Cytisine-polypeptide coupling compound and preparation method and use thereof

Abstract

The present invention provides a cytisine-peptide coupling compound and a preparation method and use thereof, and belongs to the field of medicine. The present invention also provides a cytisine-peptide coupling compound and a preparation method and use thereof. Pharmacological efficacy tests have shown that the cytisine of the present invention has the effect of treating cor pulmonale and can inhibit the proliferation of human lung fibroblasts MRC-5. The cytisine-peptide coupling compound (PDC drug) prepared by coupling cytisine with a polypeptide has a significantly reduced dosage and better effect than cytisine, can improve right heart dysfunction, reduce myocardial fibrosis, and alleviate right heart remodeling, providing a new option for the clinical treatment of cor pulmonale.

Description

Technical Field

[0001] The invention relates to a cytisine-polypeptide coupling compound and a preparation method and application thereof, belonging to the field of medicines. Background Art

[0002] Pulmonary heart disease (PHD) refers to a type of heart disease caused by lesions in the lung tissue or pulmonary artery and its branches (such as lung disease, pulmonary vascular disease, chest deformity or respiratory center disorder, etc.), which increases pulmonary circulation resistance and causes pulmonary arterial hypertension (PAH), which leads to right ventricular hypertrophy and myocardial fibrosis, and ultimately leads to decreased cardiopulmonary function, which seriously endangers people's health and increases the medical burden. Clinically, the treatment of PHD is still very limited, and it is impossible to effectively cure the remodeling of cardiopulmonary tissue and restore function. The main solution is to maintain lung function and keep the oxygen saturation level >90%. PHD mainly includes two aspects: surgical treatment and drug treatment. Surgical treatment includes oxygen therapy, percutaneous balloon pulmonary valvuloplasty, pulmonary thromboendarterectomy and lung transplantation.

[0003] At present, clinical treatment for PHD is limited. The main first-line drugs include cardiotonic agents, diuretics, antibiotics, vasodilators, expectorants and antiasthmatics, etc., which aim to delay the progression of the underlying disease and improve the quality of life of patients, but cannot repair the tissue damage that has already occurred in the heart and lungs. Therefore, it is urgent to find new targets and new drugs that can reverse the process of cardiopulmonary remodeling and effectively treat cor pulmonale.

[0004] Cytisine, also known as cytisine or cytisine, is an alkaloid isolated mainly from plants of the leguminous family. It has a grayish white to yellowish brown crystalline solid form and has specific chemical properties, such as the molecular formula C 11 H 14 N2O, molecular weight 190.242, and melting point at 154-156℃. Cytisine has a variety of biological activities in pharmacology, including smoking cessation, reducing drinking behavior, anti-tumor, cardiovascular protection, blood sugar regulation, neuroprotection and osteoporosis prevention and treatment. It also has anti-arrhythmia, anti-microbial infection, anti-ulcer, and white blood cell increase effects. In addition, cytisine has certain toxicity. For example, in acute toxicity tests, there are relevant literature reports on the lethal doses of different animals.

[0005] There is no literature report on the treatment of cor pulmonale with cytisine. Summary of the invention

[0006] The technical solution of the present invention is to provide a cytisine-polypeptide coupling compound and a preparation method and use thereof.

[0007] The invention provides a cytisine-polypeptide coupling compound, which is formed by connecting cytisine and a polypeptide via cathepsin B.

[0008] Wherein, the C-terminal 5 peptides of the polypeptide constitute a cyclic peptide, and the sequence is as follows:

[0009] Cys-Thr-Arg-Gln-Arg-Tyr-Cys;

[0010] The substrate sequence of cathepsin B includes: Phe-Lys, Val-Cit, Glu-Val-Cit, GGFG, Phe-Arg, Val-Ala, Ala-Ala, cBU-Cit, Abz-Gn VR↓AK(Dnp)-OH, Z-VR-AMC, and Z-ER-AMC.

[0011] Further preferably, the substrate sequence of cathepsin B is Val-Cit, and the structural formula of the cytisine-polypeptide coupling compound is:

[0012]

[0013] The invention provides a method for preparing the cytisine-polypeptide coupling compound, which is synthesized by solid phase synthesis.

