WO2022056897A1 - 白细胞介素37与干扰素联用在治疗病毒感染中的应用 - Google Patents

白细胞介素37与干扰素联用在治疗病毒感染中的应用 Download PDF

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WO2022056897A1
WO2022056897A1 PCT/CN2020/116399 CN2020116399W WO2022056897A1 WO 2022056897 A1 WO2022056897 A1 WO 2022056897A1 CN 2020116399 W CN2020116399 W CN 2020116399W WO 2022056897 A1 WO2022056897 A1 WO 2022056897A1
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ifn
virus
protein
vector
active
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PCT/CN2020/116399
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French (fr)
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徐建青
张晓燕
李昂
袁松华
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上海市公共卫生临床中心
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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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • 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
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application belongs to the fields of biotechnology and medicine. Specifically, the present application relates to the combination of interleukin 37 and interferon, its related products, and its application in the treatment of viral infections.
  • Virus is a very small infectious disease-causing entity with only one nucleic acid, but its impact on human society is profound and significant.
  • the 2019 novel coronavirus disease will undoubtedly have a huge impact on people's lives and lives.
  • viruses Due to the many types of viruses, they can be divided into DNA viruses (including Adenoviridae, Herpesviridae, Picornaviridae, Poxviridae and Hepadnaviridae, etc.) and RNA viruses (General Qi Including Coronaviridae, Fibroviridae, Orthomyxoviridae, Paramyxoviridae, Retroviridae, etc.).
  • DNA viruses including Adenoviridae, Herpesviridae, Picornaviridae, Poxviridae and Hepadnaviridae, etc.
  • RNA viruses General Qi Including Coronaviridae, Fibroviridae, Orthomyxoviridae, Paramyxoviridae, Retroviridae, etc.
  • the transmission route it can be divided into respiratory transmission, gastrointestinal transmission, blood transmission, etc.
  • a total of 4,958 viruses have been classified, and there are still a large number of new viruses that cannot be classified due to the imperfection
  • Clinical broad-spectrum antiviral therapy is usually antiviral drugs such as ribavirin, vidarabine, etc. These drugs can selectively inhibit by inhibiting the metabolic analogs of the virus, but long-term use will affect hematopoiesis. system, the digestive system brings serious side effects, and there is a lot of liver toxicity.
  • interferon as a broad-spectrum antiviral molecule, has been used clinically for the treatment of viral infections. It mainly activates downstream interferon-stimulated genes by interacting with cell surface receptors, thereby inhibiting the replication of viruses in cells. Its antiviral effect is superior to broad-spectrum antiviral drugs due to its fast onset and strong effect.
  • interferon also has certain toxic and side effects, which can mediate liver and kidney toxicity, especially in the process of clearing the virus, it is likely to cause excessive activation of inflammation in the body, produce a large number of inflammatory factors, and even cause inflammatory factor storms, which can easily lead to Septic shock and multiple organ failure increase mortality.
  • Interleukin-37 is the seventh member of the IL-1 family and the only anti-inflammatory cytokine in the IL-1 family. Because of its strong anti-inflammatory ability, it plays an important role in a variety of inflammatory diseases. Early researchers found that IL-37 can enter the nucleus and combine with Smad3, a key transcriptional regulator of the TGF- ⁇ signaling pathway, to form a functional complex, thereby achieving anti-inflammatory functions by affecting gene transcription. Another newly discovered anti-inflammatory mechanism of IL-37, IL-37 can bind to IL-18Ra, thereby recruiting orphan receptor IL-1R8 (SIGIRR or TIR8), and using IL-1R8 to inhibit downstream pro-inflammatory signaling kinases mTOR, MAPK Etc.
  • SIGIRR orphan receptor IL-1R8
  • the present application provides an application, product and method for the treatment of respiratory viral infection based on interleukin 37.
  • a product for the treatment of viral infectious diseases or symptoms comprising:
  • IFN interferon
  • the product is a pharmaceutical composition, a combination of formulations, or a kit.
