CN108926707B

CN108926707B - anti-RSV use of PF4

Info

Publication number: CN108926707B
Application number: CN201710384729.7A
Authority: CN
Inventors: 王健伟; 任丽丽; 韩子泊
Original assignee: National Institute of Pathogen Biology CAMS and PUMC
Current assignee: National Institute of Pathogen Biology CAMS and PUMC
Priority date: 2017-05-26
Filing date: 2017-05-26
Publication date: 2021-12-03
Anticipated expiration: 2037-05-26
Also published as: CN108926707A

Abstract

Anti-RSV application of PF4, the invention discloses that platelet factor 4 can reduce the symptoms of virus infection by inhibiting the replication of respiratory syncytial virus in the host, and is an effective intervention measure against respiratory syncytial virus infection. The present invention provides the use of platelet factor 4 in the preparation of a medicament for preventing and/or treating diseases and/or symptoms caused by respiratory syncytial virus infection, and also provides an administration route, dosage form and the medicament suitable for the medicament Combinations with other drugs, etc.; and methods for non-therapeutic purposes of inhibiting respiratory syncytial virus in cells in vitro with platelet factor 4 are also provided.

Description

anti-RSV use of PF4

Technical Field

The present invention relates to respiratory syncytial virus and in particular to antiviral interventions useful for the prevention and/or treatment of diseases and/or symptoms caused by the virus.

Background

Respiratory Syncytial Virus (RSV) belongs to the genus Pneumovirus of the subfamily Pneumovirinae of the family Paramyxoviridae, is an enveloped single-stranded negative-strand RNA Virus, the genome of which encodes 11 viral proteins, namely, a nonstructural protein 1(NS1), a nonstructural protein 2(NS2), a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a Small Hydrophobin (SH), a glycoprotein (G), a fusion protein (F), an M2ORF1/ORF2 protein (M2-1/M2-2) and a large protein (L) in sequence from the 3 'end to the 5' end. RSV is distributed worldwide, has only one serotype, and can be divided into A, B subgroups based on antigen reactivity, with subgroup A being prevalent in most regions of the world (Griffiths C, Drews S J, Marchant D J. respiratory synthetic Virus: Infection, Detection, and New Options for Prevention and Treatment [ J ]. Clin microbial Rev,2017,30(1): 277) 319). RSV is transmitted via the respiratory tract and is the most important pathogen responsible for acute lower respiratory tract infections in young children (Hall C B, Weinberg G A, Ivine M K, et al. the garden of respiratory synthetic infection in young children, 2009,360(6):588-98), and can cause severe lower respiratory tract diseases including bronchiolitis and pneumonia. Studies have shown that in hospitalized infants with respiratory illness, the RSV detection rate in bronchiolitis cases can reach 43% and the pneumonia case detection rate is 25% (Kim H W, Arrobio J O, Brandt C D, et al. epidemic of respiratory synthetic infection in Washington, D.C.I. Immunity of the virus in differential respiratory tract disease synthesis and temporal distribution of infection [ J ]. Am J epidemic, 1973,98(3): 216-225). Children are the major susceptible population to RSV, and by the age of 2 years more than 95% of children have been infected with RSV. RSV is more susceptible to lower respiratory tract infections than other common respiratory viruses, such as influenza and parainfluenza viruses (Fisher R G, Gruber W C, Edwards K M, et al. Twenty. years of outer respiratory syndrome infection: a frame for vaccine efficacy strategies [ J ]. Pediatrics,1997,99(2): E7), whereas acute lower respiratory tract infections are one of the leading causes of childhood death worldwide (Nair H, Nokes D J, Gessner B D, et al. Global viral infection of infant efficacy strategies to respiratory syndrome in bone cement n. system: a. stewart-metal and analysis [ J ]. 375, 1555). One meta-analysis showed that children under 6.6-19.9 ten thousand 5 years of age died of RSV infection and related complications worldwide in 2005, with 99% occurring in developing countries (Nair H, Nos D J, Gessner B D, et al. Global garden of acid lower respiratory infection to respiratory syndrome virus in your child: a systematic review and meta-analysis [ J ]. Lancet,2010,375 (5): 1545-1555); another Study reported 23.4 million children dying globally in 2010 from RSV infection under the age of 5 and 25.35 million deaths in the age group (Lozano R, Naghavi M, Formaman K, et al. Global and regional mortalities from 235 cases for death for 20age groups in 1990and 2010: a systematic analysis for the Global Burden of Disease Study 2010[ J ] Lancet,2012,380(9859): 2095-. In addition to children, RSV also frequently infects the elderly and people with low immunity.

Although RSV is a worldwide threat to human health, current preventive and therapeutic measures against RSV infection are very limited. However, until now, no commercial vaccine has been available for active prevention of RSV. RSV F protein-specific mAbs (palivizumab) have been commercialized in recent years and used for passive immunization of high risk groups, but have limited clinical utility due to the emergence of costly and resistant strains (Adams O, Bonzel L, Kovacevic A, et al, palivizumab-resistant human respiratory infection in efficacy [ J ]. Clin infection Dis,2010,51(2): 185-188). Furthermore, studies of RSV disease burden have shown that most children are completely healthy prior to infection with RSV, suggesting that existing passive immunization strategies for high risk groups have limited effect in reducing disease burden (Hall C B, Weinberg G a, Iwane M K, et al, the burden of respiratory synthetic infection in your children [ J ]. N Engl J Med,2009,360(6): 588-. At present, no efficient antiviral drug can be used for treating RSV infection, clinically, treatment of RSV infection mainly takes supportive treatment as main treatment, and other available treatment drugs such as ribavirin, bronchodilators, glucocorticoids and the like lack reliable evidence of evidence-based medicine in effectiveness; none of the RSV antiviral drugs in the research has entered the third phase clinical trial.

