WO2023232095A9 - 一种基于病毒阻断剂的口腔喷雾制剂及其用途 - Google Patents

一种基于病毒阻断剂的口腔喷雾制剂及其用途 Download PDF

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WO2023232095A9
WO2023232095A9 PCT/CN2023/097612 CN2023097612W WO2023232095A9 WO 2023232095 A9 WO2023232095 A9 WO 2023232095A9 CN 2023097612 W CN2023097612 W CN 2023097612W WO 2023232095 A9 WO2023232095 A9 WO 2023232095A9
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blocking agent
oral spray
spray preparation
virus blocking
virus
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PCT/CN2023/097612
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French (fr)
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WO2023232095A1 (zh
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郭敏
刘章
董海涛
伍志
谢高伟
于雪
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康码（上海）生物科技有限公司
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Publication of WO2023232095A1 publication Critical patent/WO2023232095A1/zh
Publication of WO2023232095A9 publication Critical patent/WO2023232095A9/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the invention belongs to the field of antiviral drugs, and specifically relates to an oral spray preparation based on a virus blocking agent and its use.
  • the new coronavirus (SARS-CoV-2) is a new coronavirus of the beta genus. After human infection, it can cause upper respiratory tract infection and acute distress syndrome. There is currently a lack of effective antiviral drugs. Although it is currently known that small molecule drugs such as mdesivir and Paxlovid have been confirmed to have certain efficacy against new coronavirus infection (John H Beigel.etal., N Engl J Med. 2020 Nov 5; 383(19):1813-1826.doi: 10.1056/NEJMoa2007764.), however, complete control of the new coronavirus epidemic has not yet been achieved.
  • Spray spray preparations are used for inhalation into the lungs or spraying directly into the mucous membrane, skin and space of the oral cavity for disinfection. They can directly reach the site of action.
  • the drug works quickly. Improving bioavailability can avoid gastrointestinal side effects and is effective against the new coronavirus ( For infectious diseases caused by SARS-CoV-2), it is still the primary treatment drug.
  • Patent CN113440563A discloses a compound Houttuynia cordata spray preparation. Its composition includes Houttuynia cordata, honeysuckle, forsythia suspensa, skullcap, isatis root and mint. It does not need to add auxiliary materials and can be directly applied to mucous membranes and skin parts locally.
  • the compound formula The EC50 of the in vitro inhibitory activity of Houttuynia cordata spray formulation against SARS-CoV-2 is 17 ⁇ L/m.
  • Patent CN1133332243A discloses a plant-derived antibacterial and anti-new coronavirus, which uses Polygonum cuspidatum extract, Senecio Extract, Scutellaria baicalensis extract, Sophora flavescens extract, and Cortex Phellodendron extract as plant raw materials, combined with nanofilm-forming agents and tea aromatic extracts.
  • the formulated spray spray preparation was tested for killing the new coronavirus, and it was confirmed that the antiviral activity rate was as high as over 99.99%, and it was experimentally proven to have no skin irritation.
  • Patent CN111658664A discloses an inhalant prepared with sea cucumber polysaccharide as the main active component, which can enter the nasal cavity, trachea, and lungs to prevent the virus from infecting the matrix tissue, and is used to prevent and treat the new coronavirus.
  • Patent CN111529566A discloses an anti-COVID-19 spray preparation, including Phyllanthus emblica extract, metal salts and solvents, which has been proven to have a good inhibitory effect on the COVID-19 through mouse experiments.
  • Patent CN111297838A discloses an inhalation spray preparation of antiviral drugs, using a spray selected from the group consisting of hydroxychloroquine sulfate, chloroquine phosphate, favipiravir, remdesivir, Kaletra, Arbidol, and oseltamivir phosphate.
  • a spray selected from the group consisting of hydroxychloroquine sulfate, chloroquine phosphate, favipiravir, remdesivir, Kaletra, Arbidol, and oseltamivir phosphate.
  • ribavirin, rimantadine hydrochloride or amantadine hydrochloride are used as antiviral active agents, and are used in combination with additives, and are inhaled into the patient's respiratory tract at the same time to produce a synergistic effect, eliminating the virus at the same time Treats severe respiratory and lung infections caused by COVID-19.
  • Patent CN111419787A discloses a chloroquine spray and its preparation method.
  • the spray consists of two parts: powder and solution.
  • the powder contains chloroquine or chloroquine derivatives, carrier macromolecule materials and sweeteners, and the solution A saline solution containing borneol and/or menthol.
  • the carrier macromolecule material is used to wrap chloroquine to extend the residence time of chloroquine spray on the oral mucosa or nasal mucosa, and achieve a sustained release and long-lasting effect.
  • supplemented with menthol/borneol/ethanol can significantly improve the antibacterial and disinfection effect of chloroquine.
  • Patent CN113491676 A discloses a ribavirin aerosol absorption solution, each ml solution includes: 1) 0.005-5g ribavirin or its pharmaceutically acceptable salt; 2) 0.1-0.5mg isotonic agent; 3) 0.2-0.8mg buffer salt; 4) 0.3-4.0mg penetration enhancer; 5) 0-50mg cyclodextrin; 6) 0.1-1.0mg solvent.
  • the use of phospholipids as penetration enhancers improves the solubility of the drug in liquid state
  • Penetration enhancers improve the delivery ratio in the respiratory tract, especially the alveoli, and accelerate absorption.
  • the pharmaceutical preparation provides a therapeutic drug and treatment plan that are lacking in the existing technology, including accurate dosage, high quality and stable drug quality, and safe and simple clinical application.
  • the spike protein of the Omicron strain is more prevalent, with 37 mutations compared to the original strain, 15 of which are located in the RBD, resulting in a higher affinity between Omicron and ACE2, resulting in Omicron Rong's significant spread and significant neutralizing antibody immune escape. Therefore, new strategies for drug development targeting these variants become an urgent task.
  • ACE2 mimetics are expected to compete with human ACE2 for binding to the RBD and have been reported to inhibit variants of concern in vitro and in vivo (Miller, A. et al. A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection. Sci Rep 11,10617(2021)).
  • the present invention provides an oral spray preparation based on a virus blocking agent, using a new and newly designed ACE2 mimicking protein virus blocking agent as an antiviral active agent, which is effective against all coronaviruses.
  • Viruses in tubes that infect cells through ACE2 have significant neutralizing effects, low clinical toxicity, and good storage stability.
  • the spray preparation is a preparation that can be inhaled into the lungs or sprayed directly to the mucous membranes of the oral cavity, nasal cavity and other cavities.
  • An oral spray preparation based on a virus blocking agent characterized in that it includes the following components: a therapeutically effective content of a virus blocking agent, an additive, and a solvent;
  • the virus blocking agent is a coronavirus spike protein (RBD) blocking agent
  • the additives are selected from self-solvents, stabilizers, flavoring agents, antioxidants, preservatives, osmotic pressure regulators, pH regulators or combinations thereof.
  • the therapeutically effective amount is 1-100 nM, preferably 10-50 nM.
  • the solvent is a hydroxyl-type polar solvent, selected from one or more of water, ethanol, polyethylene glycol, propylene glycol, glycerin, butanol or pivalol, preferably water, propylene glycol or glycerol. One or more of them, more preferably water.
  • the co-solvent is not particularly limited, and is preferably lecithin, poloxamer, hydroxypropyl- ⁇ -cyclodextrin, arginine, glycine, aspartic acid, glutamine, polysorbate 20 or Polysorbate 80, and/or pH 7.0-9.0.
  • the osmotic pressure regulator is sodium chloride, mannitol, glucose, phosphate, acetate, etc.
  • the pH adjuster is an acidic substance, an alkaline substance or; the acidic substance is selected from hydrochloric acid, acetic acid, citric acid, tartaric acid, malic acid, etc.; the alkaline substance is selected from strong potassium oxide or sodium hydroxide wait.
  • the preservative is not particularly limited, and is preferably parahydroxybenzoate, potassium sorbate, phenethyl alcohol, sodium propionate, benzalkonium chloride, etc.
  • the stabilizer is preferably tris salt buffer solution or phosphate buffer solution.
  • the antioxidant is selected from sodium sulfite, sodium ascorbate, potassium sulfite, potassium ascorbate, etc.
  • the flavoring agent is cyclamate, menthol, and sugar alcohol compounds, selected from one or more of xylitol, sorbitol, or mannitol, preferably one of xylitol or mannitol. or several, further preferably xylitol.
  • virus blocking agent includes the following structural domains: spike protein binding domain (SBD), rigid domain, oligomer site and membrane exclusion tail.
  • SBD spike protein binding domain
  • rigid domain oligomer site
  • membrane exclusion tail oligomer site
  • different structural domains of the virus blocking agent are connected by covalently linked linking groups.
  • the linking groups are selected from polypeptides or disulfide bonds, and there are The connection sequence is not particularly limited.
  • the spike protein binding domain (SBD), rigid domain, oligomer site and membrane exclusion tail of the blocker are formed by pressing from N The direction from segment to segment C is connected in sequence.
  • the spike protein binding domain is a region that specifically binds to the SARS-CoV-2 spike protein.
  • the present invention designs Spike binding domains of various lengths (10-20 residues) based on the ACE2 binding site.
  • the spike binding domain includes a polypeptide shown in any of the amino acid sequences of SEQ ID NO.1-3 and SEQ ID NO.6-17:
  • the spike binding domain includes at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identical polypeptides.
  • the spike binding domain includes a sequence with one or several amino acid substitutions, deletions or additions to the polypeptide shown in any of the amino acid sequences of SEQ ID NO.1-3 and SEQ ID NO.6-17.
  • the number of substituted, deleted or added amino acids may be 1-20, preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the blocking agent in order to increase the binding affinity of the blocking agent to the spike protein, contains at least one spike binding domain.
  • the virus blocking agent has two spike binding domains, preferably including More than two complementarity determining regions, such as 1-10, preferably 2-8, most preferably 4-8.
  • the rigid domain can maintain the three-dimensional structure and improve the biological activity of the blocking agent.
  • the rigid domain includes eGFP (SEQ ID NO. 4), or rigid proteins improved on the basis of eGFP such as mTagBFP2, moxCerulean3, AmCyanl, MiCy, ZsGreen, Clover, mVenus, ZsYellow 1, mKO2, TurboRFP, tdTomato, eqFP611 , mKate1.3, mNeptune2, miRFP670, mAme-trine, PAmCherry 2, mEos3.2, etc.
  • eGFP SEQ ID NO. 4
  • rigid proteins improved on the basis of eGFP such as mTagBFP2, moxCerulean3, AmCyanl, MiCy, ZsGreen, Clover, mVenus, ZsYellow 1, mKO2, TurboRFP, tdTomato, eqFP611 , mKate
  • the rigid domain comprises at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, with the polypeptide shown in the amino acid sequence of SEQ ID NO.4. More preferably a polypeptide is at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identical.
  • the membrane-repellent tail further causes the blocker complex to repel the host cell membrane, thus blocking viral entry into the host cell.
  • the modularly assembled blockers are expected to impair the cell attachment capabilities of SARS-CoV-2 variants.
  • the membrane repellent tail is a negatively charged amino acid short chain polymer, and the number of amino acids in the short chain polymer is 0-50, preferably 2-40, more preferably 3-30, most preferably 4-20 .
  • the negatively charged amino acids are aspartic acid and/or glutamic acid.
  • the oligomer sites promote blocker oligomerization and aggregation of virus-blocker complexes.
  • the oligomer site is a single-chain protein or polypeptide with a polymerization function, and the polymerization function means that the single-chain protein has the function of naturally polymerizing into a multimer.
  • the polymerization function means that the single-chain protein has the function of naturally polymerizing into multimers.
  • the multimer includes but is not limited to oligomers with a monomer number of 2-10, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, etc.
  • the single-chain protein with polymerization function is derived from natural polymeric proteins and functional variants thereof.
