Polypeptide coupling medicine and application thereof

Abstract

The invention provides a polypeptide coupling medicine and application thereof. The polypeptide coupling medicine comprises antiviral polypeptide, connecting polypeptide, amino acid and ambroxol which are connected in sequence. The polypeptide coupling drug provided by the invention is obtained by coupling a coronavirus membrane fusion inhibitor and ambroxol, and is formed by coupling polypeptide serving as a targeting carrier and a load drug through a connexon via a covalent bond. Through polypeptide coupled drug activity evaluation and pharmacokinetic research, evaluation of activity changes of polypeptide and ambroxol and research on drug exposure and half-life periods of inhalants and injections, the polypeptide coupled drug retains antiviral activity of polypeptide and phlegm eliminating / respiratory tract regulating functions of ambroxol, the drug exposure is increased, and the half-life period is prolonged.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a polypeptide-conjugated drug and its application. Background Technology

[0002] As is well known, antibody-drug conjugates (ADCs) have become a hot research topic in the field of oncology treatment in recent years, with more than ten ADC drugs approved for marketing globally to date. With more and more companies investing in ADC drugs, their R&D pipelines are becoming increasingly crowded, resulting in a concentration of targets and indications. At the same time, as the application and development of ADC drugs deepen, challenges such as toxic side effects, high production costs, and less-than-ideal efficacy against some solid tumors are gradually emerging. Therefore, peptide-drug conjugates (PDCs), as a new generation of targeted therapies following ADCs, have become a new research hotspot for conjugate drugs.

[0003] Peptide-drug conjugates (PDCs) are a novel type of conjugate drug with a structure similar to ADCs. They consist of a peptide as a target carrier, which is conjugated to a drug payload via a linker.

[0004] Compared with ADC drugs, PDC drugs have advantages such as small molecular weight, strong tumor penetration, low immunogenicity and low production cost. They also retain the function and biological activity of peptides, improve the stability and targeting of drugs in vivo, and reduce the toxic side effects of drugs.

[0005] There are four main methods for preparing PDCs: gene fusion expression of drugs and peptides using recombinant technology, covalent bonding, formation of ion-interacting complexes, and modification of drug-loaded nanoparticles with CPP.

[0006] Novel coronavirus infection is an acute respiratory infectious disease, classified as a Class B infectious disease. It is caused by infection with the novel coronavirus and is highly contagious, spreading through respiratory droplets and close contact. Common signs and symptoms after infection with the coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing.

[0007] Our company has developed a coronavirus membrane fusion inhibitor that acts on the HR1 region of the S2 subunit of the SARS-CoV-2 virus S protein. It can competitively interact with the viral HR1 region to form a heterologous six-helix bundle structure (6-HB), thereby inhibiting the formation of homologous 6-HB between the HR1 and HR2 domains of the virus itself, blocking the fusion process between the virus and the host cell, exerting antiviral activity, and has a broad spectrum.

[0008] Meanwhile, ambroxol is currently the most widely used expectorant in clinical practice. It stimulates the formation of respiratory surfactant and regulates serous and mucous secretions, simultaneously improving the clearance function of both ciliated and non-ciliated areas of the respiratory tract, reducing the adhesion of sputum and cilia, further facilitating expectoration, and alleviating coughing. Because of its rapid and reliable effects and good tolerability, it can be taken long-term. Its mechanism of action is as follows.

[0009] (1) It mainly works by stimulating type II alveolar cells to synthesize and secrete pulmonary surfactant, thereby enhancing alveolar tension, improving lung compliance and mucociliary clearance function, and relieving cough and sputum symptoms.

[0010] (2) It can inhibit the release of inflammatory mediators from mast cells and neutrophils, reduce lung inflammation, and reduce airway hyperresponsiveness.

[0011] (3) It has the effects of relieving cough, expectorating phlegm and improving lung function, and can effectively relieve symptoms such as cough and phlegm caused by chronic bronchitis, bronchial asthma and other diseases.