[0014] Specifically, the preparation method of the present invention comprises the following steps:

[0015] a. Place the resin in a solid phase reactor, add dichloromethane to soak it, add piperidine / DMF solution to the reactor, soak it, react with nitrogen bubbling, and then wash it with DMF;

[0016] b. Add cysteine, threonine, arginine, glutamine, arginine, and tyrosine in sequence;

[0017] c. cleavage of the peptide from the resin;

[0018] d. coupling cytisine, cleaving and purifying the polypeptide to obtain a cytisine-polypeptide coupling compound.

[0019] Among them, the cutting reagents described in step c are TFE and DCM, and the volume ratio is: 1:4; the cleavage reagents described are: TFA, water, EDT, Tis, and the volume ratio is: 95:1:2:2.

[0020] The present invention also provides use of the cytisine-polypeptide coupling compound in preparing a medicine for treating cor pulmonale.

[0021] The present invention also provides a pharmaceutical composition for treating cor pulmonale, which is prepared from the cytisine-polypeptide coupling compound as an active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients into a commonly used pharmaceutical preparation.

[0022] Wherein, the preparation is an oral preparation.

[0023] The beneficial effects of the present invention are:

[0024] The efficacy test proves that the cytisine of the present invention has the effect of treating cor pulmonale and can inhibit the proliferation of human lung fibroblasts MRC-5. The cytisine-polypeptide coupling compound (PDC drug) prepared by coupling cytisine with a polypeptide has a significantly lower dosage and better effect than cytisine, can improve right heart dysfunction, reduce myocardial fibrosis, and alleviate right heart remodeling, providing a new option for the clinical treatment of cor pulmonale. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 HPLC chromatogram;

[0026] Figure 2 MS spectra;

[0027] Figure 3 EdU experiments confirmed that PDC could inhibit the proliferation of lung myofibroblasts in a dose-dependent manner;

[0028] Figure 4 PDC can inhibit the migration of human lung fibroblasts (MRC-5);

[0029] Figure 5 Ultrasound results showed that PDC drugs can reduce right heart dysfunction in rats in a dose-dependent manner;

[0030] Figure 6 Masson staining results showed that PDC drugs could reduce right heart fibrosis in rats in a dose-dependent manner. DETAILED DESCRIPTION

[0031] Example 1 Preparation of the Cytisine-Polypeptide Conjugate Compound PDC of the Present Invention

[0032] 1.Solid phase synthesis of peptide coupling compounds

[0033] 1.1 Experimental equipment: 1 / 10,000 electronic balance; vertical reactor (20*250mm, No. 1 sand core); 50ml centrifuge tube; centrifuge, HPLC semi-preparative, 2cm C18 column, disposable pipette; nitrogen; circulating water vacuum pump; washing bottle; test tube; long-necked pipette; constant temperature heater.

[0034] 1.2 Experimental materials and reagents: 2-chlorotrityl chloride resin; anhydrous DCM, DIC, DIEA, methanol, industrial grade DMF; analytical grade DMF; 20% piperidine / DMF, detection reagent A (5 g ninhydrin-100 mL anhydrous ethanol), B (analytical grade pyridine), HOBT, acetonitrile, ether, TFA, TFE, EDT, TIS, cytisine;

[0035] Cytisine is a quinolizine alkaloid with a tricyclic fused structure extracted from the seeds of leguminous plants: Molecular formula C 11 H 14 N2O; molecular weight 190.24.

[0036]

[0037] Required amino acids:

[0038] FMOC-CIT-OH,FMNOC-VAL-OH,

[0039] FMOC-ARG(PBF)-OH, FMOC-THR(TBU)-OH, FMOC-CYS(TRT)-OH, FMOC-GLN(TRT)-OH, FMOC-TYR(TBU)-OH.

[0040] 1.3 Select a reactor (20*250mm, No. 1 sand core) and write the name of the polypeptide on the reactor as a mark. Weigh 0.5g of 2-chlorotrityl chloride resin with an electronic balance, put it into the reactor, add DCM and soak for 30 minutes to remove Fmoc: add 20% piperidine / DMF solution to the reactor with a washing bottle, the volume of the reagent is about 3 times the volume of the resin, so that the resin is completely soaked in the solution, react with nitrogen bubbling for 20 minutes, and then wash with DMF 5 times.