  • the single-chain protein with polymerization function has a relationship with a single peptide chain of streptavidin protein (SEQ ID NO. 5) of at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, More preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • SEQ ID NO. 5 single peptide chain of streptavidin protein
  • the virus blocking agent preferably has a similarity of at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably between the Constine TM blocking protein or its amino acid sequence.
  • the oral spray preparation optionally also contains dietary supplements;
  • the dietary supplements include but are not limited to antioxidants, vitamins, minerals, amino acids and other approved dietary supplements.
  • the vitamins are selected from, but are not limited to, one of vitamin A, vitamin B complex, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, vitamin P, vitamin PP, vitamin M, vitamin T, and vitamin U. or more.
  • the vitamin B complex includes, but is not limited to: pantothenic acid, niacin, biotin, folic acid, vitamin B1 (thiamine), vitamin B2 (riboflavin), pyridoxine (vitamin B6) and cyanocobalamin ( Vitamin B12).
  • the blocker of the present invention can be used in combination with another ACE inhibitor, and the ACE inhibitor is selected from the group consisting of Captopril, Enalapril, Benazepril, Fosinopril, Lisinopril, Quinapril, Cozaar, Valsartan, Irbesartan (Irbesartan), Candesartan (Candesartan), Eprosartan (Eprosartan), etc.
  • the ACE inhibitor is selected from the group consisting of Captopril, Enalapril, Benazepril, Fosinopril, Lisinopril, Quinapril, Cozaar, Valsartan, Irbesartan (Irbesartan), Candesartan (Candesartan), Eprosartan (Eprosartan), etc.
  • the blocker of the present invention can be used in combination with other antiviral drugs.
  • the other antiviral drugs are anti-novel coronavirus COVID-19 and are selected from the group consisting of oseltamivir, lopi Navir, ritonavir, chloroquine or hydroxychloroquine, azithromycin, clarithromycin, doxycycline, ivermectin, nelfinavir, ribavirin, favipiravir, amantaine, Amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, comparable One or more of combivir, complera, darunavir, delavirdine, didanosine interferon.
  • Most of the other antiviral drugs are broad-spectrum drugs, which are effective against diseases caused by multiple epidemic viruses at the same time.
  • virus blocking agent is produced through protein engineering technology. After encoding and optimization, the blocking agent gene is cloned into an expression vector and then transferred to host cells or in vitro cell-free reaction systems. After incubation, it is purified. Get virus blockers.
  • virus blocking agent is produced through protein engineering technology. After encoding and optimization, the blocking agent gene is cloned into an expression vector and then transferred to host cells or in vitro cell-free reaction systems. After incubation, it is purified. Get virus blockers.
  • the blocked product is preferably produced by the D2P technology of Kangma (Shanghai) Biotechnology Co., Ltd., such as the D2P system.
  • the production by the D2P technology includes the following steps: the gene is optimized for coding and cloned into the pD2P vector.
  • the plasmid was amplified using the Ampi system and then added to the Protein Factory Rapid Reaction System (Conma (Shanghai) Biotechnology Co., Ltd.) with a volume ratio of 1:30.
  • the reaction mixture was incubated at 30 °C for 4 h and then collected and purified by centrifugation.
  • Another aspect of the present invention provides a method or use for preventing and treating diseases related to coronavirus infection, which includes using the spray spray formulation of the present invention for preventing and treating diseases related to coronavirus infection.
  • Another aspect of the present invention provides a method for preventing and treating diseases associated with coronavirus infection in a subject, the method comprising administering to a subject in need thereof an effective amount of an antiviral drug inhalation spray according to the present invention Spray formulation.
  • the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2; in a more preferred embodiment, the coronavirus is SARS-CoV-2.
  • the disease associated with coronavirus infection is COVID-19 or Middle East Respiratory Syndrome (MERS) or Severe Acute Respiratory Syndrome (SARS); in a more preferred embodiment, the coronavirus infection
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the coronavirus infection is COVID-19.
  • Another aspect of the present invention provides a use of the spray spray preparation of the present invention for preparing a medicament for preventing and treating diseases related to coronavirus infection
  • the medicament is used to inhibit the fusion of coronavirus and cells, or is used to prevent and treat diseases related to coronavirus infection.
  • /Treat diseases related to coronavirus infection the medicament is used to inhibit the fusion of the coronavirus SARS-CoV, MERS-CoV or SARS-CoV-2 with cells, or to prevent the coronavirus SARS-CoV, MERS-CoV or SARS-CoV -2 Infection-related diseases.
  • the coronavirus is SARS-CoV-2.
  • the disease associated with coronavirus infection is COVID-19 or Middle East Respiratory Syndrome (MERS) or Severe Acute Respiratory Syndrome (SARS); in a more preferred embodiment, the coronavirus infection The disease associated with the infection is COVID-19.
  • the present invention uses a newly designed ACE2-mimetic protein virus blocker as an antiviral active agent, which has a significant neutralizing effect on all coronaviruses that infect cells through ACE2, and has high affinity and multi-angle capture of adherent new coronaviruses. characteristics, thereby causing the virus to lose its function and pathway of infecting the body, ultimately achieving the effect of blocking the spread and infection of the new coronavirus. It has low clinical toxicity and good storage stability, providing a new method for clinical prevention and treatment of SARS-CoV-2 infection. s solution.
  • the present invention provides a method for improving protein expression efficiency in vitro.
  • examples are given below to further illustrate the present invention. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.
  • Coronavirus in the present invention refers to coronaviruses that belong to the genus Coronavirus in the order Nidovirales, family Coronaviridae, and use ACE2 as a binding receptor, including but not Limited to SARS-CoV, MERS-CoV, SARS-CoV-2, etc.
  • ACE2 of the present invention is also called ACEH, which is called angiotensin-converting enzyme 2.
  • ACE2 consists of 805 amino acids and is a type I transmembrane glycoprotein with a single extracellular catalytic domain.
  • ACE2 is the receptor protein used by coronaviruses such as SARS-Cov-2 to infect human cells.
  • Spike protein in the present invention means that it is on the surface of coronavirus and can directly (for example, by interacting with a viral receptor (such as Ace2)) or indirectly (for example, by mediating one or more other proteins or molecules) Interaction with viral receptors (such as Ace2) mediates viral attachment to target host cells.
  • a viral receptor such as Ace2
  • Coronavirus infection in the present invention refers to infection with coronavirus, such as SARS-CoV-2, MERSCoV or SARS-CoV.
  • coronavirus respiratory infections usually in the upper respiratory tract, lower respiratory tract.
  • Complications are mainly fever, which may be combined with symptoms such as mild dry cough, fatigue, difficulty breathing, diarrhea, runny nose, and sputum production, pneumonia, renal failure, renal dysfunction, septic shock, and death in severe cases.
  • Effective amount and “therapeutically effective amount” in the present invention refer to the amount of virus blocking agent in the oral spray formulation that can effectively treat viral infection or related diseases or conditions in a subject or mammal.
  • a therapeutically effective dose of reagent has a therapeutic effect, so that the virus can lose its function and pathway of infecting the body, and ultimately achieve the effect of blocking the spread and infection of the new coronavirus.
  • this reagent prevents virus transmission and infection, it can be regarded as a neutralizing ability, using high affinity to capture the characteristics of adhering to the new coronavirus from multiple angles, thereby causing the virus to lose its function and pathway of infecting the body.
  • the therapeutically effective amount is 1-100 nM, preferably 10-50 nM.
  • the "D2P" system in the present invention includes but is not limited to IVTT reaction (in vitro transcription and translation reaction).
  • IVTT reaction is preferred.
  • the IVTT reaction corresponding to the IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also call this type of in vitro protein synthesis system, D-to-P system, D_to_P system, and DNAto-Protein.
  • the corresponding in vitro protein synthesis method is also called the D2P method, D-to-P method, D_to_P method, and DNA-to-Protein method, which is related to the "in vitro cell-free protein synthesis system", “in vitro expression system”, " Expressions such as “in vitro protein synthesis system”, “in vitro protein synthesis reaction system” and “cell-free protein synthesis system” have the same meaning.
  • in vitro protein synthesis system in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS System (cell-free protein synthesis system) and other description methods.
  • CFS system cell-free system
  • CFPS System cell-free protein synthesis system
  • RNA polymerase The protein components required in the in vitro cell-free protein synthesis system of the present invention (for example, RNA polymerase) can be provided endogenously or added exogenously.
  • endogenous means reference can be made to documents including but not limited to CN108690139A, CN109423496A, CN106978439A, CN110408635A, CN110551700A, CN110093284A, CN110845622A, CN110938649A, CN111378708A, CN1 11484998 A.
  • virus blocker An oral spray preparation based on a virus blocker, including the following mass percentages: virus blocker 1-100nM, additives, and the remainder is solvent; the additives are co-solvents, stabilizers, and flavoring agents agent, antioxidant, preservative, osmotic pressure regulator, pH regulator or a combination thereof; the virus blocking agent is a spike protein (RBD) blocker.
  • the virus blocking agent is preferably SARS-CoV-2 spike protein (RBD) blocker.
  • the spray preparation of the present invention is used to directly act on the oral cavity and/or nasal cavity and upper respiratory tract during the outbreak of novel coronavirus (SARS-CoV-2) and other coronaviruses, and then enter the lungs through breathing for precise targeted administration to achieve The purpose of preventing the infection and spread of the new coronavirus.
  • SARS-CoV-2 novel coronavirus
  • the ACE2 mimetic protein used in the present invention can inhibit the attachment of viruses to host cells through competitive binding with RBD. It has high binding ability and can significantly reduce drug dosage, thus effectively reducing side effects such as clinical toxicity caused by drugs.
  • Spray preparations are administered by inhalation using an atomizer, which not only has the characteristics of high efficiency and quick effect, but can directly reach the nose, mouth, upper respiratory tract and lung lesions, increasing the local drug concentration, and the lung absorption speed is as fast as intravenous injection; lung The absorption area is huge, with a total absorption area of 70 to 100 m2, which is equivalent to 25 times the body surface area; the alveolar sacs have thin walls, composed of a single layer of epithelial cells, close to a dense capillary network, and the thickness of the cell wall or capillary wall is only 0.5 to 1 ⁇ m; Drug enzyme activity is lower than that of the gastrointestinal tract, and the pH is suitable.
  • the drug stays in the oral and nasal cavities; when 10 ⁇ m > atomized particle size > 5 ⁇ m, the drug can reach the first 6 bronchi of the lower respiratory tract; when 5 ⁇ m > atomized particle size > 1 ⁇ m, the drug can reach It reaches the terminal alveoli of the lungs (in the terminal 5th to 6th level bronchi). If the particle size is too small, 0.1 ⁇ m ⁇ atomized particle size ⁇ 1 ⁇ m, the probability of aerosol exhalation will increase significantly, the deposition rate will decrease sharply, and the bioavailability will decrease.
  • the concentration specification of the virus blocking agent and additive is 1 mg to 300 mg/ml, preferably 1 mg/ml, 5 mg/ml, 10 mg/ml, 25 mg/ml, 50 mg/ml, 100 mg/ml, and more preferably 10 mg/ml. ml, 50mg/ml, 100mg/ml.
  • the oral spray preparation is filled in an atomizing device, and the atomizing device is preferably an air compression atomizer, a heated atomizer, a vibrating mesh micropore atomizer or an ultrasonic atomizer. Air compression atomizers and vibrating screen micropore atomizers are more suitable for the atomizer of the present invention.
  • the spray preparation is preferably a solution aerosol, a suspension aerosol or an emulsion aerosol.
  • the mass percentage of the virus blocking agent is preferably 2-50nM, most preferably 5-20nM;
  • the mass percentage of the additive is 0.001%-50%, preferably 0.01%-20%, and most preferably 0.1%-20%.
  • the solvent is a hydroxyl-type polar solvent, selected from one or more of water, ethanol, polyethylene glycol, propylene glycol, glycerol, butanol or pivalol, preferably water, propylene glycol or glycerol. One or more of them, more preferably water.