Platelet Factor 4(Platelet Factor 4, PF4) belongs to the CXC chemokine family, also known as CXCL4, and is a multifunctional secreted cytokine. The mature HUMAN PF4 protein contains 70 amino acids, has a monomer molecular weight of 7.8kDa and has the accession number P02776 in the UniProtKB database (http:// www.uniprot.org/uniprot/P02776, PLF4_ HUMAN, glutamic acid at position 32-serine at position 101). The physiological functions of PF4 include promoting blood coagulation and wound healing, regulating hematopoietic function, immunomodulation, anti-angiogenesis, etc. (Vandercapellen J, Van Damme J, Struyf S. the role of the CXC chemokines sheet Factor-4(CXCL4/PF-4) and its variable (CXCL4L1/PF-4 variable) in inflammation, angiogenesis and cancer [ J ]. Cytokine Growth Factor Rev,2011,22(1): 1-18). Researchers have used it for the treatment of tumors, transplant rejection and hematopoietic disorders and have achieved better results in animal models. There is currently little research on the antiviral function of PF 4.

Disclosure of Invention

The inventor finds that the platelet factor 4 can inhibit the replication of the respiratory syncytial virus and can be used as an effective intervention measure for resisting the respiratory syncytial virus infection.

In an in vitro experiment, the inventor treats human laryngeal cancer cells (HEp-2 cells) with recombinant human platelet factor 4, and detects the infected cells of the respiratory syncytial virus by immunofluorescence and western blot (western blot), and finds that the replication of the respiratory syncytial virus is inhibited, and the infection rate of the respiratory syncytial virus and the concentration of the platelet factor 4 are in semilog negative correlation. In an in vivo experiment, the inventor verifies the function of the platelet factor 4 against the respiratory syncytial virus by using a respiratory syncytial virus mouse infection model, and finds that the nasal administration of the platelet factor 4 can obviously reduce the lung respiratory syncytial virus load, relieve the pathological damage of the lung, and reduce the level of inflammatory cytokines such as Interleukin-6 (IL-6), Monocyte chemotactic protein-1 (Monocyte chemotactic protein-1, MCP-1) in the respiratory tract. These findings indicate that platelet factor 4 can alleviate symptoms of respiratory syncytial virus infection by inhibiting viral replication in the host, and is an effective intervention against respiratory syncytial virus infection.

The invention provides an application of platelet factor 4 in preparing a medicament for preventing and/or treating diseases and/or symptoms caused by respiratory syncytial virus infection. In addition, the invention also provides an administration route and a dosage form suitable for the medicine, and the combination of the medicine and other medicines.

The invention also provides a method of non-therapeutic purpose of inhibiting respiratory syncytial virus in cells in vitro with platelet factor 4.

Drawings

FIG. 1 recombinant PF4 protein inhibits RSV replication

HEp-2 cells were treated with 0, 125, 250, 500, 1000, 2000nM recombinant PF4 for 4h, 24h post RSV infection: a. immunofluorescence was measured for cellular RSV infection rate (oval light gray dots in the figure indicate DAPI stained nuclei, irregular shaped light gray plaques indicate cells infected with RSV, and it can be seen that the RSV infection rate of cells decreases with increasing concentration of PF 4); b. correlation of PF4 concentration with cellular RSV infection rate (semilog negative correlation, R)²0.92); c. and detecting the expression level of RSV N protein and G protein in the cell by western blot.

FIG. 2 nasal administration of PF4 reduces pulmonary viral load following RSV infection in mice

SPF grade 6-8 week old female BALB/c mice are divided into 3 groups, mice in RSV group and RSV + PF4 group are respectively administrated with DPBS or 1.8 mu g PF4 by nasal drip, and after 6h, 1 × 10 mice are inoculated by nasal drip⁶PFU RSV a2, when control mice were nasally inoculated with DPBS; mice were sacrificed 4d post infection and the biological material was taken for detection. RT-PCR detects RSV N gene copy number in lung tissue. The lung RSV N gene copy number of the mice in the RSV + PF4 group is obviously lower than that in the RSV group (p is 0.046). b. Mouse lung RSV immunohistochemistry (goat anti-RSV antibody labeled RSV, combined with horseradish peroxidase labeled secondary antibody, and brown after color development), shows that the distribution density and signal intensity of RSV signals in lung tissues of mice of RSV + PF4 group are obviously lower than those of RSV group.

FIG. 3 nasal administration of PF4 to reduce pulmonary inflammation following RSV infection in mice

SPF grade 6-8 week old female BALB/c mice are divided into 3 groups, mice in RSV group and RSV + PF4 group are respectively administrated with DPBS or 1.8 mu g PF4 by nasal drip, and after 6h, 1 × 10 mice are inoculated by nasal drip⁶PFU RSV a2, when control mice were nasally inoculated with DPBS; mice were sacrificed 4d post infection and the biological material was taken for detection. a. Pathological changes in the mouse Lung (Lung)HE staining of tissue sections). The control group of mice without RSV infection has narrow alveolar space and clear structure; the pulmonary alveolar interval of mice in the RSV group is greatly widened, partial pulmonary alveolar structures are difficult to distinguish, a large amount of inflammatory cell infiltration (marked by a solid triangle) appears around bronchi, and lymphocyte exudation is locally seen (marked by an open triangle); the pulmonary alveolar space of the mice in the RSV + PF4 group is slightly widened, the pulmonary alveolar structure is relatively complete, a small amount of inflammatory cell infiltration (marked by a solid triangle) is observed at the position of the peribronchial part, and no lymphocyte exudation is observed. b. Detection of inflammatory cytokines in mouse BALF. IL-6 and MCP-1 concentrations in BALF were lower in the RSV + PF4 group mice than in the RSV group.