[0041] 1.4 Weigh 0.15mmol of FMOC-CIT-OH (C-terminal) and HOBT into a centrifuge tube, add 10mL of anhydrous DCM and 0.5mmol of DIEA, and shake well. Use a disposable pipette to draw the solution and add it to the reactor in the previous step. Bubble nitrogen for 90 minutes. After the reaction is completed, add 2mL of methanol + 6mL of DCM and react for 20 minutes.

[0042] 1.5 Washing: After draining the liquid in the reactor with a circulating water vacuum pump, add industrial-grade DMF to the reactor with a wash bottle. The volume of the reagent should be about 3 times the volume of the resin. The resin should be completely immersed in the solution. Wash for 30 seconds, then drain the liquid in the reactor with a circulating water vacuum pump. The draining time is about 30 seconds. Repeat this operation 4 times.

[0043] 1.6 Removal of Fmoc: Use a wash bottle to add 20% piperidine / DMF solution into the reactor. The volume of the reagent should be about 3 times the volume of the resin. The resin should be completely soaked in the solution. Nitrogen bubbling reaction should be carried out for 20 minutes.

[0044] 1.7 Washing: Refer to 1.5, and replace the industrial grade DMF with analytical grade DMF during the fifth washing process;

[0045] 1.8 Resin detection: Use a long-necked pipette to take 10-20 resins from the reactor and place them at the bottom of the test tube. Then use a rubber-tipped dropper to take two drops of detection reagent A and B and drop them into the test tube to allow the resin and the detection reagent to fully contact. Then place the test tube in a constant temperature of 100℃ and heat for 2 minutes. Observe the color of the resin. If the resin shows color, it means that 1.6 Fmoc removal is successful. If it does not show color, repeat 1.6-1.8 operations.

[0046] 1.9 Coupling: Weigh 0.5mmol FMOC-VAL-OH (second position from C-terminal) and 0.5mmol HOBT in a centrifuge tube, dissolve them in 5ml DMF, add 0.5mmol DIC, mix for 1min, add to the resin after draining, and react with nitrogen bubbling for 1h. It is strictly forbidden to add DIC directly to the reactor; when AA is activated in advance, the generated crystals are not added to the reactor. If the solution in the reactor cannot make the resin shake evenly during the reaction, a little DMF can be added appropriately;

[0047] 1.10 Resin detection: Refer to 1.8 and observe the color of the resin. If it is colorless, it means the connection is complete, and proceed to 1.11; if there is color, repeat 1.9.

[0048] 1.11 Washing, same as 1.5.

[0049] 1.12 Repeat the steps 1.6-1.11, connecting the amino acid raw materials in sequence according to the peptide sequence, until the peptide connection is completed without removing Fmoc, then wash (see 1.5), add 0.5mmol BOC anhydride and 0.5mmol Diea, use DCM as solvent, react for 20 minutes, and then wash (see 1.5).

[0050] The polypeptide sequence is: the C-terminal 5 peptides of NPY constitute a cyclic peptide: Cys-Thr Arg Gln Arg Tyr-Cys.

[0051] 1.13 Resin drying: Wash the resin with methanol three times and filter dry.

[0052] 2. Cleavage of the peptide from the resin

[0053] 2.1 Preparation of cutting reagent

[0054] The formula for preparing the cutting reagent (taking 100 mL of cutting reagent as an example) is: 20 mL of TFE + 80 mL of DCM, set aside, and the preparation amount is generally 1 g of resin plus 20 mL of cutting reagent.

[0055] 2.2 Peptide cleavage

[0056] The resin was drained and weighed, and a cutting solution was added to cut for 2 h. The filtrate was then filtered and rotary evaporated to obtain the crude peptide.

[0057] 2.3 Coupling of small molecules

[0058] The fully protected peptide, HOBT and small molecule were mixed in equal moles and dissolved in DMF. An equal mole of DIC was added and stirred for 2 h, and then the mixture was lyophilized by rotary evaporation.