  • the additive is a co-solvent, a stabilizer, a flavoring agent, an antioxidant, a preservative, an osmotic pressure regulator, a pH regulator or a combination thereof.
  • the co-solvent is not particularly limited, and is preferably lecithin, poloxamer, hydroxypropyl- ⁇ -cyclodextrin, arginine, glycine, aspartic acid, glutamine, polysorbate 20 or Polysorbate 80, and/or pH 7.0-9.0.
  • the osmotic pressure regulator is sodium chloride mannitol, glucose, phosphate, acetate, etc.
  • the pH adjuster is an acidic substance, an alkaline substance or; the acidic substance is selected from hydrochloric acid, acetic acid, citric acid, tartaric acid, malic acid, etc.; the alkaline substance is selected from strong potassium oxide or sodium hydroxide wait.
  • the preservative is not particularly limited, and is preferably parahydroxybenzoate, potassium sorbate, phenethyl alcohol, sodium propionate, benzalkonium chloride, etc.
  • the stabilizer is preferably tris salt buffer solution or phosphate buffer solution.
  • the antioxidant is selected from sodium sulfite, sodium ascorbate, potassium sulfite, potassium ascorbate, etc.
  • the flavoring agent is cyclamate, menthol, and sugar alcohol compounds, selected from one or more of xylitol, sorbitol, or mannitol, preferably one of xylitol or mannitol. or several, further preferably xylitol.
  • SARS-CoV-2 spike protein The interaction between the SARS-CoV-2 spike protein and ACE2 is the first step in the virus's life cycle, so blocking this interaction is a promising treatment option for COVID-19. To this end, we designed de novo a series of potential SARS-CoV-2 spike protein blockers.
  • virus blocking agent includes the following structural domains: spike protein binding domain (SBD), rigid domain, oligomer site and membrane exclusion tail.
  • SBD spike protein binding domain
  • rigid domain oligomer site
  • membrane exclusion tail oligomer site
  • the linking group is selected from polypeptides or disulfide bonds, and there are The connection sequence is not particularly limited.
  • the spike protein binding domain (SBD), rigid domain, oligomer site and membrane exclusion tail of the blocker are composed of N The direction from segment to segment C is connected in sequence.
  • the spike protein binding domain is a region that specifically binds to the SARS-CoV-2 spike protein.
  • the present invention designs Spike binding domains of various lengths (10-20 residues) based on the ACE2 binding site.
  • the spike binding domain includes a polypeptide shown in any of the amino acid sequences of SEQ ID NO.1-3 and SEQ ID NO.6-17:
  • the spike binding domain includes at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identical polypeptides.
  • the spike binding domain includes a sequence with one or several amino acid substitutions, deletions or additions to the polypeptide shown in any of the amino acid sequences of SEQ ID NO.1-3 and SEQ ID NO.6-17.
  • the number of substituted, deleted or added amino acids may be 1-20, preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the blocking agent in order to increase the binding affinity of the blocking agent to the spike protein, contains at least one spike binding domain.
  • the virus blocking agent has two spike binding domains, preferably including More than two complementarity determining regions, such as 1-10, preferably 2-8, most preferably 4-8.
  • the rigid domain can maintain the three-dimensional structure and improve the biological activity of the blocking agent.
  • the rigid domain includes eGFP (SEQ ID NO. 4), or rigid proteins improved on the basis of eGFP such as mTagBFP2, moxCerulean3, AmCyanl, MiCy, ZsGreen, Clover, mVenus, ZsYellow 1, mKO2, TurboRFP, tdTomato, eqFP611 , mKate1.3, mNeptune2, miRFP670, mAme-trine, PAmCherry 2, mEos3.2, etc.
  • eGFP SEQ ID NO. 4
  • rigid proteins improved on the basis of eGFP such as mTagBFP2, moxCerulean3, AmCyanl, MiCy, ZsGreen, Clover, mVenus, ZsYellow 1, mKO2, TurboRFP, tdTomato, eqFP611 , mKate
  • the rigid domain comprises at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, with the polypeptide shown in the amino acid sequence of SEQ ID NO.4. More preferably a polypeptide is at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identical.
  • the membrane-repellent tail further causes the blocker complex to repel the host cell membrane, thus blocking viral entry into the host cell.
  • the modularly assembled blockers are expected to impair the cell attachment capabilities of SARS-CoV-2 variants.
  • the membrane repellent tail is a negatively charged amino acid short chain polymer, and the number of amino acids in the short chain polymer is 0-50, preferably 2-40, more preferably 3-30, most preferably 4-20 .
  • the negatively charged amino acids are aspartic acid and/or glutamic acid.
  • the oligomer sites promote blocker oligomerization and aggregation of virus-blocker complexes.
  • the oligomer site is a single-chain protein or polypeptide with a polymerization function, and the polymerization function means that the single-chain protein has the function of naturally polymerizing into a multimer.
  • the multimer includes but is not limited to oligomers with a monomer number of 2-10, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, etc.
  • the single-chain protein with polymerization function is derived from natural polymeric proteins and functional variants thereof.
  • the single-chain protein with polymerization function has a relationship with a single peptide chain of streptavidin protein (SEQ ID NO. 5) of at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, More preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • SEQ ID NO. 5 single peptide chain of streptavidin protein
  • the virus blocking agent preferably has a similarity of at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least A protein that is 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identical.
  • the virus blocking agent preferably has a similarity of at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably between the Constine TM blocking protein or its amino acid sequence.
  • the Konstin protein spike protein binding domain (SBD), rigid structural domain, oligomer site and membrane exclusion tail are connected in sequence from the N segment to the C segment, wherein the spike protein binding domain contains SEQ ID
  • the spike binding domain of NO.2 and SEQ ID NO.4 contains the sequence shown in SEQ ID NO.5, and the membrane exclusion tail is 10E (SEQ ID NO.18:EEEEEEEEEE).
  • the oral spray preparation optionally also contains dietary supplements;
  • the dietary supplements include but are not limited to antioxidants, vitamins, minerals, amino acids and other approved dietary supplements.
  • the vitamins are selected from, but are not limited to, one of vitamin A, vitamin B complex, vitamin C, vitamin D, vitamin E, vitamin K, vitamin H, vitamin P, vitamin PP, vitamin M, vitamin T, and vitamin U. or more.
  • the vitamin B complex includes, but is not limited to: pantothenic acid, niacin, biotin, folic acid, vitamin B1 (thiamine), vitamin B2 (riboflavin), pyridoxine (vitamin B6) and cyanocobalamin ( Vitamin B12).
  • the blocker of the present invention can be used in combination with another ACE inhibitor, and the ACE inhibitor is selected from the group consisting of Captopril, Enalapril, Benazepril, Fosinopril, Lisinopril, Quinapril, Cozaar, Valsartan, Irbesartan (Irbesartan), Candesartan (Candesartan), Eprosartan (Eprosartan), etc.
  • the ACE inhibitor is selected from the group consisting of Captopril, Enalapril, Benazepril, Fosinopril, Lisinopril, Quinapril, Cozaar, Valsartan, Irbesartan (Irbesartan), Candesartan (Candesartan), Eprosartan (Eprosartan), etc.
  • the blocker of the present invention can be used in combination with other antiviral drugs.
  • the other antiviral drugs are anti-novel coronavirus COVID-19 and are selected from the group consisting of oseltamivir, lopi Navir, ritonavir, chloroquine or hydroxychloroquine, azithromycin, clarithromycin, doxycycline, ivermectin, nelfinavir, ribavirin, favipiravir, amantaine, Amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, comparable One or more of combivir, complera, darunavir, delavirdine, didanosine interferon.
  • Most of the other antiviral drugs are broad-spectrum drugs, which are effective against diseases caused by multiple epidemic viruses at the same time.
  • virus blocking agent is produced through protein engineering technology. After encoding and optimization, the blocking agent gene is cloned into an expression vector and then transferred to host cells or in vitro cell-free reaction systems. After incubation, it is purified. Get virus blockers.
  • virus blocking agent is produced through protein engineering technology. After encoding and optimization, the blocking agent gene is cloned into an expression vector and then transferred to host cells or in vitro cell-free reaction systems. After incubation, it is purified. Get virus blockers.
  • the host cell is well known in the art, including but not limited to E. coli, CHO cells, Chinese hamster ovary, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 293 cells and many other cell lines. Spodoptera frugiperda or Trichoplusiani), amphibian cells, bacterial cells, plant cells and fungal cells.
  • Fungal cells include yeast and filamentous fungal cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia coklama Pichia koclamae), Pichia membranaefaciens, Pichia minuta (methanol-inducible yeast (Ogataeaminuta), Pichia lindneri), Pichia opuntiae , Pichia thermotolerans, Pichia salictaria, Pichia g uercuum, Pichia pijperi, Pichiastiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenulapolymorpha, Kluyveromyces sp.), Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
  • the in vitro cell-free protein synthesis system includes, but is not limited to, E. coli in vitro protein synthesis system, bacterial in vitro protein synthesis system, mammalian cell (such as HF9, Hela, CHO, HEK293) in vitro protein synthesis system, plant cell in vitro protein synthesis system.
  • Protein synthesis system yeast cell in vitro protein synthesis system, insect cell in vitro protein synthesis system.
  • yeast in vitro protein synthesis system Preferably it is a yeast in vitro protein synthesis system, more preferably it is a Kluyveromyces lactis in vitro protein synthesis system, and most preferably it is a Kluyveromyces lactis in vitro protein synthesis system.
  • Embodiments or implementation methods include but are not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN1094 23509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785 A.
  • the blocked product is preferably produced by the D2P technology of Kangma (Shanghai) Biotechnology Co., Ltd., such as the D2P system.
  • the production by the D2P technology includes the following steps: the gene is optimized for coding and cloned into the pD2P vector.
  • the plasmid was amplified using the Ampi system and then added to the Protein Factory Rapid Reaction System (Conma (Shanghai) Biotechnology Co., Ltd.) with a volume ratio of 1:30.
  • the reaction mixture was incubated at 30 °C for 4 h and then collected and purified by centrifugation.
  • Another aspect of the present invention provides a method or use for preventing and/or treating diseases related to coronavirus infection, which includes using the spray preparation of the present invention to prevent and/or treat diseases related to coronavirus infection.
  • Another aspect of the present invention provides a method for preventing and/or treating diseases related to coronavirus infection in a subject, the method comprising administering an effective amount of an antiviral drug inhalation of the present invention to a subject in need thereof.
  • Spray formulation In a preferred embodiment, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2; in a more preferred embodiment, the coronavirus is SARS-CoV-2.
  • the disease associated with coronavirus infection is COVID-19 or Middle East Respiratory Syndrome (MERS) or Severe Acute Respiratory Syndrome (SARS); in a more preferred embodiment, the coronavirus infection
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the coronavirus infection is COVID-19.
  • Another aspect of the present invention provides the use of the spray formulation of the present invention for preparing a medicament for inhibiting the fusion of coronavirus and cells, or for preventing and/or treating diseases related to coronavirus infection.
  • the medicament is used to inhibit the fusion of the coronavirus SARS-CoV, MERS-CoV or SARS-CoV-2 with cells, or to prevent the coronavirus SARS-CoV, MERS-CoV or SARS-CoV -2 Infection-related diseases.
  • the coronavirus is SARS-CoV-2.
  • the disease associated with coronavirus infection is COVID-19 or Middle East Respiratory Syndrome (MERS) or Severe Acute Respiratory Syndrome (SARS); in a more preferred embodiment, the coronavirus The disease associated with the infection is COVID-19.
  • Another aspect of the present invention provides the use of the spray formulation of the present invention for the disinfection of coronaviruses on skin and the environment.
  • the spray is used for the disinfection of coronaviruses SARS-CoV, MERS-CoV or SARS-CoV-2;
  • the coronavirus is SARS-CoV-2.
  • the optimized gene sequence of the virus blocking agent was inserted into the pD2P plasmid, and then added to the self-made Kluyveromyces lactis in vitro cell-free protein synthesis system, using Kluyveromyces lactis ( KluyveromyceslactisNRRL Y-1140) preparation in vitro.