[0012] (4) It can also increase the concentration of antibiotics in lung tissue, enhance the efficacy of antibiotics, and shorten the course of disease.

[0013] The core pathological feature of COVID-19 infection is that the virus replicates in the respiratory tract, causing inflammation, accompanied by increased mucus secretion in the respiratory tract (especially in severely ill patients), leading to airway obstruction, oxygenation impairment, and even progression to acute respiratory distress syndrome (ARDS).

[0014] Based on the synergistic strategy of "direct antiviral therapy + improvement of respiratory microenvironment", a peptide conjugate drug of coronavirus membrane fusion inhibitor and ambroxol can be developed. This drug retains the antiviral activity of the peptide and the expectorant / respiratory regulation function of ambroxol, thereby reducing viral replication, relieving respiratory symptoms, and forming a synergistic therapeutic effect. Summary of the Invention

[0015] This invention provides a novel peptide-conjugated drug and its application. It designs a conjugate drug of a broad-spectrum coronavirus membrane fusion inhibitor and ambroxol. Through the synergistic effect of "inhibiting viral membrane fusion and blocking infection" and "expectorant, anti-inflammatory and improving respiratory microenvironment", it can directly inhibit viral invasion and reduce respiratory damage caused by infection. At the same time, it enhances local efficacy through targeted delivery, and finally achieves the dual effect of anti-COVID-19 and symptom relief.

[0016] Its core mechanisms include:

[0017] (1) Synergistic effect of targeted antiviral therapy and microenvironment optimization

[0018] Antiviral peptides directly block viral invasion or replication, but the thick mucus in the respiratory tract may hinder the contact between the peptides and the virus / target cells. Ambroxol, by dissolving mucus and reducing its viscosity, can reduce the "retention" of peptides in the mucus, increase their local concentration at viral infection sites (such as bronchi and alveoli), and enhance the effectiveness of antiviral peptides.

[0019] (2) Synergistic effect of antiviral and inflammation control

[0020] Following COVID-19 infection, respiratory inflammatory responses (such as mucosal edema and exudation) can exacerbate tissue damage and may promote viral spread. Ambroxol's anti-inflammatory effect can reduce local inflammation and decrease the "pathological basis" of viral infection; at the same time, anti-COVID-19 peptides inhibit viral replication, which can reduce inflammatory stimulation from the source, forming a positive cycle of "antiviral-anti-inflammatory".

[0021] (3) Synergistic acceleration of virus clearance

[0022] After the anti-COVID-19 peptides neutralize or inhibit viral activity, ambroxol can accelerate the expulsion of neutralized viruses and inflammatory secretions from the body by enhancing ciliary movement, shortening the retention time of the virus in the respiratory tract, and promoting infection recovery.

[0023] (4) Potential targeted delivery optimization

[0024] After conjugation, it may enhance the affinity of ambroxol for respiratory mucosa, thereby increasing the targeted aggregation of anti-COVID-19 peptides in the respiratory tract (reducing systemic distribution), reducing off-target effects, and enhancing local antiviral activity.

[0025] To achieve this objective, the present invention adopts the following technical solution:

[0026] In a first aspect, the present invention provides a polypeptide-conjugated drug comprising an antiviral polypeptide, a linker polypeptide, an amino acid, and ambroxol connected in sequence.

[0027] Preferably, the amino acid sequence of the antiviral polypeptide is SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK.

[0028] Preferably, the antiviral polypeptide has a protecting group at its amino terminus, and the protecting group is an acetyl group.

[0029] Preferably, the amino acid sequence of the linking polypeptide is EAAAK.

[0030] Preferably, the amino acid is lysine.

[0031] Preferably, the antiviral polypeptide, the linking polypeptide, and the amino acid are linked by amide bonds.

[0032] Preferably, the amino acid is linked to ambroxol via a chemical bond.

[0033] Preferably, the chemical bond is a nitrogen-carbon single bond or an amide bond.