[0059] 2.4 Peptide cleavage

[0060] Prepare the cutting reagent (taking 100 ml of cutting reagent as an example); the formula is: 95 ml TFA + 1 ml water + 2 ml EDT + 2 ml Tis, set aside, the preparation amount is generally 10 ml of cutting reagent for 1 g of resin, 1 ml of cutting reagent is washed with 10 ml of ether and centrifuged to obtain a crude product.

[0061] 3. Peptide Purification

[0062] Chromatographic column: 20*250mm diagesol 8 micron

[0063] Mobile phase: A: 0.1% TFA water B: 0.1% TFA acetonitrile

[0064] Flow rate: 10mL / mlin

[0065] The sample was loaded through pump A, and then 10% acetonitrile water was equilibrated for 5 min to start the gradient run.

[0066]

[0067] Prepare and collect the sample peak for detection, with an analytical purity greater than 80%, and collect it in a beaker.

[0068] Disulfide bond preparation:

[0069] An equal volume of water was added to the sample in the beaker, the pH was adjusted to 8 with ammonium bicarbonate, and the mixture was stirred overnight to form disulfide bonds.

[0070] The sample was loaded through pump A and prepared and purified using the same preparation method as above to prepare and collect the sample peak for detection, with an analytical purity of greater than 95%.

[0071] 4. Sample freeze drying

[0072] The sample was transferred into a freeze-drying bottle and placed in a freeze dryer for 24 h to obtain the pure product.

[0073] Structural Characterization of Cys-Thr Arg Gln Arg Tyr-Cys-Val-Cit-Cytisine:

[0074] HPLC results are shown in Figure 1 , MS results are shown in Figure 2 .

[0075] The structural formula is:

[0076]

[0077] Val-Cit was selected as the linker to connect the peptide and cytisine, expressed as Cys-Thr Arg Gln ArgTyr-Cys-Val-Cit-Cytisine.

[0078] The beneficial effects of the present invention are demonstrated by pharmacodynamic tests as follows.

[0079] Test Example 1 In vitro experiments on cytisine and cytisine-polypeptide coupling compounds of the present invention

[0080] 1. Cell culture, grouping and drug administration

[0081] Human lung fibroblasts MRC-5 were cultured in a culture medium containing 10% fetal bovine serum, adherent cultured at 37°C and 5% CO2, and digested and passaged with 0.25% trypsin when the cell confluence reached about 90% of the flask wall area. The cell culture experiment was divided into 6 groups: Ctrl group, TGF-β+PBS group, TGF-β+cytisine CYT (20 μM) group, TGF-β+PDC-L (2.5 μM) group, TGF-β+PDC-M (5 μM) group, and TGF-β+PDC-H (10 μM) group, wherein the PDC group was the cytisine-peptide coupling compound prepared in Example 1.

[0082] 2. EDU experiment to detect the proliferation of MRC-5 stimulated by TGF-β (5 ng / mL)

[0083] (1) Cell culture and drug treatment

[0084] MRC-5 cell suspension was inoculated in a 96-well cell culture plate (cell suspension density was 5×10 4 / mL, 100μL / well), after the cells were cultured overnight and recovered to normal state, they were pretreated with cytisine CYT and PDC at low (2.5μM), medium (5μM), and high doses (10μM) for 2h, and then stimulated with TGF-β (5ng / mL) for 24h. The specific groups were as follows: Ctrl group, TGF-β+PBS group, TGF-β+cytisine CYT group, TGF-β+PDC-L group, TGF-β+PDC-M group, and TGF-β+PDC-H group.

[0085] (2) EdU labeling

[0086] According to the results of the preliminary experiment, the final concentration of EdU was set at 10μM (1×), and EdU (10mM) was diluted 1 / 500 with cell culture medium to obtain 2× EdU working solution (20μM). An equal volume of EdU working solution preheated to 37°C was added to the 96-well plate to make the final concentration of EdU in the 96-well plate 10μM, and the cells were incubated for 2h.