  • the in vitro cell-free protein synthesis system used in this example includes 50% Kluyveromyces lactis cell extract (v/v), 22mM trishydroxymethylaminomethane (pH8), 90mM potassium acetate, 4.0mM Magnesium acetate, 3.0mM nucleoside tri-19-phosphate mixture, 0.16mM amino acid mixture, 22mM potassium phosphate, 0.003mg/mL amylase, 3% (w/v) polyethylene glycol (PEG-8000), 340mM maltodextrin ( Measured in glucose units, corresponding to approximately 55mg/mL), 0.04mg/mL exogenously added RNA polymerase, and 15ng/ ⁇ L target protein DNA, where 15ng/ ⁇ L is the total concentration of each fluorescent protein DNA. Place the above reaction system in an environment of 22-30°C and incubate for about 20 hours.
  • the solution was centrifuged at 4000 rpm and 4 degrees for 10 minutes. Add 5% (w/v) ammonium sulfate powder to the supernatant, stir thoroughly to dissolve, and centrifuge at 4000 rpm and 4 degrees for 10 min. Continue to add 25% (w/v) ammonium sulfate powder to the supernatant liquid, slowly add it, and stir to dissolve while adding. Centrifuge at 12000rpm 4 degrees for 10 minutes. The supernatant was discarded, and the pellet was rinsed once with 30% (w/v) ammonium sulfate solution. Centrifuge at 12000rpm 4 degrees for 10 minutes. The pellet was resuspended in PBS and fully dissolved. Centrifuge at 12000 rpm for 10 minutes at 4 degrees, filter with a 0.22 ⁇ m syringe filter, and store at 4 degrees for later use. Determine the protein concentration by BCA method, which requires no less than 1 mg/ml.
  • This embodiment provides an oral spray preparation A, which includes the following components in terms of weight percentage: 20 nM of the virus blocking agent prepared in Example 1; 0.1%% glycerol; 0.01% menthol; 0.2% sodium citrate, 0.1 % citric acid phosphate; 0.05% cyclamate; the balance is purified water to adjust the pH to 7.2.
  • This embodiment provides an oral spray preparation B, which includes the following components in terms of weight percentage: 10 nM of the virus blocking agent prepared in Example 1; 0.1% polyethylene glycol; 0.01% menthol; 0.1% sodium citrate , 0.5% citric acid; 0.05% xylitol; 0.05% vitamin C, 0.05% vitamin B1, 0.05% vitamin B6, 0.05% vitamin B3, 0.05% provitamin B5; the balance is purified water to adjust the pH to 6.9.
  • This embodiment provides an oral spray preparation C, which includes the following components in terms of weight percentage: 50 nM of the virus blocking agent prepared in Example 1; 0.2% hydroxypropyl- ⁇ -cyclodextrin; 0.01% menthol; 0.5% trisodium phosphate, 0.5% phosphoric acid; 0.5% mannitol; 0.5% vitamin C; the balance is purified water to adjust the pH to 7.1.
  • This embodiment provides an oral spray preparation D, which includes the following components in terms of weight percentage: 80 nM of the virus blocking agent prepared in Example 1; 0.2% glutamic acid; 0.3% sodium citrate, 0.3% citric acid 0.5 % honey; 0.05% vitamin B1, 0.05% vitamin B6, 0.05% provitamin B5; the balance is purified water, adjust the pH to 7.1.
  • This embodiment provides an oral spray preparation E, which includes the following components in terms of weight percentage: 3 nM of the virus blocking agent prepared in Example 1; 0.1% lecithin; 0.01% menthol; 0.2% Tween-20; 0.05% cyclamate; 0.5% vitamin C, 0.5% vitamin B1, 0.05% vitamin B6, 0.5% vitamin B3, 0.1% provitamin B5; the balance is purified water, adjust the pH to 7.2.
  • Example 2-7 The oral spray preparation prepared in Example 2-7 was subjected to accelerated testing at 37°C for 90 days, and there was no significant change in the protein content and virus neutralizing effect.
  • ICR mice were provided by the Animal Center of Nanjing Medical University.
  • Sprague Dawley (SD) rats were purchased from Pizhou Dongfang Breeding Co., Ltd.
  • New Zealand white rabbits were provided by Yizheng Anlimao Biotechnology Co., Ltd. Animals were kept at 20°C-26°C, with a relative humidity of 40%-70% within the local barrier system.
  • SPF ICR mice (18.0-22.0g) and 20 SPF SD rats (180-220g) were used for acute oral toxicity testing. The number of males and females is equal.
  • the oral spray formulations of Examples 2-6 were administered to overnight fasted mice and rats at a dose of 1 mg/Kg. The animals were monitored weekly for the next 14 days for clinical signs of toxicity and mortality (days 0, 7 and 14). Behavior, mortality, and body weight were assessed separately and dissected at the end of the observation period.
  • Acute inhalation toxicity test 20 SPF ICR mice (18.0 ⁇ 22.0g) were used for acute inhalation toxicity test. The number of males and females is equal. 2.2g of the virus blocking agent prepared in Example 1 was placed in a 220L poison exposure cabinet, and the concentration was assumed to be 10,000 mg/m3. The exposure time for this inhalation was set at 2 hours. The symptoms and deaths of mice were recorded during the 14-day observation period (days 0, 7, and 14).
  • Acute eye irritation test Three male New Zealand rabbits (2.5 ⁇ 3.5kg) were used for the acute eye irritation test. 0.1 mL of the oral spray preparation of Examples 2-6 was dropped into the conjunctival sac of the rabbit's right eye, and physiological saline was dropped into the left eye as a control. Close your eyes for 4 seconds and rinse with saline after 30 seconds. The damage and recovery of rabbit conjunctiva, iris and cornea were observed for 21 days (1 hour, 24 hours, 48 hours, 72 hours, 7 days, 14 days and 28 days). The severity of corneal damage, iris damage, conjunctival hyperemia, and conjunctival edema was scored.
  • Mouse bone marrow polychromatic erythrocyte (PCE) micronucleus test Fifty ICR mice were used for mouse bone marrow polychromatic erythrocyte micronucleus test. The number of males equals the number of females. The animals were divided into five groups of five female and five male mice. The test group was administered Kansetin once at doses of 5000, 2500 and 1250 mg/kg ⁇ bw respectively. One group served as a negative control and was treated with solvent-purified water. The other group served as a positive control and received intraperitoneal injection of 40 mg/kg ⁇ bw cyclophosphamide (CP). Test groups were exposed to Kansetin via oral administration at 0 hours and 24 hours.
  • CP cyclophosphamide
  • mice were sacrificed 6 hours after the second exposure to kansetin, and bone marrow smears were prepared. The occurrence of micronuclei was counted in 1000 polychromatic erythrocytes (PCE) per animal. Once 200 PCEs were counted, the ratio of PCEs to normochromic erythrocytes (NCEs) was determined. Statistical analysis was performed by U test.
  • PCE polychromatic erythrocytes
  • NCEs normochromic erythrocytes
  • virus blocking agent of the present invention is considered non-toxic.

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Abstract