[0034] Preferably, the connection is achieved by linking the amino terminus of the amino acid straight chain or branched chain to the hydroxyl or bromine terminus of ambroxol, or by linking the carboxyl terminus of the amino acid straight chain or branched chain to the amino terminus of ambroxol.

[0035] Preferably, the carboxyl terminus of the amino acid that does not participate in coupling has a protecting group, and the protecting group is an amino group.

[0036] Preferably, the amino terminus of the amino acid that does not participate in coupling may or may not have a modifying group; when a modifying group is present, the modifying group is cholesterol succinate monoester.

[0037] Preferably, the polypeptide-conjugated drug is selected from the following structures:

[0038] , , , ;

[0039] X is Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAK-.

[0040] In a second aspect, the present invention provides an antiviral, expectorant, and respiratory tract regulating pharmaceutical composition, the pharmaceutical composition comprising the polypeptide-conjugated drug described in the first aspect.

[0041] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0042] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects:

[0044] The peptide-conjugated drug provided by this invention is obtained by conjugating a coronavirus membrane fusion inhibitor with ambroxol. It uses a peptide as a targeting carrier, covalently coupled to the loaded drug via a linker. This peptide-conjugated drug retains the antiviral activity of the peptide and the expectorant / respiratory regulation function of ambroxol, while also increasing drug exposure and prolonging its half-life. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise stated, all raw materials and excipients involved in the following examples are commercially available.

[0047] Example 1: Preparation of peptide-conjugated drugs

[0048] Based on the coronavirus membrane fusion inhibitor developed by our company and the structural characteristics of ambroxol, we prepared a conjugate drug.

[0049]

[0050] I. Chemical reagents required in the preparation process

[0051] All chemical reagents, such as various Fmoc amino acids, N,N-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N,N-dimethylformamide (DMF), piperidine (PIPE), ninhydrin, acetic anhydride (Ac2O), N,N-diisopropylethylamine (DIEA), hydrazine hydrate, ambroxol, trifluoroacetic acid (TFA), ethylene dithiol (EDT), anisole (TA), triisopropylsilane (TIPS), phenol, and N-fluorenemethoxycarbonyl-tetraethylene glycol-carboxylic acid (Fmoc-NH-PEG4-CH2CH2COOH), were purchased from major chemical reagent suppliers and were not further purified before use.

[0052] The protective amino acid raw materials used in peptide synthesis include Fmoc-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, and Fmoc-Arg(Pbf)-OH. The abbreviations have well-known definitions: Fmoc is 9-fluorenyloxycarbonyl, Dde is 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl, Boc is tert-butoxycarbonyl, tBu is tert-butyl, OtBu is tert-butoxy, Trt is triphenylmethyl, and Pbf is (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl.

[0053] II. Drug Synthesis Method

[0054] 1. Synthesis of peptide resins

[0055] Using Rink Amide MBHA resin as the carrier resin, peptide resins were prepared by sequentially coupling the peptides with the corresponding protected amino acids of the peptide amino acid sequence through de-Fmoc protection and coupling reactions.

[0056] 1) Integrate the first protected amino acid in the main chain

[0057] Take 0.3 mmol of the first protected amino acid Fmoc-Lys(Dde)-OH and 0.3 mmol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.3 mmol of DIC, and slowly add it to the DMF solution of the protected amino acid while shaking. Incubate the mixture at 25°C for 5 minutes with shaking to obtain the activated protected amino acid solution for later use.

[0058] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g × 0.3g), protect it with 25% PIPE / DMF solution (volume ratio) for 20 minutes (twice), wash and filter to obtain Fmoc-free resin.

[0059] The activated solution of the first protected amino acid was added to the resin after Fmoc removal, and the coupling reaction was carried out for 60 minutes. After filtration and washing, a resin containing the first protected amino acid Fmoc-Lys(Dde) was obtained.