[0087] (3) Cell fixation and permeabilization

[0088] a. After EdU labeling, remove the culture medium and wash with PBS 1-2 times, 5 minutes each time. Then add 50 μL 4% paraformaldehyde to each well and fix at room temperature for 15 minutes;

[0089] b. Remove the fixative solution, add 50 μL of 2 mg / mL glycine to each well, incubate on a decolorizing shaker for 5 minutes, and then discard the glycine solution;

[0090] c. Add 100 μL PBS to each well and wash the cells 3 times, 3-5 min each time;

[0091] d. Remove PBS, add 50 μL permeabilization solution (PBS containing 0.3% TritonX-100) to each well, and incubate at room temperature for 10-15 minutes;

[0092] e. Remove the permeabilization solution and add 100 μL PBS to each well to wash the cells 1-2 times, each time for 3-5 minutes.

[0093] (4) Apollo staining:

[0094] a. Prepare Apollo staining reaction solution according to the instructions of the kit;

[0095] b. Remove the washing solution from the previous step, add 50 μL of Apollo staining reaction solution to each well, gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample, and incubate at room temperature in the dark for 30 minutes;

[0096] c. Aspirate the Apollo staining reaction solution and wash three times with PBS, each time for 3-5 minutes.

[0097] (4) Nuclear staining:

[0098] a. After removing the washing solution, add 50 μL of ready-to-use DAPI staining solution to each well and incubate at room temperature in the dark for 10 minutes;

[0099] b. Remove the DAPI staining solution and wash with PBS three times, 3-5 minutes each time;

[0100] Subsequently, the high-content cell drug analysis system can be used for fluorescence detection and analysis of the proportion of EdU-positive cells; DAPI is blue fluorescence with a maximum excitation wavelength of 346nm and a maximum emission wavelength of 460nm; Apollo 643 is red fluorescence with a maximum excitation wavelength of 653nm and a maximum emission wavelength of 667nm.

[0101] 3. Cell migration (Transwell) assay to detect the migration effect of PDC on MRC-5 stimulated by TGF-β (5 ng / mL) The cell scratch assay is to study cell migration, repair ability and cell-cell interaction by scratching the cell monolayer and capturing images regularly under a microscope.

[0102] The specific steps of the scratch test are as follows:

[0103] (1) Use a marker to draw even horizontal lines on the back of a 6-well plate (with a ruler for assistance), crossing the wells and drawing three horizontal lines in each well.

[0104] (2) Cell suspension (MRC-5 cell suspension density is 1×10 5 cells / mL, 2 mL / well; the density of HPMEC cell suspension was 2×10 5 / mL, 2mL / well) were inoculated in a 6-well plate. After the cells adhered overnight, the cell fusion rate was observed under a microscope. When it reached 100%, scratching could be performed.

[0105] (3) Use a 200 μL pipette tip to scratch perpendicular to the horizontal line on the back, with the tip perpendicular to the bottom of the plate, and make two vertical scratches in each well. Discard the culture medium, rinse twice with PBS to remove the scratched cells, add serum-free medium and treat with drugs.

[0106] (4) Take photos under a 4x microscope. Take one photo above and below the intersection of the scratch and the horizontal line at 0 h. Take 12 photos per well in total. Ensure that the scratch is centered and vertical, and pay attention to the consistency of the background. After taking photos, continue to place the cells in the incubator for routine culture. Take samples and take photos again after 24 h.

[0107] (5) Use ImageJ software to analyze the scratch area using the find-edges method. The specific steps are as follows:

[0108] a. Open the image in ImageJ software, click Image, and select the image type as 8bit;

[0109] b. Select Process-Enhance Contrast, check Normalize, and generally use 0.5% as the value to make the boundary between the cell edge and the background at the scratch more obvious;

[0110] c. Select Process-Find Edges, which will greatly enhance the contrast between cells and background through binarization;

[0111] d. Click Image-Adjust-Threshold in sequence to adjust the threshold so that the mask fills the scratches; then click Apply to perform binarization, resulting in the image being either black or white;

[0112] e. After the binarization process, the background color is uniform, and you can directly use the magic wand tool to select the scratches, create a selection, and select the actual scratch area;

[0113] f. Click Analyze-Measure to calculate the area of ​​the selected area in the image (i.e. the scratch area).