本发明提供一种基于病毒阻断剂的口腔喷雾制剂及其用途，采用一种ACE2模拟蛋白类病毒阻断剂作为抗病毒活性剂，对冠状病毒所有通过ACE2感染细胞的管中病毒均具有中和作用，临床毒性低、储存稳定，为临床预防和治疗SARS-CoV-2感染提供解决方案。

Description

一种基于病毒阻断剂的口腔喷雾制剂及其用途 技术领域
本发明属于抗病毒药物领域，具体涉及一一种基于病毒阻断剂的口腔喷雾制剂及其用途。
 背景技术
新冠病毒(SARS-CoV-2)是一种β属新冠病毒，人体感染后，会引起上呼吸道感染、急性窘迫综合征，目前尚缺少有效的抗病毒药物。虽然目前道了mdesivir、Paxlovid等小分子药物被证实对新冠病毒的感染具有一定的疗效(John H Beigel.etal.,N Engl J Med.2020 Nov 5；383(19):1813-1826.doi:10.1056/NEJMoa2007764.)，然而仍未实现对新冠疫情的彻底控制。
喷雾剂喷雾制剂用于肺部吸入或直接喷至腔道粘膜、皮肤及空间消毒的制剂，可直接到达作用部位，药物奏效快，提高生物利用度可避免胃肠道的副作用，对新冠病毒(SARS-CoV-2)引起的感染病症而言，仍是首要考虑的治疗药物。
专利CN113440563A公开了一种复方鱼腥草喷雾剂喷雾制剂，其组方包括鱼腥草、金银花、连翘、黄芩、板蓝根和薄荷，无需添加辅料，可直接局部作用于黏膜及皮肤部位，该复方鱼腥草喷雾剂喷雾制剂对SARS-CoV-2体外抑制活性的EC50为17μL/m。专利CN1133332243A公开了一种植物源抗菌抗新冠状病毒，以虎杖提取液、千里光提取液、黄芩提取液、苦参提取液、黄柏提取液为植物原材料与纳米成膜剂、茶叶芳香提取物复配成的喷雾剂喷雾制剂，进行了新冠病毒的杀灭试验，证实抗病毒活性率高达99.99％以上，且经过实验证实无皮肤刺激性。专利CN111658664A则公开一种用海参多糖作为主要活性组分制备而成的吸入剂，进入鼻腔、气管、肺部，防止病毒的感染基体组织，用于预防治疗新冠病毒。专利CN111529566A公开了一种抗新冠病毒喷雾剂喷雾制剂，包括余甘子提取物、金属盐和溶媒，通过小鼠实验证明了对新冠病毒具有很好的抑制作用。
专利CN111297838A公开了一种抗病毒药物的吸入喷雾剂喷雾制剂，使用选自硫酸羟氯喹、磷酸氯喹、法匹拉韦、瑞德西韦、克立芝、阿比朵儿、磷酸奥司他韦、利巴韦林、盐酸金刚乙胺或盐酸金刚烷胺中的一种或几种作为抗病毒活性剂，并与附加剂联合用药，同时吸入到患者呼吸道内，产生协同作用，清除病毒的同时治疗有新冠病毒引起的严重呼吸道和肺部感染。专利CN111419787A公开了一种氯喹喷剂及其制备方法，将所述的喷剂由粉末和溶液两部分组成，所述粉末含有氯喹或氯喹衍生物以及载体大分子材料和甜味剂，所述溶液为含有冰片和/或薄荷脑的盐溶液。利用载体大分子材料包裹氯喹，延长氯喹喷剂在口腔黏膜或鼻黏膜的停留时间，并达到缓释长效的效果，同时辅以薄荷脑/冰片/乙醇可显著提高氯喹的抑菌消毒效果。本发明氯喹喷剂温和不刺激、口感好、使用方便，在新型冠状病毒等病毒防护方面，能为健康人群提供正常护理及感染或潜伏人群提供更为有效的防治措施。专利CN113491676 A公开了一种利巴韦林的雾化吸收用溶液，每ml溶液中包括：1)0.005-5g利巴韦林或者其可用药用盐；2)0.1-0.5mg等渗剂；3)0.2-0.8mg缓冲盐；4)0.3-4.0mg渗透促进剂；5)0-50mg环糊精；6)0.1-1.0mg溶媒使用磷脂作为渗透促进剂提高了药物在液态下的溶解性、渗透促进剂提高了在呼吸道尤其是肺泡内的递送比例、加速吸收。该药物制剂提供了一种现有技术所缺乏的药用剂量准确，药品质量优质、稳定，临床应用安全、简捷的治疗药品和治疗方案。
奥密克戎毒株的刺突蛋白具有更强的流行性，相对于原始毒株有37个突变，其中15个位于RBD，造成了奥秘克戎与ACE2更高的亲和力，从而导致奥密克戎的显著传播和显著地中和抗体免疫逃逸。因而，针对这些变异的药物开发的新策略成为一项紧迫的任务。ACE2模拟物有望与人类ACE2竞争结合RBD，据报道在体外和体内抑制种关注变异(Miller,A.et al.A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection.Sci Rep 11,10617(2021))。
然而，这些基于ace2的诱饵也存在中和效果差、制备工艺复杂、无法大规模生产应用且存在临床毒性和存储稳定性等缺陷，使得具有临床应用价值的全新的抗新冠病毒的喷雾制剂类药物的出现受到了极大的限制。
发明内容
本发明针对现有技术存在的上述问题，提供一种基于病毒阻断剂的口腔喷雾制剂，采用一种全新的全新设计的ACE2模拟蛋白类病毒阻断剂作为抗病毒活性剂，对冠状病毒所有通过ACE2感染细胞的管中病毒均具有显著的中和作用，临床毒性低、储存稳定性好。所述的喷雾制剂为可用作肺部吸入或直接喷至口腔、鼻腔等腔道黏膜的制剂。
本发明的目的可以通过以下技术方案来实现：
1.一种基于病毒阻断剂的口腔喷雾制剂，其特征在于：包括以下组分：治疗有效含量的病毒阻断剂，附加剂、和溶剂；
优选地，所述病毒阻断剂为冠状刺突蛋白(RBD)阻断剂；
所述附加剂选自助溶剂、稳定剂、矫味剂、抗氧剂、防腐剂、渗透压调节剂、pH调节剂或其组合。
所述治疗有效量为1-100nM，优选为10-50nM。
所述溶剂为羟基型极性溶剂，选自水、乙醇、聚乙二醇、丙二醇、丙三醇、丁醇或特戊醇的一种或几种，优选为水、丙二醇或丙三醇中的一种或几种，进一步优选为水。
所述的助溶剂没有特别地限制，优选为卵磷脂、泊洛沙姆、羟丙基-β-环糊精、精氨酸、甘氨酸、天冬氨酸、谷氨酰胺、聚山梨醇20或聚山梨酯80，和/或pH为7.0-9.0。
所述的渗透压调节剂为氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。
所述的pH调节剂为酸性物质、碱性物质或；所述的酸性物质选自盐酸、醋酸、柠檬酸、酒石酸、苹果酸等；所述的碱性物质选自强氧化钾、或氢氧化钠等。
所述的防腐剂没有特别的限制，优选为对羟基苯甲酸酯、山梨酸钾、苯乙醇、丙酸钠、苯扎氯铵等。
所述的稳定剂优选为tris盐缓冲溶液或磷酸盐缓冲溶液。
所述的抗氧化剂选自亚硫酸钠、抗环血酸钠、亚硫酸钾、抗环血酸钾等。
所述的所述矫味剂为甜蜜素、薄荷醇、糖醇类化合物，选自木糖醇、山梨醇或甘露醇中的一种或几种，优选木糖醇或甘露醇中的一种或几种，进一步优选为木糖醇。
进一步地，所述病毒阻断剂包括如下结构域：刺突蛋白结合域(SBD)、刚性结构域、寡聚体位点和膜排斥尾。
进一步地，所述病毒阻断剂的不同的结构域之间由共价连接的连接基团连接，优选地所述的所述的连接基团选自多肽或二硫键，且个结构域间的连接顺序没有特别的限制，如在一种优选的实施方案中，所述的阻断剂的刺突蛋白结合域(SBD)、刚性结构域、寡聚体位点和膜排斥尾由按从N段到C段的方向依次连接。
所述刺突蛋白结合域是与SARS-CoV-2刺突蛋白特异性性结合区域，本发明基于ACE2的结合位点设计了多种长度的Spike结合域(10-20个残基)。
进一步地，所述刺突结合域的包含如SEQ ID NO.1-3和SEQ ID NO.6-17任一氨基酸序列所示的多肽：
进一步地，所述刺突结合域包含与如SEQ ID NO.1-3和SEQ ID NO.6-17任一氨基酸序列所示的多肽具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的多肽。
进一步地，所述的刺突结合域包含与如SEQ ID NO.1-3和SEQ ID NO.6-17任一氨基酸序列所示的多肽具有一个或几个氨基酸的置换、缺失或添加的序列，所述置换、缺失或添加的氨基酸的数量可以为1-20个，优选为1-10个，如1、2、3、4、5、6、7、8、9、10。
优选地，为了增加阻断剂与刺突蛋白的结合亲和力，所述的阻断剂包含至少一个刺突结合域，最优选地，所述病毒阻断剂具有两个刺突结合域，优选包含两个以上的互补决定区，如1-10个，优选2-8个，最优选4-8个。
所述的刚性结构域可以保持三维结构并提高阻断剂的生物活性。所述的刚性结构域包含eGFP(SEQ ID NO.4)，或在eGFP基础上改进的刚性蛋白如mTagBFP2、moxCerulean3、AmCyanl、MiCy、ZsGreen、Clover、mVenus、ZsYellow 1、mKO2、TurboRFP、tdTomato、eqFP611、mKate1.3、mNeptune2、miRFP670、mAme-trine、PAmCherry 2、mEos3.2等。
进一步地，所述的刚性结构域包含与如SEQ ID NO.4氨基酸序列所示的多肽具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的多肽。
SEQ ID NO.4:
膜排斥尾巴进一步使阻断剂复合物排斥宿主细胞膜，从而阻碍病毒进入宿主细胞。组合起来，模块化组装的阻断剂预计会削弱SARS-CoV-2变体的细胞附着能力。所述的膜排斥尾巴为带有负电的氨基酸短链聚合物，所述的短链聚合物的氨基酸的个数为0-50，优选2-40，更优选3-30，最优选4-20。所述的带有负电的氨基酸为天冬氨酸和/或谷氨酸。
进一步地，所述寡聚体位点促进阻断剂的寡聚化和病毒-阻断剂复合物的聚集。所述寡聚体位点为具有聚合功能的单链蛋白、多肽，所述聚合功能是指所述单链蛋白具有天然聚合为多聚体的功能。所述聚合功能是指所述单链蛋白具有天然聚合为多聚体的功能。进一步地，所述多聚体包括但不限于单体数量为2-10的寡聚体，如二聚体、三聚体、四聚体、五聚体、六聚体、七聚体、八聚体、九聚体、十聚体及其混合物，单体数量为11-20的低聚体以及单体数量为20以上的高聚体。在一种可行实施方案中，所述具有聚合功能的单链蛋白来源于天然聚合蛋白及其功能性变体。
优选地，所述具有聚合功能的单链蛋白与链霉亲和素蛋白单条肽链(SEQ ID NO.5)具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。
SEQ ID NO.5:
进一步，所述的病毒阻断剂优选康斯汀TM阻断蛋白或与其氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的蛋白。
进一步地，所述的口腔喷雾制剂，任选地还包含膳食补充剂；所述膳食补充剂包括但不限于抗氧剂、维生素、矿物质、氨基酸及其他得到批准的膳食补充剂。
所述的维生素选自但不限于维生素A，维生素B复合体，维生素C，维生素D，维生素E，维生素K，维生素H，维生素P，维生素PP，维生素M，维生素T，维生素U中的一种或多种。
所述的维生素B复合体包括但不限于：泛酸、烟酸、生物素、叶酸、维生素B1(硫胺素)、维生素B2(核黄素)、吡哆醇(维生素B6)和氰钴胺(维生素B12)。
进一步地，本发明所述的阻断剂可以与另外的ACE抑制剂联用，所述的ACE抑制剂选自ACE抑制剂选自卡托普利(Captopril)、依那普利(Enalapril)、贝那普利(Benazepril)、福辛普利(Fosinopril)、赖诺普利(Lisinopril)、喹那普利(Quinapril)、洛沙坦(Cozaar)、缬沙坦(Valsartan)、厄贝沙坦(Irbesartan)、坎替沙坦(Candesartan)、依普沙坦(Eprosartan)等。
进一步地，本发明所述的阻断剂可以与另外的抗病毒药物联用，所述的另外的抗病毒药物为抗新型冠状病毒COVID-19，选自奥司他韦(oseltamivir)、洛匹那韦、利托那韦、氯喹或羟氯喹、阿奇霉素、克拉霉素、强力霉素、伊维菌素、奈非那韦、利巴韦林、法匹拉韦、金刚烷胺(amantaine)、安普那韦(amprenavir)、安普利近(ampligen)、阿比朵尔(arbidol)、阿扎那韦、立普妥(atripla)、波西普韦(boceprevir)、西多福韦、可比韦(combivir)、康普莱(complera)、地瑞那韦、地拉韦啶、地达诺新干扰素中的一种或几种。另外的抗病毒药物大多为广谱药物，即同时对多种流行性病毒引发的疾病产生药效。
进一步地，所述的病毒阻断剂通过蛋白质工程技术进行生产，阻断剂的基因经过编码优化后，克隆到表达载体中，然后转载到宿主细胞或体外无细胞反应体系中，孵育后纯化即获得病毒阻断剂。
进一步地，所述的病毒阻断剂通过蛋白质工程技术进行生产，阻断剂的基因经过编码优化后，克隆到表达载体中，然后转载到宿主细胞或体外无细胞反应体系中，孵育后纯化即获得病毒阻断剂。
进一步地，所阻断优选通过康码(上海)生物科技有限公司D2P技术进行生产，如D2P系统，所述D2P技术进行生产，如包括如下步骤：该基因经过编码优化并克隆到pD2P载体中。质粒是使用Ampi系统扩增，然后添加到蛋白质工厂快速反应中系统(康码(上海)生物科技有限公司)，体积比为1:30。这将反应混合物在30℃下孵育4小时，然后通过离心收集纯化。将无细胞混合物的上清液与磁性His Monster Beads(Kangma Healthcode(Shanghai)Biotech)在4℃下旋转1小时。用洗涤缓冲液(50mM Tris-HCl,pH 8.0,500mM NaCl，10mM咪唑)并用洗脱缓冲液(50mM Tris-HCl，pH 8.0，500mM NaCl、250mM咪唑)。
本发明另一个方面提供一种预防和治疗冠状病毒感染相关疾病的方法或用途，包括将本发明所述的喷雾剂喷雾制剂用于预防和治疗冠状病毒感染相关的疾病。
本发明另一个方面提供一种用于在对象中预防和治疗冠状病毒感染相关的疾病的方法，所述方法包括向有此需要的对象施用有效量的本发明所述的抗病毒药物吸入喷雾剂喷雾制剂。在优选的实施方案中，所述冠状病毒是SARS-CoV，MERS-CoV或SARS-CoV-2；在更优选的实施方案中，所述冠状病毒是SARS-CoV-2。
在某些优选的实施方案中，所述冠状病毒感染相关的疾病为新冠肺炎或中东呼吸综合征(MERS)或重症急性呼吸综合征(SARS)；在更优选的实施方案中，所述冠状病毒感染相关的疾病是新冠肺炎。
本发明另一个方面提供一种将本发明的喷雾剂喷雾制剂用于制备预防和治疗冠状病毒感染相关的疾病药物的用途，所述药物用于抑制冠状病毒与细胞的融合，或用于预防和/治疗冠状病毒感染相关的疾病。在优选的实施方案中，所述药物用于抑制冠状病毒SARS-CoV、MERS-CoV或SARS-CoV-2与细胞的融合，或用于预防冠状病毒SARS-CoV、MERS-CoV或SARS-CoV-2感染相关的疾病。
在更优选的实施方案中，所述冠状病毒是SARS-CoV-2。在某些优选的实施方案中，所述冠状病毒感染相关的疾病为新冠肺炎或中东呼吸综合征(MERS)或重症急性呼吸综合征(SARS)；在更优选的实施方案中，所述冠状病毒感染相关的疾病是新冠肺炎。
本发明用一种全新设计的ACE2模拟蛋白类病毒阻断剂作为抗病毒活性剂，对所有通过ACE2感染细胞的冠状病毒均具有显著的中和作用，具备高亲和力、多角度捕获黏连新冠病毒的特性，进而使病毒丧失侵染机体的功能和途径，最终达到阻断新冠病毒传播及感染的效果，临床毒性低、储存稳定性好，为临床预防和治疗SARS-CoV-2感染提供了新的解决方案。
 具体实施方式
[根据细则91更正 09.06.2023]
本发明提供了一种提高蛋白体外表达效率的方法，为使本发明的目的、技术方案及效果更加清楚、明确，以下列举实例对本发明作进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
术语
本发明中的“冠状病毒”是指冠状病毒在系统分类上属套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)且以ACE2作为结合受体的冠状病毒，包括但不限于SARS-CoV、MERS-CoV、SARS-CoV-2等。
本发明所述的ACE2也称为ACEH，称为血管紧张素转化酶2。ACE2由805个氨基酸组成，是具有单一胞外催化结构域的I型跨膜糖蛋白。ACE2为SARS-Cov-2等冠状病毒侵染人体细胞的受体蛋白。
本发明中的“刺突蛋白”是指在冠状病毒表面上，并且能够直接(例如通过与病毒受体(例如Ace2)相互作用)或间接(例如通过介导一种或多种其它蛋白或分子与病毒受体(例如Ace2)的相互作用)介导病毒附着于靶宿主细胞.