  • the IL-37 active substance is selected from the group consisting of: native IL-37 protein, recombinant IL-37 protein, nucleic acid molecules encoding the aforementioned IL-37 proteins, expression vectors or cells comprising the nucleic acid molecules, including Cell membrane components of the IL-37 protein, synthetic IL-37 protein.
  • the nucleic acid molecule encoding the IL-37 protein is selected from: (i) a nucleic acid molecule having the nucleotide sequence shown in any one of Gene ID: 27178 or SEQ ID NO: 6-10 or its degenerate sequence or its spliceosome nucleic acid molecule; (ii) comprising one or more nucleotide substitutions, deletions and/or additions in the nucleic acid molecule of (i) and having the ability to encode activity with the nucleic acid molecule of (i) A nucleic acid molecule of IL-37; (iii) having at least 80% homology (eg, at least 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) with the nucleic acid molecule in (i) homology) and a nucleic acid molecule capable of encoding IL-37 protein.
  • the IL-37 protein is selected from: (a) an IL-37 protein having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 5, or a spliceosome molecule thereof; (b) (a) a protein containing one or more amino acid substitutions and/or deletions and/or additions in the IL-37 protein or its spliceosome molecule and having an anti-inflammatory function; and/or (c) having at least 80% of the same as the aforementioned polypeptides % homology (e.g.
  • IL-37 protein that inhibits inflammation; (d) by ( The IL-37 protein or polypeptide expressed by the nucleic acid molecule described in i), (ii) or (iii).
  • the IFN active substance is selected from the group consisting of: native IFN protein or recombinant IFN protein (eg, recombinant human IFN), encoding molecules of the aforementioned IFN proteins, expression vectors or cells comprising the encoded molecules, comprising the IFN Cell membrane components of proteins.
  • the IFN is one or more selected from the group consisting of one or more of the IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ families, preferably, the The IFN is one or more of IFN- ⁇ 1b, IFN- ⁇ 2a, IFN- ⁇ 2b, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ 1 and IFN- ⁇ 2, more preferably, the interferon is IFN- ⁇ (eg IFN- ⁇ 2b) or IFN- ⁇ .
  • the expression vector is one or more selected from the group consisting of adenovirus vector, adeno-associated virus vector, poxvirus vector, retrovirus vector, herpes virus vector, RNA virus vector, EB Viral vector, baculovirus vector, phage vector, animal virus vector, plant virus vector, DNA plasmid vector, RNA vector.
  • the amount of the IL-37 active substance is 0.01-10 ⁇ g, 0.05-5 ⁇ g, 0.1-4 ⁇ g, 0.5-3 ⁇ g; and/or, the amount of the IFN active substance is 1 ⁇ 10 4 to 1 ⁇ 10 8 Activity units, 5x10 4 to 5x10 7 activity units, 1x10 5 to 1x10 7 activity units, and 5x10 5 to 5x10 6 activity units.
  • the product is in a form suitable for administration of the IL-37 active substance and the IFN active substance by the same or different routes selected from the group consisting of aerosol inhalation, nasal instillation, spray, intravenous administration, intra-target tissue administration medicine or oral administration.
  • the product is in a form suitable for simultaneous, sequential or spaced administration of the IL-37 active substance and the IFN active substance.
  • the article of manufacture further comprises: one or more containers containing the IL-37 active substance and the IFN active substance, such as one or more containers containing a unit dose of the IL-37 active substance and the IFN active substance; Apparatus for drug administration; instructions for use, etc.
  • the IL-37 active substance and the interferon active substance are provided simultaneously or separately in the form of an active protein (eg, a separate active protein and/or a fusion protein form of two or more active proteins) , a nucleic acid molecule encoding an active protein or a fusion protein thereof, an expression vector comprising the nucleic acid molecule (e.g., a separate expression vector or an expression vector comprising both nucleic acid molecules) or a cell form, a cell membrane group comprising the active protein point.