[0114] Data processing was performed using Excel, and the cell migration rate was calculated as follows: Cell migration rate (scratch healing rate) = (initial scratch area - scratch area at time t) / initial scratch area × 100%

[0115] 4. In vitro experimental results: PDC drug treatment can inhibit the proliferation and migration of MRC-5 stimulated by TGF-β. The specific results are as follows:

[0116] 4.1. The results of EDU experiment showed that compared with the PBS group, administration of 20 μM cytisine CYT could reduce the number of EDU-positive MRC-5 cells, and 2.5, 5, and 10 μM PDC could dose-dependently reduce the number of EDU-positive MRC-5 cells, indicating that the proliferation of MRC-5 cells was inhibited, and the effect of 10 μM PDC was better than that of 20 μM cytisine, and the differences were statistically significant (P<0.05). Figure 3 .

[0117] 4.2. The results of cell migration experiment showed that compared with the PBS group, administration of 20 μM cytisine CYT increased the distance of Transwell tubules, and 2.5, 5, and 10 μM PDC increased the distance of Transwell tubules in a dose-dependent manner, indicating that the migration of MRC-5 cells was inhibited, and the effect of 10 μM PDC was better than that of 20 μM cytisine, and the differences were statistically significant (P<0.05). Figure 4 .

[0118] The above experiments prove that PDC has better effects with less dosage than cytisine alone.

[0119] Experimental Example 2 In vivo experiment of cytisine-polypeptide coupling compound of the present invention

[0120] 1. Animal modeling, grouping and drug administration

[0121] SPF male SD rats (150-180g), monocrotaline MCT was dissolved in anhydrous ethanol solution, and then diluted with saline (anhydrous ethanol: saline = 2:8) to a 10mg / ml LMCT suspension. The suspension was ultrasonically dissolved in an ultrasonic cleaner for 1 hour, shaken overnight at 37°C for full dissolution, and then filtered with a 0.22μm sterile filter. MCT solution (50mg / kg) was injected into the abdomen of the rats as a model group (PH group), and the same volume of saline was injected as a control group (Sham group). The modeled animals were raised under normoxic and normal pressure conditions for 14 days. All operations on experimental animals were in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (National Institutes of Health Release No. 85-23, revised in 1996). PDC drugs were prepared with 0.9% saline. Fourteen days after modeling, the rats in the PH group were randomly divided into a normal saline group, a low-dose PDC group (5 mg / kg), a medium-dose PDC group (10 mg / kg), and a high-dose PDC group (20 mg / kg). The drugs were administered by oral gavage every day, and the animals continued to be raised under normoxic and normobaric conditions for 14 days.

[0122] 2. Animal ultrasound detection of rat heart function

[0123] After the animals were raised under normoxic and normobaric conditions for 28 days, the cardiac function of the rats was detected using a high-resolution small animal ultrasound imaging system. The rats were anesthetized with 3% inhaled isoflurane and maintained with 1.5% isoflurane, with spontaneous breathing and a heart rate of 300 to 350 beats per minute. The rats were fixed on the operating table in the supine position and cleaned by first removing the hair on the chest surface with a shaver, and then removing the remaining hair with a depilatory cream. Transthoracic cardiac ultrasound examination was performed using the Vevo3100 small animal ultrasound imaging system equipped with a 20-MHz probe.

[0124] Right heart function ultrasound is mainly completed by collecting pulmonary artery section, right heart long axis section and four-chamber heart section. The probe positioning of the pulmonary artery short axis section is based on the standard parasternal short axis section, and the probe is moved toward the aorta until the aortic valve enters the field of view. The pulmonary artery short axis section can obtain ultrasound data of pulmonary artery diameter and pulmonary artery blood flow through B-ultrasound mode and pulsed Doppler mode, and calculate pulmonary artery acceleration time (PAT) and pulmonary artery ejection time (PET). The probe positioning of the right heart long axis section is based on the parasternal long axis section and is translated toward the right heart until the RV view appears. Under the right heart long axis section, RVFW and right heart inner diameter (RVID) can be obtained through M-ultrasound mode. The probe positioning of the four-chamber heart section is based on the parasternal short axis section, and the animal's head is tilted to the lower left corner, and the tilted probe angle is rotated to obtain the coronary section view of the heart cut from the apex. RV area and tricuspid annular systolic excursion (TAPSE) can be obtained by B-mode and M-mode ultrasound in the four-chamber view.