本发明中“冠状病毒感染”是指感染冠状病毒，如SARS-CoV-2、MERSCoV或SARS-CoV。所述术语包含通常在上呼吸道、下呼吸道中的冠状病毒呼吸道感染。并发症主要是发烧，可合并轻度干咳、乏力、呼吸不畅、腹泻、流涕、咳痰等症状、肺炎、肾衰竭、肾功能障碍、脓毒性休克和严重病例的死亡。
本发明中“有效量”和“治疗有效量”是指口腔喷雾制剂中的病毒阻断剂含量能够有效治疗受试者或哺乳动物的病毒感染或相关疾病或病症的量。在感染病毒的情况下，治疗有效量的试剂具有治疗作用，因此可病毒丧失侵染机体的功能和途径，最终达到阻断新冠病毒传播及感染的效果。在该试剂预防病毒传播和感染的情况下，其可视为中和能力，利用高亲和力多角度捕获黏连新冠病毒的特性，进而使病毒丧失侵染机体的功能和途径。所述治疗有效量为1-100nM，优选为10-50nM。
本发明中“D2P”体系包括但不限于IVTT反应(体外转录翻译反应)。本发明中，优选IVTT反应。IVTT反应，对应IVTT体系，是在体外将DNA转录翻译为蛋白质(Protein)的过程，因此，我们还将这类的体外蛋白合成体系称为、D-to-P体系、D_to_P体系、DNAto-Protein体系；相应的体外蛋白合成方法，还称为D2P方法、D-to-P方法、D_to_P方法、DNA-to-Protein方法，其与“体外无细胞蛋白合成体系”、“体外表达系统”、“体外蛋白合成体系”、“体外蛋白质合成反应体系”、“无细胞蛋白合成体系”等表述具有相同的含义。蛋白质体外合成系统、体外蛋白合成体系、无细胞系统、无细胞蛋白合成体系、无细胞体外蛋白合成体系、体外无细胞蛋白合成体系、体外无细胞合成体系、CFS体系(cell-free system)、CFPS体系(cell-free protein synthesis system)等描述方式。包括体外翻译体系、体外转录翻译体系(IVTT体系)等。我们还将体外蛋白合成系统称为“蛋白质合成工厂”(Protein Factory)。体外蛋白合成反应，是指在体外无细胞合成体系中合成蛋白的反应，至少包括翻译过程。
本发明所述体外无细胞蛋白合成体系中需要的蛋白组分(举例如RNA聚合酶)，可以通过内源方式提供，也可以通过外源方式添加。通过内源方式提供时，可以参考包括但不限于文献CN108690139A、CN109423496A、CN106978439A、CN110408635A、CN110551700A、CN110093284A、CN110845622A、CN110938649A、CN111378708A、CN111484998 A、“Molecular andCellular Biology,1990,10(1):353-360”等现有文献及其引用文献提供的基因改造方法，具体地，包括但不限于：将编码序列插入到细胞内游离型质粒，将编码基因整合入细胞基因组，及其组合方式。通过外源方式提供时，用量可以根据体系所需进行控制和调节。
一种基于病毒阻断剂的口腔喷雾制剂，包括以下质量百分含量的组成：病毒阻断剂1-100nM，附加剂，余量为溶剂；所述附加剂为助溶剂、稳定剂、矫味剂、抗氧剂、防腐剂、渗透压调节剂、pH调节剂或其组合；所述病毒阻断剂为刺突蛋白(RBD)阻断剂，优选地，所述的病毒阻断剂优选为SARS-CoV-2刺突蛋白(RBD)阻断剂。
在冠状病毒感染初期，主要是在口腔和鼻腔以及上呼吸道复制繁殖活跃，一段时间后才会展到肺部，有些只是保持在上呼吸道持续感染。本发明的喷雾制剂用途是在新型冠状病毒(SARS-CoV-2)等冠状病毒爆发期间，直接作用于口腔和/或鼻腔、上呼吸道，然后通过呼吸进入肺部，精准靶向给药，达到预防新冠病毒传染和传播的目的。本发明的所采用的ACE2模拟蛋白能够通过与RBD的竞争性结合来抑制病毒与宿主细胞的附着具有高结合结合能力，可显著减少药物剂量，因此可有效降低药物引起的临床毒性等副作用。
喷雾制剂使用雾化器吸入给药，不仅具有高效速效的特点，可直达鼻口腔，上呼吸道和肺部病灶，增加局部药物浓度，且肺部的吸收速度快，不亚于静脉注射；肺部吸收面积巨大，总吸收面积70～100m2，相当于体表面积的25倍；肺泡囊壁薄，由单层上皮细胞构成，紧靠致密的毛细血管网，细胞壁或毛细血管壁厚度仅0.5～1μm；药酶活性较胃肠道低，pH适宜，胃肠道难以吸收大分子药物可在肺部吸；肺部吸收可直接进入大循环，避免肝脏首过作用。因此抗病毒药物的雾化剂对预防新冠病毒的早期传染和传播，中期和后期的治疗具有广泛应用前景。且所述喷雾制剂药物浓度一般不需较大，给药的剂量可显著降低，因此大大降低了药物的临床毒性。
当15μm>雾化粒径>10μm，药物停留在口鼻腔内；当10μm>雾化粒径>5μm，药物可达下呼吸道前6级支气管内；当5μm>雾化粒径>1μm，药物可达肺部末端肺泡(终末5～6级支气管内)。粒径太小0.1μm＜雾化粒径＜1μm，气溶胶呼出概率显著增大，沉积率急剧下降，生物利用度减少。
所述病毒阻断剂和附加剂的浓度规格为1mg～300mg/ml，优选为1mg/ml、5mg/ml、10mg/ml、25mg/ml、50mg/ml、100mg/ml，进一步优选为10mg/ml、50mg/ml、100mg/ml。
所述口腔喷雾制剂灌装于雾化装置中，所述雾化装置优选为空气压缩式雾化器、加热雾化器，振动筛微孔雾化器或超声波雾化器。空气压缩式雾化器和振动筛微孔雾化器更适用本发明的雾化剂。
进一步地，所述的喷雾制剂优选为溶液型气雾剂、混悬型气雾剂或乳剂型气雾剂。
进一步地，所述病毒阻断剂的质量百分含量，优选为2-50nM，最优选5-20nM；
进一步地，所述附加剂的质量百分含量为0.001％-50％，优选为0.01％-20％，最优选为0.1％-20％。
所述溶剂为羟基型极性溶剂，选自水、乙醇、聚乙二醇、丙二醇、丙三醇、丁醇或特戊醇的一种或几种，优选为水、丙二醇或丙三醇中的一种或几种，进一步优选为水。
进一步地，所述附加剂为助溶剂、稳定剂、矫味剂、抗氧剂、防腐剂、渗透压调节剂、pH调节剂或其组合。
所述的助溶剂没有特别地限制，优选为卵磷脂、泊洛沙姆、羟丙基-β-环糊精、精氨酸、甘氨酸、天冬氨酸、谷氨酰胺、聚山梨醇20或聚山梨酯80，和/或pH为7.0-9.0。
所述的渗透压调节剂为氯化钠甘露醇、葡萄糖、磷酸盐、醋酸盐等
所述的pH调节剂为酸性物质、碱性物质或；所述的酸性物质选自盐酸、醋酸、柠檬酸、酒石酸、苹果酸等；所述的碱性物质选自强氧化钾、或氢氧化钠等。
所述的防腐剂没有特别的限制，优选为对羟基苯甲酸酯、山梨酸钾、苯乙醇、丙酸钠、苯扎氯铵等。
所述的稳定剂优选为tris盐缓冲溶液或磷酸盐缓冲溶液。
所述的抗氧剂选自亚硫酸钠、抗环血酸钠、亚硫酸钾、抗环血酸钾等。
所述的所述矫味剂为甜蜜素、薄荷醇、糖醇类化合物，选自木糖醇、山梨醇或甘露醇中的一种或几种，优选木糖醇或甘露醇中的一种或几种，进一步优选为木糖醇。
新冠病毒(SARS-CoV-2)刺突蛋白与ACE2的相互作用是病毒生命周期的第一步，因此阻断这种相互作用是一种很有前景的针对COVID-19的治疗方案。为此，我们从头设计了一系列潜在的SARS-CoV-2刺突蛋白阻断剂。
进一步地，所述病毒阻断剂包括如下结构域：刺突蛋白结合域(SBD)、刚性结构域、寡聚体位点和膜排斥尾。
进一步地，所述病毒阻断剂的不同的结构域之间由共价连接的连接基团连接，优选地所述的所述的连接基团选自多肽或二硫键，且个结构域间的连接顺序没有特别的限制，如在一种优选的实施方案中，所述的阻断剂的刺突蛋白结合域(SBD)、刚性结构域、寡聚体位点和膜排斥尾由按从N段到C段的方向依次连接。
所述刺突蛋白结合域是与SARS-CoV-2刺突蛋白特异性性结合区域，本发明基于ACE2的结合位点设计了多种长度的Spike结合域(10-20个残基)。
进一步地，所述刺突结合域的包含如SEQ ID NO.1-3和SEQ ID NO.6-17任一氨基酸序列所示的多肽：
SEQ ID NO.1:
SEQ ID NO.2:
SEQ ID NO.3:
SEQ ID NO.6:
SEQ ID NO.7:
SEQ ID NO.8:
SEQ ID NO.9:
SEQ ID NO.10:
SEQ ID NO.11:
SEQ ID NO.12:
SEQ ID NO.13:
SEQ ID NO.14:
SEQ ID NO.15:
SEQ ID NO.16:
SEQ ID NO.17:
进一步地，所述刺突结合域包含与如SEQ ID NO.1-3和SEQ ID NO.6-17任一氨基酸序列所示的多肽具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的多肽。
进一步地，所述的刺突结合域包含与如SEQ ID NO.1-3和SEQ ID NO.6-17任一氨基酸序列所示的多肽具有一个或几个氨基酸的置换、缺失或添加的序列，所述置换、缺失或添加的氨基酸的数量可以为1-20个，优选为1-10个，如1、2、3、4、5、6、7、8、9、10。
优选地，为了增加阻断剂与刺突蛋白的结合亲和力，所述的阻断剂包含至少一个刺突结合域，最优选地，所述病毒阻断剂具有两个刺突结合域，优选包含两个以上的互补决定区，如1-10个，优选2-8个，最优选4-8个。
所述的刚性结构域可以保持三维结构并提高阻断剂的生物活性。所述的刚性结构域包含eGFP(SEQ ID NO.4)，或在eGFP基础上改进的刚性蛋白如mTagBFP2、moxCerulean3、AmCyanl、MiCy、ZsGreen、Clover、mVenus、ZsYellow 1、mKO2、TurboRFP、tdTomato、eqFP611、mKate1.3、mNeptune2、miRFP670、mAme-trine、PAmCherry 2、mEos3.2等。
进一步地，所述的刚性结构域包含与如SEQ ID NO.4氨基酸序列所示的多肽具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的多肽。
SEQ ID NO.4:
膜排斥尾巴进一步使阻断剂复合物排斥宿主细胞膜，从而阻碍病毒进入宿主细胞。组合起来，模块化组装的阻断剂预计会削弱SARS-CoV-2变体的细胞附着能力。所述的膜排斥尾巴为带有负电的氨基酸短链聚合物，所述的短链聚合物的氨基酸的个数为0-50，优选2-40，更优选3-30，最优选4-20。所述的带有负电的氨基酸为天冬氨酸和/或谷氨酸。
进一步地，所述寡聚体位点促进阻断剂的寡聚化和病毒-阻断剂复合物的聚集。所述寡聚体位点为具有聚合功能的单链蛋白、多肽，所述聚合功能是指所述单链蛋白具有天然聚合为多聚体的功能。进一步地，所述多聚体包括但不限于单体数量为2-10的寡聚体，如二聚体、三聚体、四聚体、五聚体、六聚体、七聚体、八聚体、九聚体、十聚体及其混合物，单体数量为11-20的低聚体以及单体数量为20以上的高聚体。在一种可行实施方案中，所述具有聚合功能的单链蛋白来源于天然聚合蛋白及其功能性变体。
优选地，所述具有聚合功能的单链蛋白与链霉亲和素蛋白单条肽链(SEQ ID NO.5)具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。
进一步，所述的病毒阻断剂优选康斯汀阻断蛋白或与其氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的蛋白。
SEQ ID NO.5:
进一步，所述的病毒阻断剂优选康斯汀TM阻断蛋白或与其氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的蛋白。