  • an active protein eg, a separate active protein and/or a fusion protein form of two or more active proteins
  • an expression vector comprising the nucleic acid molecule (e.g., a separate expression vector or an expression vector comprising both nucleic acid molecules) or a cell form, a cell membrane group comprising the active protein point.
  • the IL-37 active substance and the interferon active substance exist or express separately, and are used in combination; fused and expressed to form a multifunctional molecule; or the IL-37 active substance and an interferon After fusion, it is used in combination with another or more interferons.
  • the IL-37 active substance is used in combination with one or more IFN active substances, or the IL-37 active substance and the one or more IFN active substances are expressed from the same or different vectors, or The method of applying the cell membrane together after being expressed on different cell membranes respectively, or the method of applying the cell membrane after the IL-37 active substance and one or more IFN active substances are expressed on the same cell membrane.
  • the IL-37 active substance is fused and expressed with one or more IFN active substances, including constructing a fusion gene through gene recombination technology for protein expression, expressing the fusion gene through a vector, and passing through the cytoplasmic membrane.
  • the fusion recombinant protein is expressed on the cell membrane and linked by chemical methods.
  • IL-37 is expressed in fusion with IFN- ⁇ or IFN- ⁇ .
  • the product also includes other drugs for the treatment of viral infections, such as traditional Chinese medicine, chemical drugs, biological drugs, including but not limited to antiviral drugs, antiviral preparations, antiviral serum, anti-inflammatory drugs Drugs, hormones, cytokines, antibodies, etc.
  • drugs for the treatment of viral infections such as traditional Chinese medicine, chemical drugs, biological drugs, including but not limited to antiviral drugs, antiviral preparations, antiviral serum, anti-inflammatory drugs Drugs, hormones, cytokines, antibodies, etc.
  • the viruses are DNA viruses and RNA viruses.
  • the DNA virus is selected from the group consisting of: adenovirus, herpes virus, pox virus, hepadnavirus, HBV, HPV, and the like.
  • the RNA virus is selected from the group consisting of: coronaviruses (such as SARS, SARS-CoV-2, MERS), fiber viruses (such as Ebola virus), flaviviruses (such as Zika virus, yellow fever virus) , dengue virus, West Nile virus, Japanese encephalitis virus, HCV), orthomyxovirus (influenza virus (such as H1N1, H3N2, H7N9, H5N1), avian influenza virus), paramyxovirus (measles virus, human respiratory fusion virus ), retroviruses (HIV), and picornaviruses (eg, rhinoviruses, enteroviruses).
  • coronaviruses such as SARS, SARS-CoV-2, MERS
  • fiber viruses such as Ebola virus
  • flaviviruses
  • the virus is a novel coronavirus
  • its viral infectious diseases or symptoms include: novel coronavirus pneumonia; one or more conditions associated with novel coronavirus infection selected from the group consisting of: dyspnea, Hypoxemia, acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction, multiple organ failure, pulmonary fibrosis, persistent chronic inflammation, fever, dry cough, fatigue, nasal congestion, runny nose , sore throat, myalgia and diarrhea.
  • the virus-infected subject is a mammal, preferably a human.
  • IL-37 active substances and IFN active substances in the manufacture of a product for the treatment of viral infections as described herein is provided.
  • a method of treating a viral infectious disease or condition in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a combination of an IL-37 active substance and an IFN active substance or A product as described herein or made by a method described herein.
  • the features involved in this aspect may be as previously described.
  • FIG. 1 Establishment of an influenza virus infection model in H1N1-PR8-challenged mice
  • Figure 1A Schematic diagram of the monitoring process of mouse survival and body weight change: three groups of 8 mice were challenged with H1N1-PR8 influenza virus at a dose of 250, 500 and 1000 TCID50 via intranasal injection, and were continuously weighed for 14 days. And monitor mouse survival and body weight changes;
  • Figure 1B The change rate of mouse body weight over time in 14 days, where the ordinate represents the decrease rate of mouse body weight
  • FIG. 1C Survival of mice in each group at the end of the 14-day observation.