[0060] 2) Incorporate other protective amino acids into the main chain

[0061] Using the same method as described above for adding the first protecting amino acid to the main chain, other protecting amino acids corresponding to the peptide were sequentially added to obtain a resin containing the main chain amino acids. Finally, the N-terminus was acetylated and capped with 0.3 mmol Ac₂O + 0.6 mmol DIEA to complete the synthesis of the main chain. Each reaction step was monitored using the Kaiser Test; if the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide was obtained.

[0062] 3) Sidechain integration

[0063] (1) Treat the resin with the smallest possible volume of 2% hydrazine hydrate / DMF solution (volume ratio) to remove the Dde protecting group of the C-terminal lysine side chain (10 minutes, twice), filter and wash to obtain the Dde-free resin for later use.

[0064] (2) Ambroxol modification (hydroxyl group removal): Dissolve 0.3 mmol of ambroxol and 0.3 mmol of HOBt in an appropriate amount of DMF; slowly add 0.3 mmol of DIC to the solution containing ambroxol and HOBt, and shake the mixture at 25°C for 5 minutes. Add the prepared solution containing ambroxol, HOBt and DIC to the Dde-removed resin obtained in step (1), and couple the mixture for 60 minutes. Filter, wash and dry to obtain the peptide resin.

[0065] 2. Preparation of crude product

[0066] Take the above-mentioned peptide resin, add lysis reagent (15 mL / g resin), mix well, and react with shaking at 30°C for 3 hours to lyse the target peptide from the resin and remove the side chain protecting groups. Collect the filtrate of the reaction mixture, wash the resin three times with a small amount of TFA / DCM, combine the filtrates, add anhydrous diethyl ether to precipitate, and centrifuge. Wash the filter cake twice with cold anhydrous diethyl ether, and dry to obtain a white powder, which is the crude lipopeptide.

[0067] The lysis reagent is composed of the following: trifluoroacetic acid: 1,2-ethylenedithiol: benzyl sulfide: phenol: H2O: triisopropylsilane = 68.5:10:10:5:3.5:1 (volume ratio).

[0068] 3. Preparation of pure products

[0069] The crude lipopeptide was dissolved in water / acetonitrile by stirring, and the insoluble matter was removed by centrifugation. Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). An Agela C18 column (10 μm, 100 Å, 50 × 250 mm) was used. The mobile phase consisted of mobile phase A (0.05% TFA and 2% acetonitrile aqueous solution) and mobile phase B (90% acetonitrile / aqueous solution). The flow rate was 25 mL / min. The UV detection wavelength was 220 nm. The crude solution was loaded onto the column and subjected to gradient elution. The corresponding purified fractions were collected and directly freeze-dried to remove the solvent, yielding the loose, pure trifluoroacetate peptide.

[0070] The trifluoroacetate polypeptide was redissolved in water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added and stirred for 3 hours. After filtration and rinsing the ion exchange resin with a water / acetonitrile mixture, the filtrates were combined and lyophilized to obtain a loose, pure polypeptide acetate.

[0071] The chemical structure of drug 1 was characterized by MALDI-TOF mass spectrometry, and its purity was determined by analytical high-performance liquid chromatography (Agela C18-4.6×250 mm, flow rate 1 mL / min). The results showed that the purity of the synthesized drugs was greater than 95%.

[0072] The structural formula of drug 1 is as follows.

[0073]

[0074] Note: X represents Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAK-

[0075] Mass spectrometry analysis showed that the high-resolution mass spectrometry data of the sample was consistent with the structure of drug 1. The mass spectrometry analysis data are as follows.

[0076]

[0077] III. Drug 2 Synthesis Method

[0078] Based on the above synthesis method, the steps "1. Synthesis of peptide resin - 3) Incorporation of side chain - (2) Ambroxol modification" in the synthesis method of drug 1 are replaced with the following steps:

[0079] Ambroxol modification (bromine removal at the linking site): Dissolve 0.3 mmol of ambroxol and 0.6 mmol of DIEA in an appropriate amount of DMF, and slowly add it to the Dde-removed resin obtained in step (1). The coupling reaction is carried out for 60 minutes, and the mixture is filtered, washed and dried to obtain the peptide resin.