所述的康斯汀蛋白刺突蛋白结合域(SBD)、刚性结构域、寡聚体位点和膜排斥尾由按从N段到C段的方向依次连接，其中刺突蛋白结合域包含SEQ ID NO.2和SEQ ID NO.4的刺突结合域，寡居位点包含SEQ ID NO.5所示序列，所述膜排斥尾为10E(SEQ ID NO.18:EEEEEEEEEE)。
进一步地，所述的口腔喷雾制剂，任选地还包含膳食补充剂；所述膳食补充剂包括但不限于抗氧剂、维生素、矿物质、氨基酸及其他得到批准的膳食补充剂。
所述的维生素选自但不限于维生素A，维生素B复合体，维生素C，维生素D，维生素E，维生素K，维生素H，维生素P，维生素PP，维生素M，维生素T，维生素U中的一种或多种。
所述的维生素B复合体包括但不限于：泛酸、烟酸、生物素、叶酸、维生素B1(硫胺素)、维生素B2(核黄素)、吡哆醇(维生素B6)和氰钴胺(维生素B12)。
进一步地，本发明所述的阻断剂可以与另外的ACE抑制剂联用，所述的ACE抑制剂选自ACE抑制剂选自卡托普利(Captopril)、依那普利(Enalapril)、贝那普利(Benazepril)、福辛普利(Fosinopril)、赖诺普利(Lisinopril)、喹那普利(Quinapril)、洛沙坦(Cozaar)、缬沙坦(Valsartan)、厄贝沙坦(Irbesartan)、坎替沙坦(Candesartan)、依普沙坦(Eprosartan)等。
进一步地，本发明所述的阻断剂可以与另外的抗病毒药物联用，所述的另外的抗病毒药物为抗新型冠状病毒COVID-19，选自奥司他韦(oseltamivir)、洛匹那韦、利托那韦、氯喹或羟氯喹、阿奇霉素、克拉霉素、强力霉素、伊维菌素、奈非那韦、利巴韦林、法匹拉韦、金刚烷胺(amantaine)、安普那韦(amprenavir)、安普利近(ampligen)、阿比朵尔(arbidol)、阿扎那韦、立普妥(atripla)、波西普韦(boceprevir)、西多福韦、可比韦(combivir)、康普莱(complera)、地瑞那韦、地拉韦啶、地达诺新干扰素中的一种或几种。另外的抗病毒药物大多为广谱药物，即同时对多种流行性病毒引发的疾病产生药效。
进一步地，所述的病毒阻断剂通过蛋白质工程技术进行生产，阻断剂的基因经过编码优化后，克隆到表达载体中，然后转载到宿主细胞或体外无细胞反应体系中，孵育后纯化即获得病毒阻断剂。
进一步地，所述的病毒阻断剂通过蛋白质工程技术进行生产，阻断剂的基因经过编码优化后，克隆到表达载体中，然后转载到宿主细胞或体外无细胞反应体系中，孵育后纯化即获得病毒阻断剂。
所述宿主细胞宿主细胞是本领域中众所周知的，包括但不限于大肠杆菌、CHO细胞、中国仓鼠卵巢、NS0、SP2细胞、海拉细胞(HeLa cell)、小仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，Hep G2)、A549细胞、293细胞和许多其它细胞系。草地贪夜蛾(Spodoptera frugiperda)或粉纹夜蛾(Trichoplusiani))、两栖动物细胞、细菌细胞、植物细胞和真菌细胞。真菌细胞包含酵母和丝状真菌细胞，所述丝状真菌细胞包含例如，毕赤酵母、芬兰毕赤酵母(Pichia finlandica)、喜海藻糖毕赤酵母(Pichia trehalophila)、科克拉马毕赤酵母(Pichia koclamae)、膜醭毕赤酵母(Pichia membranaefaciens)、微小毕赤酵母(Pichia minuta)(甲醇诱导型酵母(Ogataeaminuta)、林氏毕赤酵母(Pichia lindneri))、仙人掌毕赤酵母(Pichia opuntiae)、耐热毕赤酵母(Pichia thermotolerans)、柳毕赤酵母(Pichia salictaria)、松栎毕赤酵母(Pichia g uercuum)、皮杰普毕赤酵母(Pichia pijperi)、树干毕赤酵母(Pichiastiptis)、甲醇毕赤酵母(Pichia methanolica)、毕赤酵母菌(Pichia sp.)、酿酒酵母(Saccharomyces cerevisiae)、酵母菌(Saccharomyces sp.)、多形汉逊酵母(Hansenulapolymorpha)、克鲁维酵母菌(Kluyveromyces sp.)、乳酸克鲁维酵母(Kluyveromyceslactis)、白色念珠菌(Candida albicans)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)、卢克诺文思金孢子菌(Chrysosporium lucknowense)、镰刀菌(Fusarium sp.)、禾谷镰刀菌(Fusarium gramineum)、镰孢霉(Fusarium venenatum)、小立碗藓(Physcomitrellapatens)以及粗糙脉孢菌(Neurospora crassa)。
进一步地，所述体外无细胞蛋白合成体系，包括但不限于大肠杆菌体外蛋白合成体系、细菌体外蛋白合成体系、哺乳动物细胞(如HF9、Hela、CHO、HEK293)体外蛋白合成体系、植物细胞体外蛋白合成体系、酵母细胞体外蛋白合成体系、昆虫细胞体外蛋白合成体系。优选为酵母体外蛋白合成体系，更优选即为克鲁维酵母体外蛋白合成体系，最优选地为乳酸克鲁维酵母体外蛋白合成体系。
本发明的体外蛋白合成体系、模板、质粒、目标蛋白、体外蛋白合成反应(孵育反应)、各种制备方法、各种检测方法等技术要素，还可以各自独立地从下述文献中选择合适的实施方式或实施方法，包括但不限于CN111484998A、CN106978349A、CN108535489A、CN108690139A、CN108949801A、CN108642076A、CN109022478A、CN109423496A、CN109423497A、CN109423509A、CN109837293A、CN109971783A、CN109988801A、CN109971775A、CN110093284A、CN110408635A、CN110408636A、CN110551745A、CN110551700A、CN110551785A、CN110819647A、CN110845622A、CN110938649A、CN110964736A等文献。除非和本发明目的相冲突，否则，这些文献及其引用文献以全部内容、全部目的被引用。
进一步地，所阻断优选通过康码(上海)生物科技有限公司D2P技术进行生产，如D2P系统，所述D2P技术进行生产，如包括如下步骤：该基因经过编码优化并克隆到pD2P载体中。质粒是使用Ampi系统扩增，然后添加到蛋白质工厂快速反应中系统(康码(上海)生物科技有限公司)，体积比为1:30。这将反应混合物在30℃下孵育4小时，然后通过离心收集纯化。将无细胞混合物的上清液与磁性His Monster Beads(Kangma Healthcode(Shanghai)Biotech)在4℃下旋转1小时。用洗涤缓冲液(50mM Tris-HCl,pH 8.0,500mM NaCl，10mM咪唑)并用洗脱缓冲液(50mM Tris-HCl，pH 8.0，500mM NaCl、250mM咪唑)。
本发明另一个方面提供一种预防和/或治疗冠状病毒感染相关疾病的方法或用途，包括将本发明所述的喷雾制剂用于预防和/或治疗冠状病毒感染相关的疾病。
本发明另一个方面提供一种用于在对象中预防和/或治疗冠状病毒感染相关的疾病的方法，所述方法包括向有此需要的对象施用有效量的本发明所述的抗病毒药物吸入喷雾制剂。在优选的实施方案中，所述冠状病毒是SARS-CoV，MERS-CoV或SARS-CoV-2；在更优选的实施方案中，所述冠状病毒是SARS-CoV-2。
在某些优选的实施方案中，所述冠状病毒感染相关的疾病为新冠肺炎或中东呼吸综合征(MERS)或重症急性呼吸综合征(SARS)；在更优选的实施方案中，所述冠状病毒感染相关的疾病是新冠肺炎。
本发明另一个方面提供将本发明的喷雾制剂用于制备药物的用途，所述药物用于抑制冠状病毒与细胞的融合，或用于预防和/或治疗冠状病毒感染相关的疾病。在优选的实施方案中，所述药物用于抑制冠状病毒SARS-CoV、MERS-CoV或SARS-CoV-2与细胞的融合，或用于预防冠状病毒SARS-CoV、MERS-CoV或SARS-CoV-2感染相关的疾病。
在更优选的实施方案中，所述冠状病毒是SARS-CoV-2。在某些优选的实施方案中，所述冠状病毒感染相关的疾病为新冠肺炎或中东呼吸综合征(MERS)或重症急性呼吸综合征(SARS)；在更优选的实施方案中，所述冠状病毒感染相关的疾病是新冠肺炎。
本发明另一个方面提供将本发明的喷雾制剂用于皮肤和环境中冠状病毒的消杀的用途，在优选的实施方案中，所述喷雾用于消杀冠状病毒SARS-CoV、MERS-CoV或SARS-CoV-2；
在更优选的实施方案中，所述冠状病毒是SARS-CoV-2。
[根据细则91更正 09.06.2023]
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于阐述说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方，通常按照常规条件，例如“Sambrook等人，分子克隆：实验室手册(New York:Cold SpringHarbor LaboratoryPress,1989)”、《无细胞蛋白合成实验手册》“Edited by AlexanderS.Spirin and James R.Swartz.Cell-free protein ynthesis:methods and protocols[M].2008”等文献中所述的实验条件，或者按照制造厂商所建议的条件，或者按照、参考上文所述的具体实施方式指引的条件。除非另外说明，否则本发明中提及的百分比和份数是重量百分比和重量份数。
实施例1病毒阻断剂的制备
将优化后的病毒阻断剂(康斯汀阻断蛋白)的基因序列插入到pD2P质粒中，然后加入到自制的乳酸克鲁维酵母体外无细胞蛋白合成体系中，以乳酸克鲁维酵母(KluyveromyceslactisNRRL Y-1140)制备体外。本实施例所用体外无细胞蛋白合成体系(总体积30μL)：包括乳酸克鲁维酵母细胞提取物50％(v/v)，22mM三羟甲基氨基甲烷(pH8)，90mM醋酸钾，4.0mM醋酸镁，3.0mM核苷三19磷酸混合物，0.16mM氨基酸混合物，22mM磷酸钾，0.003mg/mL淀粉酶，3％(w/v)聚乙二醇(PEG-8000)，340mM麦芽糊精(以葡萄糖单元计量，对应约55mg/mL)，0.04mg/mL外源添加的RNA聚合酶，以及15ng/μL目标蛋白DNA，这里的15ng/μL为各荧光蛋白DNA的总浓度。将上述反应体系置于22-30℃的环境中，静置孵育约20h。
反应后的溶液，4000rpm 4度离心10min。上清液加入5％(w/v)的硫酸铵粉末，充分搅拌溶解，4000rpm 4度离心10min。上清液中继续加入25％(w/v)的硫酸铵粉末，缓慢加入，边加入边搅拌使其溶解。12000rpm 4度离心10min。弃上清，沉淀用30％(w/v)的硫酸铵溶液漂洗1次。12000rpm 4度离心10min。沉淀用PBS重悬，充分溶解。12000rpm，4度离心10min，用0.22μm的针筒滤器过滤后，4度保存待用，BCA法测定蛋白浓度，要求不低于1mg/ml。
实施例2
本实施例提供一种口腔喷雾制剂A，以重量百分数计，包括如下组分：实施例1所制备的病毒阻断剂20nM；0.1％％甘油；0.01％薄荷醇；0.2％柠檬酸钠、0.1％柠檬酸磷酸；0.05％甜蜜素；余量为纯化水调节pH为7.2。