  • Figure 2 H1N1-PR8 mouse model to test the protective effect of the combination of IL-37 and IFN ⁇ 2b
  • Figure 2A Schematic diagram of the experimental process: the challenge dose of 500TCID50 H1N1-PR8 in Example 1 was selected. After the challenge, PBS, IL-37, IFN ⁇ 2b and the combination of IL-37 and IFN ⁇ 2b were aerosolized in each group, and the body weight was recorded continuously for 14 days , and monitor survival at the same time;
  • Figure 2B The rate of change of the body weight of the mice over time within 14 days, where the ordinate represents the rate of decrease in the body weight of the mice;
  • Figure 2C The survival of mice in each group at the end of the 14-day observation is shown in Figure 2C.
  • Figure 3 Relationship between plasma IL-37 levels and inflammatory factors in COVID-19 patients
  • Figure 3A Correlation between plasma IL-37 levels and inflammatory factor IL-6 levels in COVID-19 patients;
  • Figure 3B Correlation between plasma IL-37 levels and inflammatory factor IL-8 levels in COVID-19 patients;
  • Figure 3C Correlation between plasma IL-37 levels and inflammation-related marker high-sensitivity C-reactive protein (HS-CRP) levels in COVID-19 patients.
  • HSP high-sensitivity C-reactive protein
  • Figure 3F COVID-19 patients with high plasma IL-37 secretion levels had significantly shorter days to cough clearance.
  • IL-37 According to the median plasma IL-37 level, the patients were divided into two groups: low secretion and high secretion of IL-37. Compared with patients with low secretion of IL-37, patients with high secretion of IL-37 had higher levels of the inflammatory factor IL-6. level (Fig. 3A), the level of inflammatory factor IL-8 (Fig. 3B) and the level of inflammation-related marker high-sensitivity C-reactive protein (HS-CRP) (Fig. 3C) were significantly lower. Furthermore, COVID-19 patients with high plasma IL-37 secretion levels were also found to have significantly shorter hospital stays (Fig. 3D), significantly shorter lung CT improvement days (Fig. 3E), and significantly shorter cough clearance days (Fig. 3F).
  • Figure 4A Comparison of hospitalization days in COVID-19 patients with high IL-37 and high IFN ⁇ versus low IL-37 and low IFN ⁇ ;
  • Figure 4B Comparison of viral nucleic acid negative days in COVID-19 patients with high IL-37 and high IFN ⁇ and low IL-37 and low IFN ⁇ ;
  • Figure 4C Comparison of lung CT improvement days in COVID-19 patients with high IL-37 and high IFN ⁇ versus low IL-37 and low IFN ⁇ ;
  • Figure 4D Comparison of days of cough improvement in COVID-19 patients with high IL-37 and high IFN ⁇ versus low IL-37 and low IFN ⁇ .
  • the COVID-19 patients were divided into two groups: high IL-37 and high IFN ⁇ and low IL-37 and low IFN ⁇ groups.
  • FIG. 5 Interleukin 37 treatment significantly reduces lung inflammation in ACE2 transgenic mice infected with SARS-CoV-2 virus
  • Figure 5A The left picture is the staining of the whole lung lobe tissue section in the PBS control group, and the right picture is an enlarged display of the intercepted area of the left picture;
  • Figure 5B The left picture is the staining of the whole lung lobe tissue section in the treatment group, and the right picture is an enlarged display of the intercepted area of the left picture;
  • Figure 5C Pathological scoring data by pathological analysis software, showing significantly lower pathological scores in the interleukin-37 treatment group
  • Figure 5D The number of inflammatory cell infiltration per unit area of lung tissue calculated by pathological analysis software, showing that the number of inflammatory cell infiltration in the interleukin 37 treatment group was significantly lower.
