acyl group, a silicon group, an acyloxy group, an oxyacyl group, a borono group, a hydroxyamino group, a nitroso group, a silyl group; the substituents may also include C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Cycloalkyl, aryl, heteroaryl, heterocyclyl- (CH2) n-, aryl-C1-6Alkyl-, heteroaryl-C1-6Alkyl-, aryl- (CH)2) n-O-, heteroaryl- (CH)2)n-O-、C3-8cycloalkyl-C (O) -, heterocyclyl-C (O) -, aryl-C (O) -, or heteroaryl-C (O) -, wherein C1-6Alkyl radical, C1-6Alkoxy radical, C3-8Cycloalkyl, aryl, heterocyclyl- (CH)2) n-, aryl-C1-6Alkyl-, heteroaryl-C1-6Alkyl-, aryl- (CH)2) n-O-, heteroaryl- (CH)2)n-O-、C3-8cycloalkyl-C (O) -, heterocyclyl-C (O) -, aryl-C (O) -;
preferably, the substituents are non-polar groups;
preferably, said hydrocarbyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl.
2. The use of claim 1, wherein the substituent is any one or more of hydrocarbyl, guanidino, phenyl, and o-tolyl.
3. The use of claim 2, wherein the guanidine-containing compound comprises metformin, 1-methylguanidine, 1-dimethylguanidine, 1,3, 3-tetramethylguanidine, 1-ethylguanidine, 1-phenylbiguanide, 1- (o-tolyl) biguanide, streptomycin, cimetidine;
preferably, the guanidine-containing compound is one or more of metformin, 1-methylguanidine, 1-dimethylguanidine, 1,3, 3-tetramethylguanidine, 1-ethylguanidine, 1-phenylbiguanide, 1- (o-tolyl) biguanide, streptomycin, cimetidine.
4. The use according to any one of claims 1 to 3, wherein said Gal-10 crystal-related disease comprises an infectious disease, an inflammatory disease.
5. The use according to claim 4, wherein the pathogens of infectious diseases include bacteria, mycoplasma, chlamydia, mycobacteria, fungi, viruses and parasites;
preferably, the infectious diseases include suppurative lymphadenitis, eosinophilic cystitis, liver abscess;
preferably, the inflammation comprises inflammation that causes an increase in IL-1 β, IL-6, TNF- α, CCL-2 following stimulation by Gal-10 crystals;
preferably, the inflammation includes asthma, rhinitis, colitis, etc.;
preferably, the inflammation comprises that caused by allergy;
preferably, the colitis is eosinophilic colitis.
6. A pharmaceutical composition comprising the guanidine-containing compound of any one of claims 1-3;
preferably, the mode of administration of the pharmaceutical composition comprises: oral, aerosol inhalation, rectal, nasal, buccal, parenteral, such as tracheal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracardiac, intrasternal or intravenous administration;
preferably, the mode of use is oral or tracheal administration.
7. The pharmaceutical composition of claim 6, in a dosage form comprising: tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations, aerosols, films, injections, intravenous drip, transdermal preparations, ointments, lotions, adhesive preparations, suppositories, pellets, nasal preparations, pulmonary preparations, eye drops and the like, oral or parenteral preparations;
preferably, the pharmaceutical composition is a liquid and the solvent is PBS;
more preferably, the pH of the PBS is 7.4.
8. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable carrier, diluent or excipient;
preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the national food and drug administration for use in humans or livestock.
9. A method of solubilizing crystals of Gal-10, said method comprising contacting crystals of Gal-10 with the guanidine-containing compound of any of claims 1-3.
10. The method of claim 9, wherein the method is performed in vitro;
preferably, the method is non-therapeutic.
Priority Applications (2)
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| CN202210534642.4A CN114681438B (en) | 2022-05-17 | 2022-05-17 | Use of metformin and other guanidine-containing compounds for reversing the crystallization tendency of Gal-10 and for alleviating the associated diseases |
| CN202410259292.4A CN118141793A (en) | 2022-05-17 | 2022-05-17 | Use of metformin and other guanidine-containing compounds to solubilize summer-lyone crystals and to alleviate inflammation in acute lung injury |
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| CN202210534642.4A CN114681438B (en) | 2022-05-17 | 2022-05-17 | Use of metformin and other guanidine-containing compounds for reversing the crystallization tendency of Gal-10 and for alleviating the associated diseases |
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| CN202410259292.4A Pending CN118141793A (en) | 2022-05-17 | 2022-05-17 | Use of metformin and other guanidine-containing compounds to solubilize summer-lyone crystals and to alleviate inflammation in acute lung injury |
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| CN118141793A (en) | 2024-06-07 |
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