实施例3
本实施例提供一种口腔喷雾制剂B，以重量百分数计，包括如下组分：实施例1所制备的病毒阻断剂10nM；0.1％聚乙二醇；0.01％薄荷醇；0.1％柠檬酸钠、0.5％柠檬酸；0.05％木糖醇；0.05％维生素C、0.05％维生素B1、0.05％维生素B6、0.05％维生素B3、0.05％维生素原B5；余量为纯化水调节pH为6.9。
实施例4
本实施例提供一种口腔喷雾制剂C，以重量百分数计，包括如下组分：实施例1所制备的病毒阻断剂50nM；0.2％羟丙基-β-环糊精；0.01％薄荷醇；0.5％磷酸三钠、0.5％磷酸；0.5％甘露醇；0.5％维生素C；余量为纯化水调节pH为7.1。
实施例5
本实施例提供一种口腔喷雾制剂D，以重量百分数计，包括如下组分：实施例1所制备的病毒阻断剂80nM；0.2％谷氨酸；0.3％柠檬酸钠、0.3％柠檬酸0.5％蜂蜜；0.05％维生素B1、0.05％维生素B6、0.05％维生素原B5；余量为纯化水，调节pH为7.1。
实施例6
本实施例提供一种口腔喷雾制剂E，以重量百分数计，包括如下组分：实施例1所制备的病毒阻断剂3nM；0.1％卵磷脂；0.01％薄荷醇；0.2％吐温-20；0.05％甜蜜素；0.5％维生素C、0.5％维生素B1、0.05％维生素B6、0.5％维生素B3、0.1％维生素原B5；余量为纯化水，调节pH为7.2。
实施例7
[根据细则91更正 09.06.2023]
功效实验：分别将等体积的人工唾液、口腔喷雾制剂A-E、人工唾液与口腔喷雾剂A-E分别混合的混合物三者与一定体积的新冠病毒模拟病毒混合，静置15分钟，用培养的细胞来进行病毒培养，观察三者对于假病毒的效果。
结果显示：新冠病毒模拟病毒能侵染细胞，加入口腔喷雾制剂A-E后病毒对细胞的侵染水平降低，病毒活力显著下降。
实施例8
稳定性评价
对实施例2-7制备的口腔喷雾制剂加速实验法37℃90天，蛋白含量和病毒中和效果没有明显改变。
实施例9
毒性实验
毒理学试验按《消毒技术标准》进行(2002年版)第二部分《消毒产品检验技术标准》2.3.1急性经口毒性试验。异常毒性试验按《中华人民共和国药典2020年版(第四部分)》《通则》《生物制品原则、检验方法》，异常毒性试验。
ICR小鼠由南京医科大学动物中心提供。
Sprague Dawley(SD)大鼠购自邳州东方繁育有限公司。
新西兰大白兔由益正安里茂生物科技有限公司提供。动物饲养于20℃-26℃，局部屏障系统内相对湿度为40％-70％。
一、急性经口毒性
20只SPF ICR小鼠(18.0～22.0g)和20只SPF SD大鼠(180～220g)用于急性经口毒性试验。雄性和雌性的数量是相等的。对过夜禁食的小鼠和大鼠施用实施例2-6的口腔喷雾制剂，剂量1mg/Kg。接下来每周监测动物的毒性和死亡率的临床症状14天(第0天、第7天和第14天)。行为、死亡人数和体重分别为评估，并在观察期结束时对它们进行解剖。
二、急性吸入毒性试验20只SPF ICR小鼠(18.0～22.0g)用于急性吸入毒性试验。雄性和雌性的数量是相等的。将2.2g实施例1制备的病毒阻断剂置于220L毒物暴露柜，浓度假定为10,000mg/m3。这吸入的暴露时间设定为2小时。老鼠的症状和死亡是在14天观察期间(0天、7天和14天)记录。
三、急性眼刺激试验三只雄性新西兰兔(2.5～3.5kg)用于急性眼刺激试验。将0.1mL实施例2-6的口腔喷雾制剂滴入兔右眼结膜囊，左眼滴入生理盐水作为对照。闭眼4s，30s后用生理盐水冲洗。观察家兔结膜、虹膜和角膜的损伤和恢复情况，为期21天(1小时、24小时、48小时、72小时、7天、14天和28天)。对角膜损伤、虹膜损伤、结膜充血和结膜水肿的严重程度进行评分。小鼠骨髓多染红细胞(PCE)微核试验50只ICR小鼠用于小鼠骨髓多染红细胞微核试验。男性的数量等于女性的数量。将动物分成五组，每组五只雌性小鼠和五只雄性小鼠。试验组分别以5000、2500和1250mg/kg·bw的剂量分别给予一次Kansetin。一组作为阴性对照并用溶剂纯化水处理。另一组作为阳性对照，腹腔注射40mg/kg·bw的环磷酰胺(CP)。测试组在0小时和24小时通过口服给药暴露于Kansetin。第二次暴露于Kansetin后6小时处死小鼠，制备骨髓涂片。计算每只动物1000个多染红细胞(PCE)中微核的出现。一旦计数了200个PCE，就确定了PCE与正色素红细胞(NCE)的比率。通过U检验进行统计分析。
在目前的实验条件下，本发明的病毒阻断剂被认为是无毒的。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (20)

  1. 一种基于病毒阻断剂的口腔喷雾制剂，其特征在于：包括以下组分：治疗有效含量的病毒阻断剂，附加剂、和溶剂；
    优选地，所述病毒阻断剂为冠状刺突蛋白(RBD)阻断剂；
    所述附加剂选自助溶剂、稳定剂、矫味剂、抗氧剂、防腐剂、渗透压调节剂、pH调节剂或其组合。
  2. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述的口腔喷雾制剂为溶液型气雾剂、混悬型气雾剂或乳剂型气雾剂。
  3. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述溶剂为羟基型极性溶剂，选自水、乙醇、聚乙二醇、丙二醇、丙三醇、丁醇或特戊醇的一种或几种，优选为水、丙二醇或丙三醇中的一种或几种，进一步优选为水。
  4. 根据权利要求3所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述的助溶剂为卵磷脂、泊洛沙姆、羟丙基-β-环糊精、精氨酸、甘氨酸、天冬氨酸、谷氨酰胺、聚山梨醇20或聚山梨酯80，和/或pH为7.0-9.0。
  5. 根据权利要求3所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述渗透压调节剂为氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐。
  6. 根据权利要求3所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述防腐剂为对羟基苯甲酸酯、山梨酸钾、苯乙醇、丙酸钠或苯扎氯铵。
  7. 根据权利要求3所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述抗氧化剂选自亚硫酸钠、抗环血酸钠、亚硫酸钾、抗环血酸钾。
  8. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述矫味剂为蜂蜜、甜蜜素、薄荷醇、木糖醇、山梨醇或甘露醇中的一种或几种。
  9. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述病毒阻断剂包括如下结构域：刺突蛋白结合域(SBD)、刚性结构域、寡聚体位点和膜排斥尾。
  10. 根据权利要求9所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述病毒阻断剂的不同的结构域之间由共价连接的连接基团连接，优选地所述的所述的连接基团选自多肽或二硫键，且各个结构域间的连接顺序没有特别的限制，如在一种优选的实施方案中，所述的阻断剂的刺突蛋白结合域(SBD)、 刚性结构域、寡聚体位点和膜排斥尾由按从N端到C端的方向依次连接。
  11. 根据权利要求10所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述刺突结合域包含与如SEQ ID NO.1-3和SEQ ID NO.6-17任一氨基酸序列所示的多肽具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的多肽。
  12. 根据权利要求10所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述的刚性结构域包含与如SEQ ID NO.4氨基酸序列所示的多肽具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性的多肽。
  13. 根据权利要求10所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述的膜排斥尾巴为带有负电的氨基酸短链聚合物，所述的短链聚合物的氨基酸的个数为0-50，优选2-40，更优选3-30，最优选4-20；所述的带有负电的氨基酸为天冬氨酸和/或谷氨酸。
  14. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：所述的口腔喷雾制剂，任选地还包含膳食补充剂；所述膳食补充剂包括但不限于抗氧剂、维生素、矿物质、氨基酸及其他得到批准的膳食补充剂。
  15. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：任选地，所述的喷雾制剂还包含另外的ACE抑制剂，所述的ACE抑制剂选自ACE抑制剂选自卡托普利(Captopril)、依那普利(Enalapril)、贝那普利(Benazepril)、福辛普利(Fosinopril)、赖诺普利(Lisinopril)、喹那普利(Quinapril)、洛沙坦(Cozaar)、缬沙坦(Valsartan)、厄贝沙坦(Irbesartan)、坎替沙坦(Candesartan)、依普沙坦(Eprosartan)。
  16. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：任选地，所述的口腔喷雾制剂还包含另外的抗病毒药物，所述的另外的抗病毒药物为抗新型冠状病毒COVID-19，选自奥司他韦(oseltamivir)、洛匹那韦、利托那韦、氯喹或羟氯喹、阿奇霉素、克拉霉素、强力霉素、伊维菌素、奈非那韦、利巴韦林、法匹拉韦、金刚烷胺(amantaine)、安普那韦(amprenavir)、安普利近(ampligen)、阿比朵尔(arbidol)、阿扎那韦、立普妥(atripla)、波西普韦(boceprevir)、 西多福韦、可比韦(combivir)、康普莱(complera)、地瑞那韦、地拉韦啶、地达诺新干扰素中的一种或几种。
  17. 根据权利要求1所述的基于病毒阻断剂的口腔喷雾制剂，其特征在于：口腔喷雾制剂所用的雾化装置为空气压缩式雾化器、加热雾化器，振动筛微孔雾化器或超声波雾化器，更优选地为空气压缩式雾化器和振动筛微孔雾化器。
  18. 一种预防和治疗冠状病毒感染相关疾病的方法或用途，其特征在于：包括将权利要求1-17任一项所述的喷雾制剂用于预防和治疗冠状病毒感染相关的疾病。
  19. 一种将权利要求1-17任一项所述的口腔喷雾制剂用于制备预防和/或治疗冠状病毒感染相关的疾病药物的用途，其特征在于：所述药物用于抑制冠状病毒与细胞的融合，或用于预防和/或治疗冠状病毒感染相关的疾病。
  20. 根据权利要求18所述的用途，其特征在于：所述药物用于抑制冠状病毒SARS-CoV、MERS-CoV或SARS-CoV-2与细胞的融合，或用于预防冠状病毒SARS-CoV、MERS-CoV或SARS-CoV-2感染相关的疾病。
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