[0080] The other synthesis steps are consistent with the synthesis method of drug 1.

[0081] The structural formula of drug 2 is as follows.

[0082]

[0083] Note: X represents Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAK-

[0084] Mass spectrometry analysis showed that the high-resolution mass spectrometry data of the sample was consistent with the structure of drug 2. The mass spectrometry analysis data are as follows.

[0085]

[0086] IV. Drug Synthesis Methods

[0087] Based on the above synthesis method, step "1. Synthesis of peptide resin" in the synthesis method of drug 1 is replaced with the following step:

[0088] C-terminal ambroxol linkage: Using 2-Cl resin as the carrier resin, peptide resin is prepared by sequentially coupling the peptide with the corresponding protected amino acids of the peptide amino acid sequence through de-Fmoc protection and coupling reaction.

[0089] 1) Introduce ambroxol

[0090] Dissolve 0.3 mmol of ambroxol and 0.6 mmol of DIEA in an appropriate amount of DMF. Take 0.1 mmol of 2-Cl resin and add the dissolved solution to the resin. Perform a coupling reaction for 90 minutes, filter and wash, add 0.9 mmol of methanol, perform a coupling reaction for 30 minutes, filter and wash to obtain a resin containing ambroxol.

[0091] 2) The first protected amino acid

[0092] Take 0.3 mmol of the first protected amino acid Fmoc-Lys(Dde)-OH and 0.3 mmol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.3 mmol of DIC, and slowly add it to the DMF solution of the protected amino acid while shaking. Incubate the mixture at 25°C for 5 minutes with shaking to obtain the activated protected amino acid solution for later use.

[0093] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g × 0.3 g), protect it with 25% PIPE / DMF solution (volume ratio) for 20 minutes (twice), wash and filter to obtain Fmoc-free resin.

[0094] The activated solution of the first protected amino acid was added to the resin after Fmoc removal, and the coupling reaction was carried out for 60 minutes. After filtration and washing, a resin containing the first protected amino acid Fmoc-Lys(Dde) was obtained.

[0095] 3) Integrate other protective amino acids into the main chain

[0096] Using the same method as described above for adding the first protecting amino acid to the main chain, other protecting amino acids corresponding to the peptide were sequentially added to obtain a resin containing the main chain amino acids. Finally, the N-terminus was acetylated and capped with 0.3 mmol Ac₂O + 0.6 mmol DIEA to complete the synthesis of the main chain. Each reaction step was monitored using the Kaiser Test; if the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide was obtained.

[0097] 4) Sidechain integration

[0098] (1) Treat the resin with the smallest possible volume of 2% hydrazine hydrate / DMF solution (volume ratio) to remove the Dde protecting group of the C-terminal lysine side chain (10 minutes, twice), filter and wash to obtain the Dde-free resin for later use.

[0099] (2) Cholesterol modification of the peptide: Dissolve 0.3 mmol cholesterol succinate monoester and 0.3 mmol HOBt in an appropriate amount of DMF; separately add 0.3 mmol DIC to the solution containing cholesterol succinate monoester and HOBt, and shake the mixture at 25°C for 5 minutes. Add the prepared solution containing cholesterol succinate monoester, HOBt and DIC to the Dde-free resin obtained in step (1), and couple the mixture for 60 minutes. Filter, wash and dry to obtain the peptide resin. (Adding cholesterol can yield another peptide containing ambroxol but without cholesterol.)

[0100] The other synthesis steps are consistent with the synthesis method of drug 1.

[0101] The structural formula of drug 3 is as follows.

[0102]

[0103] Note: X represents Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAK-

[0104] Mass spectrometry analysis showed that the high-resolution mass spectrometry data of the sample was consistent with the structure of drug 2. The mass spectrometry analysis data are as follows.