Description of sequence listing

SEQ ID NO 1 shows the protein sequence of human platelet factor 4;

SEQ ID NO 2 shows the sequence of the forward primer for detection of RSV N;

SEQ ID NO 3 shows the sequence of the reverse primer for detection of RSV N;

SEQ ID NO 4 shows the sequence of a fluorescent probe for detection of RSV N.

Detailed Description

In a first aspect, the present invention provides the use of platelet factor 4 in the manufacture of a medicament for the prevention and/or treatment of diseases and/or conditions caused by respiratory syncytial virus infection.

In a particular embodiment of the invention, the platelet factor 4 is native platelet factor 4 purified from mammalian platelets.

In a preferred embodiment, the platelet factor 4 is native human platelet factor 4 purified from human platelets.

Human platelet factor 4 can be purified by methods known in the art, see for example, Handin, et al, j.biol.chem.,251: 4273-; or purified human platelet factor 4 is commercially available, for example from Sigma in the united states.

In a particular embodiment of the invention, the platelet factor 4 is recombinant platelet factor 4.

In a preferred embodiment, the platelet factor 4 is recombinant human platelet factor 4.

The Amino acid sequence of human platelet factor 4 was disclosed by Deuel, TF et al in 1977 (Deuel, TF et al, "Amino acid sequence of human planar factor 4," Proc. Natl. Acad. Sci. USA Biochemistry,74(6),2256-2258, 1977). Thereafter, scientists have sequentially identified and sequenced platelet factor 4 from different sources.

The protein sequence of human platelet factor 4 is shown in SEQ ID NO:1 (EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES). One skilled in the art can clone, express and isolate recombinant human platelet factor 4 according to known methods, see, for example, Myers et al, Protein Ex. Purif, 2:136-143 (1991); recombinant human platelet factor 4 is commercially available, for example, from ExCell Bio, Inc., Shanghai Gittacosi technologies, Inc., China.

It will be appreciated by those skilled in the art that in addition to human platelet factor 4, the present invention encompasses other mammalian native platelet factor 4 or recombinant platelet factor 4.

Those skilled in the art know that for proteins of known structure and function, proteins can be constructed by altering the amino acid sequence to either retain the original function or to increase or decrease the original function. Thus, the present invention also covers the use of a variant of platelet factor 4 showing the same, increased or decreased effect against respiratory syncytial virus relative to platelet factor 4 for the preparation of a medicament for the prevention and/or treatment of diseases and/or symptoms caused by respiratory syncytial virus infection. For example, a platelet factor 4 variant against respiratory syncytial virus can be constructed by: variants of platelet factor 4 known in the art are obtained (as described in PCT international publication WO 94/07524), or by random or site-directed mutagenesis (e.g., by replacing codons for one or more specific amino acids in the amino acid sequence of platelet factor 4 with a sequence encoding a different amino acid) and then testing whether the obtained variants have an effect against respiratory syncytial virus, as described in examples 1-3 herein.

It is contemplated that variants of platelet factor 4 encompassed by the present invention may be obtained from one or more conservative amino acid substitutions, so long as the resulting platelet factor 4 variant still has an effect against respiratory syncytial virus. Herein, conservative amino acid substitution refers to substitution between amino acids of the same type. Amino acids can be classified into the following types: basic, hydrophobic, acidic, polar, amide. A variant of platelet factor 4 produced by a substitution falls within the scope of the invention if one or more amino acids are substituted for an amino acid belonging to the same class as the one to which the substitution is made, and the substitution still produces a variant of platelet factor 4 that has an anti-respiratory syncytial virus effect. Table 1 illustrates examples of amino acids belonging to the same class:

TABLE 1

Furthermore, the invention also encompasses polypeptide fragments of platelet factor 4, or of variants of platelet factor 4, which are part of platelet factor 4 or of variants of platelet factor 4 and show the same, increased or decreased effect against respiratory syncytial virus relative to platelet factor 4. For example, a polypeptide fragment having an anti-respiratory syncytial virus effect can be constructed by: obtaining a polypeptide fragment of platelet factor 4 or a variant thereof known in the art (as described in Maine et al, Science,247:77-79 (1990)) or by protein degradation (chemically, enzymatically) and then determining whether the obtained polypeptide fragment has an effect against respiratory syncytial virus, as described in examples 1-3 herein.

In a particular embodiment of the invention, the medicament is for the prevention and/or treatment of diseases and/or symptoms caused by respiratory syncytial virus infection in a mammal or a human.

In a particular embodiment of the invention, the medicament is for preventing diseases and/or symptoms caused by respiratory syncytial virus infection in a high-risk infected person of respiratory syncytial virus, wherein the high-risk infected person of respiratory syncytial virus comprises: an elderly person over 60 years old, a child under 5 years old, a premature infant, an infant with chronic lung disease, an infant with congenital heart disease, an infant with immunodeficiency disease, an adult with congestive heart failure, an adult with chronic obstructive pulmonary disease, an adult with pneumonia, an adult with asthma, a patient receiving hematopoietic stem cells, a patient receiving lung transplantation, a patient with leukemia.

As used herein, "prevention" refers to prophylactic intervention that reduces the risk of a subject developing a disease or delays the subject becoming diseased or delays the onset of symptoms.

As used herein, "treatment" refers to a therapeutic intervention to improve the state of development of a disease or symptom in a subject, including causing regression of the disease or symptom in the subject, slowing the progression of the disease or symptom in the subject, lessening the severity of the disease or symptom in the subject, or reducing the cellular, physiological, or biochemical etiology or mechanism that causes the disease or symptom.

In a particular embodiment of the invention, the disease comprises acute upper respiratory tract infection, acute lower respiratory tract infection, bronchiolitis, pneumonia, tracheobronchitis, otitis media.

In a specific embodiment of the invention, said symptoms comprise nasal obstruction, rhinorrhea, cough, asthma, sneezing, rale, low fever at 37.3-38.3 ℃, loss of appetite, cyanosis, tachypnea, dyspnea, asphyxia, bronchitis, pneumonia.