[0125] 3. Masson staining

[0126] After the ultrasound, rats were anesthetized by intraperitoneal injection of 4% tribromoethanol solution (350-400 mg / kg), and the hearts were taken for Masson staining to detect cardiac fibrosis. The chest cavity was exposed with surgical scissors, and the heart tissue was removed. The residual blood was rinsed with pre-cooled 1×PBS and fixed in 4% paraformaldehyde solution for 24 hours. After the tissue was trimmed, it was placed in a dehydrator for dehydration with gradient alcohol in sequence. The trimmed tissue blocks were dipped in wax and embedded in an embedding machine. After further trimming of the wax block, it was placed in a paraffin slicer for sectioning (4μm). After the slices were dried in a 60°C oven, the slices were taken out and placed at room temperature for storage.

[0127] Paraffin sections were dewaxed in xylene and washed with alcohol gradient until hydrated. The sections were immersed in Masson A solution overnight and washed with water; then immersed in a mixture of Masson B and C in equal proportions for 1 minute and washed with water; differentiated with 1% hydrochloric acid alcohol and washed with water; then immersed in Masson D solution for 6 minutes and washed with water; immersed in Masson E solution for 1 minute, slightly drained and directly immersed in Masson F solution for 2-30 seconds. The sections were rinsed and differentiated with 1% glacial acetic acid, dehydrated with anhydrous ethanol, transparentized and sealed. The sections were placed under a microscope for observation, and image acquisition and data analysis were performed.

[0128] 4. Experimental results

[0129] PDC treatment for 14 days can improve right heart dysfunction, reduce myocardial fibrosis, and alleviate right heart remodeling in a dose-dependent manner. The specific results are as follows:

[0130] 1. Ultrasound results showed that the pulmonary artery acceleration time (PAT), PAT / pulmonary artery ejection time (PET), right ventricular free wall (RVFW) and tricuspid valve systolic displacement (TAPSE) of rats in the normal saline group, which reflected the pulmonary artery and right heart function, were significantly reduced, accompanied by an increase in RVFW, indicating that the right heart of PH rats was in a state of pressure and volume load, and accompanied by right heart failure. The administration of PDC drugs can increase PAT, PAT / PET and TAPSE in a dose-dependent manner and reduce RVFW, and the differences are statistically significant (P<0.05). Figure 5 .

[0131] 2. Masson staining results showed that compared with the saline group, PDC improved RV tissue fibrosis in a dose-dependent manner in PH rats, and the differences were statistically significant (P<0.05). Figure 6 .

Claims

1. A cytisine-polypeptide coupling compound, characterized in that: It is composed of cytisine and polypeptide linked by cathepsin B; The C-terminal 5 peptides of the polypeptide constitute a cyclic peptide, and the sequence is as follows: Cys-Thr-Arg-Gln-Arg-Tyr-Cys; The substrate sequence of cathepsin B is Val-Cit, and the structural formula of the cytisine-polypeptide coupling compound is: 。 2. The method for preparing the cytisine-polypeptide coupling compound according to claim 1, characterized in that: It is synthesized by solid phase synthesis; It includes the following steps: a. Place the resin in a solid phase reactor, add dichloromethane to soak it, add piperidine / DMF solution to the reactor, soak it, react with nitrogen bubbling, and then wash it with DMF; b. Add cysteine, threonine, arginine, glutamine, arginine, and tyrosine in sequence; c. cleavage of the peptide from the resin; d. coupling cytisine, cleaving and purifying the polypeptide to obtain a cytisine-polypeptide coupling compound.

3. The method for preparing the cytisine-polypeptide coupling compound according to claim 2, characterized in that: The cutting reagents described in step c are TFE and DCM in a volume ratio of 1:4; the cleavage reagents described are TFA, water, EDT, and Tis in a volume ratio of 95:1:2:

2.

4. Use of the cytisine-polypeptide coupled compound according to claim 1 in the preparation of a medicament for treating cor pulmonale.

5. A pharmaceutical composition for treating cor pulmonale, characterized in that: The cytisine-polypeptide coupling compound described in claim 1 is used as an active ingredient, and pharmaceutically acceptable excipients or auxiliary ingredients are added to prepare a commonly used pharmaceutical preparation.

6. The pharmaceutical composition for treating cor pulmonale according to claim 5, characterized in that: The preparation is an oral preparation.

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