[0105]

[0106] V. Drug Synthesis Methods

[0107] Based on the above synthesis method, step "1. Synthesis of peptide resin" in the synthesis method of drug 1 is replaced with the following step:

[0108] C-terminal ambroxol linkage: Using 2-Cl resin as the carrier resin, peptide resin is prepared by sequentially coupling the peptide with the corresponding protected amino acids of the peptide amino acid sequence through de-Fmoc protection and coupling reaction.

[0109] 1) Introduce ambroxol

[0110] Dissolve 0.3 mmol of ambroxol and 0.6 mmol of DIEA in an appropriate amount of DMF. Take 0.1 mmol of 2-Cl resin and add the dissolved solution to the resin. Perform a coupling reaction for 90 minutes, filter and wash, add 0.9 mmol of methanol, perform a coupling reaction for 30 minutes, filter and wash to obtain a resin containing ambroxol.

[0111] 2) The first protected amino acid

[0112] Take 0.3 mmol of the first protected amino acid Fmoc-Lys(Boc)-OH and 0.3 mmol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.3 mmol of DIC, and slowly add it to the DMF solution of the protected amino acid while shaking. Incubate the mixture at 25°C for 5 minutes with shaking to obtain the activated protected amino acid solution for later use.

[0113] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g × 0.3 g), protect it with 25% PIPE / DMF solution (volume ratio) for 20 minutes (twice), wash and filter to obtain Fmoc-free resin.

[0114] The activated solution of the first protected amino acid was added to the resin after Fmoc removal, and the coupling reaction was carried out for 60 minutes. After filtration and washing, a resin containing the first protected amino acid Fmoc-Lys(Dde) was obtained.

[0115] 3) Integrate other protective amino acids into the main chain

[0116] Using the same method as described above for adding the first protecting amino acid to the main chain, other protecting amino acids corresponding to the peptide were sequentially added to obtain a resin containing the main chain amino acids. Finally, the N-terminus was acetylated and capped with 0.3 mmol Ac₂O + 0.6 mmol DIEA to complete the synthesis of the main chain. Each reaction step was monitored using the Kaiser Test; if the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide was obtained.

[0117] The other synthesis steps are consistent with the synthesis method of drug 1.

[0118] The structural formula of drug 4 is as follows.

[0119]

[0120] Note: X represents Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAK-

[0121] Mass spectrometry analysis showed that the high-resolution mass spectrometry data of the sample was consistent with the structure of drug 2. The mass spectrometry analysis data are as follows.

[0122]

[0123] Example 2 Evaluation of peptide activity of conjugated drugs (viral invasion inhibition experiment)

[0124] 1) Experimental cells: 293T / ACE2 cells, cultured in DMEM medium containing 10% fetal bovine serum.

[0125] 2) SARS-CoV-2 strain: Omicron BA.2, from Kunming Institute of Zoology.

[0126] 3) Test drugs: peptides, peptide-conjugated drugs

[0127] 4) Test kit: CCK8 test kit

[0128] 5) Experimental methods

[0129] (1) The test drug was dissolved in deionized water and the concentration was determined. Then, the test drug was diluted to the initial concentration with DMEM medium and serially diluted 3-fold in a 96-well cell culture plate. Finally, each well contained 50 µL of lipopeptide solution. Nine dilutions were set up, with three replicates for each dilution. Control wells were set up with DMEM medium (50 µL per well).

[0130] (2) After completing step (1), add 50µL (virus amount of 500 TCID50) to each well and incubate at 25°C for 30 minutes.

[0131] (3) Adjust the concentration of the pre-cultured test cells to 10 × 10⁻⁶. 4 Cell suspension of cells / mL was added to DEAE-dextran to a concentration of 15µg / mL, and then added to a 96-well plate (100µL / well) after step (2) was completed. The plate was then incubated at 37°C in a 5% CO2 cell culture incubator for 48 hours.