In a specific embodiment of the invention, the medicament further comprises pharmaceutically acceptable excipients.

In the present invention, "pharmaceutically acceptable excipients" include all solvents, excipients, buffers, stabilizers, bactericides, fungicides and other excipients known in the art which are suitable for pharmaceutical applications. These adjuvants should be relatively non-toxic and harmless, and any side effects caused by the adjuvants do not impair the beneficial effects of the active ingredient.

Platelet factor 4 may be administered by any route, for example, suitable routes of administration include respiratory, parenteral (e.g., intravenous, intradermal, or subcutaneous), oral, topical, ocular, oral, nasal, sublingual, transmucosal, or rectal administration.

One skilled in the art can select appropriate excipients to formulate platelet factor 4 into a suitable dosage form according to the desired route of administration, for example, see Remington's Pharmaceutical Sciences, E.W. Martin, Mack Publishing, Easton, PA,19^th Edition (1995)；A.R.Gennaro,Remington:The Science and Practice of Pharmacy,Lippincott Williams&Wilkins,21st Edition, 2005; and L.V.Allen, Jr.et al, Ansel's Pharmaceutical Delivery Forms and Drug Delivery Systems,8th Edition, Philadelphia, PA: Lippincott, Williams&Wilkins,2004。

For platelet factor 4, suitable dosage forms include: sterile aqueous solutions for injection, dispersions, sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, tablets, pills, lozenges, emulsions, capsules, ointments, gels, transdermal patches, inhalants, nasal drops, suppositories and quick-, sustained-and timed-release dosage forms.

In a preferred embodiment, platelet factor 4 is formulated as a dry powder inhaler, spray or aerosol for oral or nasal administration.

The dry powder inhaler may be formulated with platelet factor 4 alone, or may contain a carrier acceptable for inhalation. Examples of carriers include, but are not limited to: inorganic salts such as sodium chloride or calcium carbonate; organic salts, such as sodium tartrate or calcium lactate; organic compounds such as urea; monosaccharides such as lactose, arabinose or dextrose; disaccharides, such as maltose or sucrose; polysaccharides, such as starch and dextran.

In addition to carriers, the dry powder inhalants may also contain other ingredients including, but not limited to, excipients (e.g., talc, lactose), flavoring agents (e.g., saccharin), antistatic agents (e.g., poloxamers); other pharmaceutical ingredients may also be included, such as bronchodilators (e.g., ipratropium bromide, albuterol sulfate).

The dry powder inhaler may be delivered by a dedicated powder inhalation device. The dry powder inhaler may be filled in a hard capsule or blister pack which is ruptured or drilled by an inhaler or insufflator so that the dry powder inhaler is inhaled upon oral or nasal inhalation.

The spray may be in the form of a solution, suspension, emulsion, gel, microsphere, liposome, or the like. Spray can contain ingredients including but not limited to solvents (e.g. ethanol, propylene glycol, polyethylene glycol, glycerol), surfactants (e.g. tweens, lecithin), activity protectors (e.g. human serum albumin, amino acids, polyols, cyclodextrins, lecithin), osmo-regulators (e.g. human serum albumin, sodium chloride, mannitol, glucose), preservatives (e.g. benzalkonium chloride, benzyl alcohol, phenol, cresol, paraben), adsorption inhibitors (e.g. human serum albumin, surfactants such as tweens), pH stabilizing regulators (e.g. Dunbeck Phosphate Buffered Saline (DPBS), phosphate buffers, citrate buffers, ascorbic acid buffers); other pharmaceutical ingredients may also be included, such as bronchodilators (e.g., ipratropium bromide, albuterol sulfate).

The aerosol can be filled in a special device (such as a jet nebulizer, an ultrasonic nebulizer, and a vibrating screen nebulizer) and released as a mist by pressure of a manual pump, high-pressure gas, ultrasonic vibration, or the like when used, thereby achieving oral or nasal administration.

The aerosol may be in the form of a solution, suspension, emulsion, gel, microsphere, liposome, or the like. In addition to optionally containing the above-described ingredients for sprays, aerosols also contain a suitable propellant. Examples of propellants include, but are not limited to: fluorocarbons or hydrogen-containing chlorofluorocarbons or mixtures thereof, especially hydrofluorocarbons, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, especially 1,1,1, 2-tetrafluoroethane, 1,1,1,2,3,3, 3-heptafluoro-n-propane or mixtures thereof; suitable gases are, for example, carbon dioxide.

The aerosol can be packaged in a pressure-tight container (e.g., a pressurized metered dose inhaler) with a specially designed valve system, and the aerosol can be sprayed into the mouth or nose as a mist by the pressure of the propellant.

In a preferred embodiment, platelet factor 4 is formulated as nasal drops for nasal administration.

The nasal drops may be in the form of solutions, suspensions, emulsions, gels, microspheres, liposomes, and the like. The nasal drops are preferably prepared using a suitable buffer (e.g., Dulbecco's phosphate buffered saline, phosphate buffer, citrate buffer, ascorbate buffer, acetate buffer, citrate buffer, borate buffer) or physiological saline. In addition, other ingredients that may be included in the nasal drops include, but are not limited to, preservatives (e.g., benzalkonium chloride, chlorobutanol, thimerosal, mercuric phenylacetate, mercuric phenylnitrate), surfactants (e.g., tween 80, lecithin), tonicity adjusting agents (e.g., sodium chloride, potassium chloride, mannitol, glycerin), antioxidants (e.g., sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene), absorption enhancers (e.g., cyclodextrin, taurine dihydrofusidate), chelating agents (e.g., edetate disodium); other pharmaceutical ingredients may also be included, such as bronchodilators (e.g., ipratropium bromide, albuterol sulfate).

The nasal drops may be filled into a conventional drop bottle, dropped into the nose by squeezing the drop bottle, or administered by a nasal pump.