[0132] (4) After completing step (3), discard the supernatant, then add 30 µL of cell lysis buffer per well, lyse at 25 °C for 15 minutes, then add luciferase substrate, measure the relative fluorescence units (RLU) using a microplate chemiluminescence detector, and calculate the half-maximal inhibitory concentration (IC50) of the drug.

[0133] 6) Experimental Results

[0134]

[0135] The experimental results showed that the IC50 values ​​of the peptide and conjugate drugs 1, 2, 3, and 4 for inhibiting SARS-CoV-2 infection of 293T / ACE2 cells were 0.57 nM, 0.21 nM, 0.32 nM, 0.27 nM, and 0.35 nM, respectively. Compared with the peptide, the half-maximal inhibitory concentration of the conjugate drugs was lower, and the antiviral activity was improved.

[0136] Example 3 Evaluation of the expectorant activity of the conjugated drug

[0137] 1) Sample preparation

[0138] Artificial mucus preparation: Weigh appropriate amounts of mucin (such as porcine gastric mucin), DNA (salmon sperm DNA), NaCl, CaCl2, phosphate buffer (PBS, pH 7.4), etc., dissolve them in PBS, stir magnetically until completely dissolved, and let stand at 4°C for 24 hours to allow the molecules to fully hydrate and form a uniform mucus.

[0139] 2) Drug treatment

[0140] Take equal volumes of simulated mucus (e.g., 5 mL) and place them in centrifuge tubes. Then process them into groups:

[0141] Blank control group: Add an equal volume of PBS;

[0142] Positive control group: Ambroxol (consistent with the conjugate drug) was added;

[0143] Peptide control group: Uncoupled peptides (concentration of 0.5 mg / mL, calculated as peptides) were added.

[0144] Drug conjugate group: Add peptide conjugate (0.5 mg / mL, based on peptide).

[0145] Each group of samples was incubated in a constant temperature water bath at 37°C.

[0146] 3) Viscosity measurement

[0147] Immediately after incubation, the viscosity of each group of samples was measured using a viscometer (unit: mPa·s).

[0148] Measurement conditions: Set the rotation speed (e.g., 50-200 rpm, adjust according to the sample viscosity to avoid excessive shear force damaging the mucus structure), measure each sample 3 times, and take the average value.

[0149] 4) Data Calculation

[0150] Viscosity reduction rate (%) = [(Viscosity of blank group - Viscosity of drug-treated group) / Viscosity of blank group] × 100%

[0151]

[0152] 5) Experimental Results

[0153] If the viscosity reduction rate is positive, it indicates that the drug has a viscosity-reducing effect; the higher the value, the stronger the effect. In terms of viscosity reduction effect, the order is: conjugate drug 1 > conjugate drug 3 > conjugate drug 2 > conjugate drug 4 > ambroxol > peptide. The expectorant activity of conjugate drugs is significantly higher than that of peptide drugs.

[0154] Example 4: Study on drug exposure of peptide-conjugated drugs

[0155] 1) Experimental materials:

[0156] Experimental animals: SD rats

[0157] Test drugs: peptides, peptide-conjugated drugs

[0158] 2) Experimental Grouping:

[0159] Test drug group:

[0160] I. Polypeptide group: 5mg / kg

[0161] II. Peptide-conjugated drug group: 5 mg / kg (peptide content)

[0162] 3) Route of administration: inhalation, injection

[0163] 4) Experimental steps:

[0164] As mentioned earlier, the experiment was divided into 3 groups, with 6 animals in each group.

[0165] Animals in group I were given the polypeptide drug at a dose of 5 mg / kg;

[0166] Animals in group II of the test drug group were given the polypeptide conjugate at a dose of 5 mg / kg (based on polypeptide content).

[0167] Blood samples were collected before drug administration (0 h) and at 0.5, 1, 1.5, 2, 4, 8, 12, 24, and 36 h after drug administration to measure blood drug concentrations. The area under the curve (AUC) and half-life (t) were calculated. 1 / 2 .

[0168] 5) Experimental results:

[0169]

[0170] Comparing inhalation and intravenous administration: Intravenous administration increases drug exposure (AUC) compared to inhalation, but both peptides and ambroxol act on the lungs, while inhalation can act directly on the target site, reducing systemic side effects.

[0171] Compare the half-life (t) of conjugates and peptides 1 / 2 The half-life of conjugated drugs is significantly longer than that of single peptides, and conjugated drugs prolong drug retention time.

[0172] Comparison of drug exposure (AUC) between conjugates and peptides: When both are administered by inhalation, conjugates, compared with peptides alone, can prolong the drug half-life, greatly increase the overall drug exposure, and reduce the number of administrations.

[0173] Summarize

[0174] Novel coronavirus infection is an acute respiratory infectious disease. Common signs include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing.

[0175] Ambroxol has a natural affinity for respiratory epithelial cells (its target is concentrated in the respiratory mucosa). After conjugation, it can "directly deliver" membrane fusion inhibitors to the core areas of viral infection (such as the nasopharynx, trachea, and alveolar epithelium), increasing local drug concentration and enhancing the direct inhibitory effect of the membrane fusion inhibitor on the virus. Simultaneously, ambroxol inhibits viral spread by clearing mucus, reducing inflammation, and disrupting the viral survival and transmission environment in the respiratory tract (e.g., reducing viral load in mucus and decreasing inflammation-induced "cellular susceptibility"). Furthermore, the two components have completely independent targets (one targets the viral S protein, and the other regulates host respiratory function). Conjugation avoids the selective pressure of single-target drugs and reduces the possibility of viral resistance due to mutation.

[0176] In summary, the developed peptide-conjugated drugs are more potent than the monopeptides and exhibit significant synergistic effects.

[0177] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A polypeptide-conjugate drug, characterized by, The polypeptide conjugated drug comprises, in sequence, an anti-viral polypeptide, a connecting polypeptide, an amino acid, and ambroxol.

2. The polypeptide-conjugate drug according to claim 1, wherein The amino acid sequence of the anti-viral polypeptide is SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK. Preferably, a protecting group exists at the amino terminal of the anti-viral polypeptide, and the protecting group is acetyl.

3. The polypeptide-conjugate drug according to claim 1 or 2, characterized in that, The amino acid sequence of the connecting polypeptide is EAAAK. Preferably, the amino acid is lysine. Preferably, the anti-viral polypeptide, the connecting polypeptide, and the amino acid are connected by an amide bond.

4. The polypeptide-conjugate drug according to any one of claims 1 to 3, characterized in that, The amino acid is connected to ambroxol by a chemical bond. Preferably, the chemical bond is a nitrogen-carbon single bond or an amide bond.

5. The polypeptide-conjugate drug according to claim 4, wherein The connection is either through the amino terminal of the amino acid straight chain or branched chain to the hydroxyl terminal or bromine terminal of ambroxol, or through the carboxyl terminal of the amino acid straight chain or branched chain to the amino terminal of ambroxol.

6. The polypeptide-conjugate drug according to any one of claims 1 to 5, wherein A protecting group exists at the carboxyl terminal of the amino acid not involved in conjugation, and the protecting group is amino.

7. The polypeptide-conjugate drug according to any one of claims 1 to 6, wherein A modifying group exists or not at the amino terminal of the amino acid not involved in conjugation, and when the modifying group exists, the modifying group is cholesterolic succinic acid monolipid.

8. The polypeptide-conjugate drug according to any one of claims 1 to 7, wherein The polypeptide conjugated drug is selected from the following structures: 、 、 、 ； X is Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAK-.

9. An antiviral, expectorant, respiratory modulating pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide conjugated drug of any one of claims 1-8.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Preferably, the pharmaceutically acceptable excipient comprises any one or a combination of at least two of a carrier, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure adjusting agent, a surfactant, a coating material, a coloring agent, a pH adjusting agent, an antioxidant, a bacteriostatic agent, or a buffer.

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