In a particular embodiment of the invention, the medicament is administered in combination with other medicaments.

The combination does not cause unacceptable side effects.

In a preferred embodiment, the further medicament comprises a further medicament active against a virus infection of the Pneumovirinae subfamily, in particular a respiratory syncytial virus infection.

In a more preferred embodiment, the other drugs include: ribavirin, palivizumab, Movizumab, RSV-IGIV, MEDI-557, RSV604, MDT-637, BMS-433771, ALN-RSV01, ALX-0171 and mixtures thereof.

In a preferred embodiment, the other drugs include other drugs commonly used to treat respiratory symptoms and sequelae of respiratory infections.

In a more preferred embodiment, the other drugs include bronchodilators and glucocorticoids.

Bronchodilators which may be administered in combination with the medicaments of the present invention include β 2-adrenoceptor agonists bronchodilators such as formoterol, salbutamol or salmeterol, anticholinergics such as antagonists of muscarinic receptors, particularly the M3 subtype, l- { 4-hydroxy-l- [3,3, 3-tris- (4-fluoro-phenyl) -propionyl ] -pyrrolidine-2-carbonyl } -pyrrolidine-2-carboxylic acid (1-methyl-piperidin-4-ylmethyl) -amide, 3- [3- (2-diethylamino-acetoxy) -2-phenyl-propionyloxy ] -8-isopropyl-8-methyl-8-azocation-bicyclo [3.2.1] octane (ipratropium-N, diethyl N-glycinate), 1-cyclohexyl-3, 4-dihydro-1H-isoquinoline-2-carboxylic acid 1-aza-bicyclo [2.2.2] oct-3-yl ester (solifenacin); 2-hydroxymethyl-4-methanesulfinyl-2-phenyl-butyric acid 1-aza-bicyclo [2.2.2] oct-3-yl ester (revatonyl), 2- {1- [2- (2, 3-dihydro-benzofuran-5-yl) -ethyl ] -pyrrolidin-3-yl } -2, 2-diphenyl-acetamide (darifenacin), 4-cycloheximide-1-yl-2, 2-diphenyl-butyramide (Buzepide), 7- [3- (2-diethylamino-acetoxy) -2-phenyl-propionyloxy ] -9-ethyl-9-methyl-3-oxa-9-azocation-tricyclo [3.3.1.02,4] nonane (methylbromoxysaddle-N, N-glycine diethyl ester), 7- [2- (2-diethylamino-acetoxy) -2, 2-di-thiophen-2-yl-acetoxy ] -9, 9-dimethyl-3-oxa-9-azonian-tricyclo [3.3.1.02,4] nonane (Tornammonium-N, N-glycine diethyl ester), dimethylamino-acetic acid 2- (3-diisopropylamino-1-phenyl-propyl) -4-methyl-phenyl ester (tolterodine-N, N-glycine dimethyl ester), 3- [4, 4-bis- (4-fluoro-phenyl) -2-oxo-imidazolidin-1-yl ] -1-methyl-imidazolidin-1-yl ester -1- (2-oxo-2-pyridin-2-yl-ethyl) -pyrrolidinium, 1- [1- (3-fluoro-benzyl) -piperidin-4-yl ] -4, 4-bis- (4-fluoro-phenyl) -imidazolidin-2-one, 1-cyclooctyl-3- (3-methoxy-1-aza-bicyclo [2.2.2] oct-3-yl) -l-phenyl-propan-2-yn-l-ol, 3- [2- (2-diethylamino-acetoxy) -2, 2-di-thiophen-2-yl-acetoxy ] -l- (3-phenoxy-propyl) -1-azonium cation -bicyclo [2.2.2] octane (aclidinium-N, N-glycine diethyl ester) or (2-diethylamino-acetoxy) -di-thiophen-2-yl-acetic acid 1-methyl-l- (2-phenoxy-ethyl) -piperidin-4-yl ester.

Glucocorticoid drugs that can be administered in combination with the drugs of the present invention include dexamethasone, dexamethasone sodium phosphate, fluoromethalone acetate, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone acetonide, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinolone acetonide, flunisolide, fluocortefuran-21-butylate, flumethasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide, or pharmaceutically acceptable salts thereof.

In addition, the agents that may also be administered in combination with the agents described herein are anti-inflammatory signal transduction modulators that act through an anti-inflammatory cascade mechanism, such as phosphodiesterase inhibitors (e.g., PDE-4, PDE-5 or PDE-7 specific inhibitors), transcription factor inhibitors (e.g., blocking NF-. kappa.B by IKK inhibition) or kinase inhibitors (e.g., blocking P38MAP, JNK, PI3K, EGFR, Syk), and specifically such modulators include 5- (2, 4-difluoro-phenoxy) -1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylamino-ethyl) -amide (P38MAP kinase inhibitor ARRY-797), 3-cyclopropylmethoxy-N- (3, 5-dichloro-pyridin-4-yl) -4-difluoromethoxy-benzamide (PDE-4 inhibitor) Formulation roflumilast), 4- [2- (3-cyclopentyloxy-4-methoxyphenyl) -2-phenyl-ethyl ] -pyridine (PDE-4 inhibitor CDP-840), N- (3, 5-dichloro-4-pyridinyl) -4- (difluoromethoxy) -8- [ (methylsulfonyl) amino ] -1-dibenzofurancarboxamide (PDE-4 inhibitor Oglemilast), N- (3, 5-dichloro-pyridin-4-yl) -2- [ l- (4-fluorobenzyl) -5-hydroxy-1H-indol-3-yl ] -2-oxo-acetamide (PDE-4 inhibitor AWD 12-281), and pharmaceutically acceptable salts thereof, 8-methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid (3, 5-dichloro-1-oxo-pyridin-4-yl) -amide (PDE-4 inhibitor Sch351591), 4- [5- (4-fluorophenyl) -2- (4-methanesulfinyl-phenyl) -1H-imidazol-4-yl ] -pyridine (P38 inhibitor SB-203850), 4- [4- (4-fluoro-phenyl) -1- (3-phenyl-propyl) -5-pyridin-4-yl-1H-imidazol-2-yl ] -but-3-yn-1-ol (P38 inhibitor RWJ-67657), 4-cyano-4- (3-cyclopentyloxy-4-methoxy-phenyl) -cyclohexanecarboxylic acid 2-diethylamino-ethyl ester (2-diethyl-ethyl ester prodrug of cilomilast, PDE-4 inhibitor); (3-chloro-4-fluorophenyl) - [ 7-methoxy-6- (3-morpholin-4-yl-propoxy) -quinazolin-4-yl ] -amine (gefitinib, EGFR inhibitor); and 4- (4-methyl-piperazin-1-ylmethyl) -N- [ 4-methyl-3- (4-pyridin-3-yl-pyrimidin-2-ylamino) -phenyl ] -benzamide (imatinib, EGFR inhibitor).

In addition, the drugs which can be administered in combination with the drugs of the present invention are useful as mucolytic, expectorant, etc. for the treatment of respiratory infections and symptoms, such as ambroxol, guaifenesin.

In addition, the medicaments that can also be administered in combination with the medicaments described herein are nebulized hypertonic saline, e.g., about 3% nebulized hypertonic saline, for improving the immediate and long term clearance of the small airways of patients with pulmonary disease.

In a preferred embodiment of the invention, the combined administration may be simultaneous or sequential.

The dosage, sequence and number of administrations required for the administration of the agents of the invention, either alone or in combination with other agents, can be determined by one skilled in the art by routine therapeutic testing.

It is contemplated that the agents of the present invention may produce a "synergistic effect" when administered in combination with other agents, i.e., the effect achieved when the combination is greater than the sum of the effects achieved when each is administered alone.

A second aspect of the invention provides a method of non-therapeutic interest for inhibiting respiratory syncytial virus in cells in vitro with platelet factor 4, the method comprising: contacting the platelet factor 4 with the cell before and/or after the respiratory syncytial virus is contacted with the cell; preferably, the concentration of platelet factor 4 is 5-5000 nM; more preferably, the concentration of platelet factor 4 is 125-2000 nM.

Herein, said inhibition comprises inhibiting infection or replication of respiratory syncytial virus.

In a particular embodiment of the invention, the cell is a human cell.

In a preferred embodiment, the cell is a human laryngeal carcinoma cell HEp-2.

The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The specific experimental procedures not indicated in the examples below are followed according to conventional conditions and methods, such as those described in the handbook of molecular cloning laboratories (Sambrook, et al. New York: Cold Spring Harbor Laboratory Press,1989) or those provided by the reagent manufacturer.

Example 1 PF4 inhibition of RSV infection and replication in HEp-2 cells

1.1 protocol

HEp-2 cells (purchased from ATCC, accession number: ATCC CCL-23, cell concentration 1.8X 10)⁵One/ml) in 96-well and 24-well cell plates, 5% CO at 37 ℃₂Culturing for 16h, treating HEp-2 cells with recombinant human PF4 protein (ExCell Bio, cat # CB055-492) dissolved in Opti-MEM medium (Gibco, cat # 31985-₂After 2h incubation, the mixture of RSV A2 strain (purchased from ATCC, No. ATCC VR-1540) and PF4 at MOI (multiplicity of infection) of 1 (96-well plate) or 2 (24-well plate) was incubated for 2h, and the final concentration of PF4 in the mixture was the same as before; the culture was continued until 24 hours after infection by changing to a maintenance solution (Opti-MEM medium containing 2% fetal bovine serum and 1% penicillin, wherein fetal bovine serum was purchased from Hyclone under the accession number SH 30396.03; penicillin was purchased from Gibco under the accession number 15140-.

1.2 detection of RSV infection Rate

The culture medium was aspirated from a 96-well plate, the cells were fixed with 4% paraformaldehyde, washed with PBS 1 time, stained with 1:1000 diluted DAPI dye (Invitrogen, Cat. D1306) for 30min, washed with PBS 1 time, treated with a transmembrane buffer (PBS containing 0.5% Triton X-100) for 15min, washed with PBS 2-3 times, treated with a blocking buffer (PBS containing 5% bovine serum albumin) for 90min, and washed with PBS 1:10The mouse anti-RSV N protein antibody (product of Merck Millipore Co., Ltd.; product No.: MAB858-3) was treated at 00 dilution for 2 hours and then allowed to stand at 4 ℃ overnight. The plate is washed 3-5 times by PBS, the Alexa Fluor 488-labeled goat anti-mouse IgG antibody (product of China fir gold bridge biotechnology company, product number: ZF-0512) diluted 1:500 is treated for 1h, the plate is washed 4-5 times by PBST, and the plate is washed 1-2 times by PBS. The Operetta high content microscopy imaging instrument (Perkinelmer) scans the cell image and RSV infected cells emit a green fluorescent signal. In fig. 1a, DAPI stained nuclei appear as oval, light gray dots, and RSV infected cells appear as irregular, light gray plaques. As can be seen in fig. 1a, with increasing concentration of PF4, RSV infected cells gradually decreased. And analyzing the cell image by using matched computer software, identifying the cell position through cell nucleus DAPI signals, and calculating the green fluorescence signal intensity of each cell, wherein the RSV positive cell is more than 1 time higher than the background signal. Calculating the RSV infection rate of the cells: the infection rate was 100% of RSV positive cells/total cells, and as shown in fig. 1b, the RSV infection rate of the cells gradually decreased with increasing concentration of PF4, and was linearly and negatively correlated with the logarithm of the concentration of PF4 (R)²＝0.92)。

1.3RSV protein expression assay

The 24-well plate was discarded, the plate was washed 1 time with DPBS (product of Gibco, cat # 14190-. Collecting liquid, centrifuging at 10000g and 4 ℃ for 10min, and respectively taking supernatant with proper volume from each sample to ensure that the total protein content is 50 mu g; 1/5 volumes of 6 XSDS sample buffer (ultrapure aqueous solution containing 300mM Tris-HCl (pH 6.8), 600mM dithiothreitol, 12% SDS, 0.6% bromophenol blue and 60% glycerol) were added thereto, and the mixture was mixed, and then the protein was denatured by boiling water bath for 5 to 10min, followed by electrophoresis (SDS-PAGE). The protein was transferred from the gel to a nitrocellulose membrane (product of Pall, cat # S80209) by rotating the membrane at 80V under constant pressure for 2 hours in an ice bath, and the membrane was placed in 5% skim milk and blocked for 2 hours. The membrane was washed with PBST by incubating a mouse anti-RSV N protein antibody (Abcam Co., Ltd., cat # ab94806) diluted 1:2000 and a mouse anti-RSV G protein antibody (Abcam Co., cat # ab94966) diluted 1:500 overnight at 4 ℃ and incubating for 30min in the dark using an IRDye 800-labeled goat anti-mouse IgG antibody (Li-Cor Co., cat # 926-32210) and washing the membrane with PBST. GAPDH was used as an internal reference, and a rabbit anti-GAPDH antibody (Sigma-Aldrich, cat # G9545) diluted at a ratio of 1:10000 was incubated for 1 hour in the absence of light, PBST was washed, and then a goat anti-rabbit IgG antibody (Li-Cor, cat # 925-68021) labeled with IRDye 680 was incubated for 30 minutes in the absence of light, and PBST was washed. After washing the membrane with deionized water, the membrane was scanned and imaged using an Odyssey near infrared imaging system (Li-Cor). As shown in FIG. 1c, the expression levels of RSV N and G proteins in cells gradually decreased with increasing concentration of PF4, indicating a dose-response relationship.

Example 2PF4 reduction of pulmonary viral load and pulmonary inflammation levels following RSV infection in mice

2.1 protocol

After being qualified by quarantine, SPF-grade 6-8 week-old female BALB/c mice (Beijing Huafukang Biotechnology limited) enter BSL-2-grade environment for feeding, 4-6 mice are fed in each cage, and the food and drinking water supply is sufficient; the mice need to be raised for more than 3 days before the experiment begins, so that the mice are fully adaptive to the environment. Mice were randomly divided into 3 groups, RSV + PF4 group 8 mice, RSV group 8 mice, control group 5 mice. Mice in the RSV + PF4 group and RSV group were intraperitoneally injected with 125. mu.l/mouse of 1% sodium pentobarbital, and after anesthetizing the mice, 1.8. mu.g (about 0.1mg PF4/kg body weight) of recombinant human PF4 protein solubilized in DPBS was nasally administered to each mouse in the RSV + PF4 group in a volume of 18. mu.l; in the RSV group, 18. mu.l DPBS was administered nasally to each mouse. After 6h, all mice were intraperitoneally injected with 125 μ l/mouse of 1% sodium pentobarbital, and after anesthetizing the mice, the RSV + PF4 group and RSV group were nasally inoculated with 1 × 10⁶PFU RSV a2 strain virus, diluted to 20 μ l with DPBS at inoculation; control groups each mouse was given 20. mu.l DPBS by nasal drip. All mice were kept on stock, and 4 days after infection, mice were sacrificed by cervical dislocation, and lungs were taken from some mice, fixed in 4% paraformaldehyde, and used for pathological section detection (3 RSV + PF4 groups, 3RSV groups, 2 control groups); the remaining mice were prepared for Bronchoalveolar lavage fluid (BALF) and lungs were stored at-80 ℃ for lung homogenate.

2.2PF4 treatment reduces pulmonary viral load following RSV infection in mice

A mouse lung tissue block was taken and 500. mu.l of 4 ℃ pre-cooled DPBS was added and ground on ice to prepare a homogenate. Mu.l of the homogenate was taken, 1ml of TRIzol (Invitrogen, product: 15596-. 100ng of total RNA of lung tissue was used as a test sample, 10-fold serial dilution of RNA standard of RSV N gene was used as a standard curve, and AgPath-ID was used^TMThe RSV N gene copy number is detected by a one-step RT-PCR detection kit (product of Ambion company, cat # AM 1005). Reverse transcription and PCR reactions were performed according to the instructions of the kit, with primers and fluorescent probes for detection of RSV N as follows:

a forward primer:

5'-AGATCAACTTCTGTCATCCAGCAA-3', the sequence is shown in SEQ ID NO. 2;

reverse primer:

5'-TTCTGCACATCATAATTAGGAGTRTCAAT-3' with the sequence shown in SEQ ID NO. 3;

a fluorescent probe:

5'ROX-AATACACCATCCAACGGAGCACAGGAGA-3' BHQ2, the sequence is shown in SEQ ID NO. 4.

The detection of fluorescence signal, the calculation of Ct value and the storage and analysis of experimental data during the amplification process are all CFX96^TMA fluorescent quantitative PCR detection system (BIO-RAD company) and self-contained software. And calculating the copy number of the RSV N gene. The results are shown in FIG. 2a, the RSV N gene copy number (2.27X 10) per 100ng total lung RNA of RSV + PF4 group mice⁶±1.05×10⁶) Lower than RSV group (6.62X 10)⁶±2.01×10⁶) The difference has statistical significance (p ═ 0.046); PF4 treatment was shown to significantly reduce the viral load in the lungs of the host after infection.

Mouse lungs fixed in 4% paraformaldehyde were prepared into tissue sections by paraffin embedding. Distribution of RSV in lung tissue was examined by immunohistochemistry using a goat anti-RSV antibody (Abcam Co., Ltd., cat # ab20745) and a horseradish peroxidase-labeled secondary antibody (Invitrogen Co., cat # 81-1620) and observed and photographed with an 80i upright microscope (Nikon Co.). As shown in FIG. 2b, a large number of RSV signals were observed in lung tissues of mice in the RSV group, and the distribution density and signal intensity of RSV signals in lung tissues of mice in the RSV + PF4 group were significantly reduced compared to those in the RSV group, indicating that PF4 treatment reduced RSV replication in lung tissue cells after mice were infected with RSV.

2.3PF4 treatment reduces pulmonary inflammation levels following RSV infection in mice

Mouse lungs fixed in 4% paraformaldehyde were prepared into tissue sections by paraffin embedding. Pathological changes in mouse lung tissue were detected by hematoxylin-eosin (HE) staining, observed microscopically and photographed. The results are shown in FIG. 3a, in which the control mice not infected with RSV had narrow alveolar spaces and clear structures; the pulmonary alveolar interval of mice in the RSV group is seriously widened, partial pulmonary alveolar structures are difficult to distinguish, a large amount of inflammatory cell infiltration appears around bronchi, and local lymphocyte exudation can be seen; the pulmonary alveolar space of the mice in the RSV + PF4 group is slightly widened, the pulmonary alveolar structure is relatively complete, a small amount of inflammatory cell infiltration is observed at the peripheral part of the bronchus, and lymphocyte exudation is not observed. The above results indicate that PF4 treatment can significantly reduce viral pneumonic lung pathology following RSV infection in mice.

The concentration of Interleukin-6 (Interleukin-6, IL-6) which is an important inflammatory cytokine in mouse BALF was measured using a mouse IL-6ELISA kit (product of ExCelBiol Inc., cat # EM 004-96). Using BD^TMThe CBA mouse inflammation assay kit (BD Biosciences, cat # 101310) measures the concentration of Monocyte chemotactic protein-1 (MCP-1), an important inflammatory cytokine in mouse BALF. The results are shown in fig. 3b, IL-6 concentration (8.2 ± 1.0pg/ml) in BALF of RSV + PF4 group mice is lower than that of RSV group mice (12.5 ± 2.3pg/ml), indicating that PF4 treatment significantly reduced the intra-respiratory level of inflammatory cytokine IL-6 following RSV infection in mice; MCP-1 concentration (0.51 +/-0.24 pg/ml) in BALF of mice in the RSV + PF4 group was lower than that in mice in the RSV group (1.78 +/-0.69 pg/ml), indicating that PF4 treatment was able to reduce the intra-respiratory level of the inflammatory cytokine MCP-1 after RSV infection in mice.

The above results indicate that PF4 treatment can reduce the level of lung inflammation following RSV infection in mice.

The above embodiments are exemplary embodiments and should not be construed as limiting the invention. Those skilled in the art can recognize that any modification, equivalent replacement, improvement or the like which comes within the spirit and principle of the present invention is included in the scope of the present invention.

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Claims

1. The application of the platelet factor 4 in preparing the medicine for preventing and/or treating diseases and/or symptoms caused by respiratory syncytial virus infection.

2. The use of claim 1, wherein said platelet factor 4 is native platelet factor 4; alternatively, the platelet factor 4 is recombinant platelet factor 4.

3. The use of claim 2, wherein said platelet factor 4 is native human platelet factor 4.

4. The use of claim 2, wherein said platelet factor 4 is recombinant human platelet factor 4.

5. Use according to claim 1 or 2, wherein the medicament is for the prevention and/or treatment of diseases and/or conditions caused by respiratory syncytial virus infection in a mammal or a human.

6. The use according to claim 1 or 2, wherein the medicament is for preventing diseases and/or symptoms caused by respiratory syncytial virus infection in those at high risk of respiratory syncytial virus infection, wherein the high risk of respiratory syncytial virus infection comprises: an elderly person over 60 years old, a child under 5 years old, a premature infant, an infant with chronic lung disease, an infant with congenital heart disease, an infant with immunodeficiency disease, an adult with congestive heart failure, an adult with chronic obstructive pulmonary disease, an adult with pneumonia, an adult with asthma, a patient receiving hematopoietic stem cells, a patient receiving lung transplantation, a patient with leukemia.

7. The use of claim 1 or 2, wherein the disease comprises acute upper respiratory tract infection, acute lower respiratory tract infection, bronchiolitis, pneumonia, tracheobronchitis, otitis media, asthma.

8. The use of claim 1 or 2, wherein said symptoms comprise nasal congestion, nasal discharge, cough, sneezing, rale, low fever at 37.3-38.3 ℃, loss of appetite, cyanosis, tachypnea, dyspnea, asphyxia.

9. The use of claim 1 or 2, wherein platelet factor 4 is formulated as a dry powder inhaler, spray or aerosol for oral or nasal administration; alternatively, platelet factor 4 is formulated as nasal drops for nasal administration.

10. The use of claim 1 or 2, wherein the medicament is administered in combination with another medicament.

11. The use of claim 10, wherein the other medicament comprises ribavirin, palivizumab, mevizumab, RSV-IGIV, MEDI-557, a-60444, MDT-637, BMS-433771, ALN-RSV01, ALX-0171, and mixtures thereof; bronchodilators and glucocorticoids.

12. A method for the non-therapeutic purpose of inhibiting respiratory syncytial virus in cells in vitro with platelet factor 4, the method comprising: the platelet factor 4 is contacted with the cells before and/or after the respiratory syncytial virus is contacted with the cells.

13. The method of claim 12, wherein the concentration of platelet factor 4 is 5-5000 nM.

14. The method of claim 13, wherein the concentration of platelet factor 4 is 125 nM and 2000 nM.

15. The method of claim 12, wherein the cell is a human cell.

16. The method of claim 15, wherein the cell is a human laryngeal carcinoma cell HEp-2.