  • the inventors unexpectedly found in their research that the combination of interleukin 37 and interferon (eg interferon alpha 2b) was applied to a mouse model of H1N1 influenza infection, and found that the combination had a significant protective effect on mice, including mice The rate of weight loss was reduced and the survival rate was significantly improved. Since the interleukin 37 in the composition can antagonize the excessive activation of inflammation, combined with the inhibitory effect of interferon on virus replication, the combination of the two has obvious therapeutic and protective effects on virus infection, and has great clinical application value.
  • interleukin 37 and interferon eg interferon alpha 2b
  • the present application provides the combined use of interleukin 37 and interferon for the treatment of viral infections, especially respiratory viral infections.
  • the combination provides a safer and more effective drug and/or regimen for treating viral infections.
  • interferon broadly inhibits viral replication
  • IL-37 inhibits viral infection and the inflammatory pathogenic effects of interferon use.
  • This application contains at least a combination of interleukin 37 and an interferon, which has important antiviral advantages and can simultaneously act on the two targets of virus replication and inflammation and pathogenesis, and has a synergistic effect; it has a rapid and broad-spectrum antiviral effect At the same time, it can antagonize the excessive response of inflammatory factors that may be caused by interferon in the antiviral process, and achieve a safe and effective treatment effect. It has important therapeutic value for viral infections, especially new and refractory viral infections. Broad application prospects.
  • unit dose and “unit dosage form” refer to a single drug administration entity.
  • 0.1-2.5 mg/day includes 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, etc. up to 2.5 mg/day. The same goes for the ratio range.
  • Interleukin 37 active substances and interferon active substances are interleukin 37 active substances and interferon active substances
  • interleukin 37 active substance or "IL-37 active substance” refers to an interleukin 37 protein or a substance that can be used to generate or supplement the interleukin 37 protein.
  • the IL-37 active substances include, but are not limited to: native IL-37 protein, recombinant IL-37 protein, nucleic acid molecules encoding the aforementioned IL-37 protein, expression vectors or cells comprising the nucleic acid molecules, Cell membrane components of 37 protein, synthetic IL-37 protein, etc.
  • interleukin 37 protein or polypeptide
  • the interleukin 37 protein is known in the art as a member of the interleukin 1 family, eg, the sequence of human interleukin 37 can be shown in Gene ID:27178.
  • interferon active refers to an interferon protein or a substance that can be used to produce or supplement interferon.
  • the IFN active substances include, but are not limited to: natural IFN proteins, recombinant IFN proteins, nucleic acid molecules encoding the aforementioned IFN proteins, expression vectors or cells comprising the nucleic acid molecules, cell membrane components comprising the IFN proteins, synthetic IFN protein etc.
  • IFN protein or polypeptide refers to a protein or polypeptide encoded by an IFN gene, conservatively variant polypeptides thereof, or homologous proteins or polypeptides thereof, or active fragments thereof.
  • Useful interferons include, but are not limited to: one or more of the IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ families; preferably IFN- ⁇ 1b, IFN- ⁇ 2a, IFN- ⁇ 2b, IFN- ⁇ , One or more of IFN- ⁇ , IFN- ⁇ 1, IFN- ⁇ 2; more preferably, IFN- ⁇ 2b or IFN- ⁇ .
  • interferon IFN- ⁇ 2b or IFN- ⁇ is a recombinant human interferon, which can be obtained through commercial channels.
  • Interleukin 37 actives and interferon actives that can be used in the present application can be as described in detail above.
  • the full-length sequence of the active substance of the present disclosure or a fragment thereof can generally be obtained by a PCR amplification method, a recombinant method or an artificial synthesis method.
  • primers can be designed, for example, based on the relevant nucleotide sequences, especially open reading frame sequences, disclosed in the present disclosure or in other databases, and using commercially available cDNA libraries or as already known by those skilled in the art
  • a cDNA library prepared by a known conventional method is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
  • proteins or polypeptides of the present disclosure may be glycosylated, or may be non-glycosylated.
  • the term "treating" includes: (1) in an animal (especially a mammal and In humans, in particular, preventing or delaying the onset of clinical symptoms of the condition, disorder or condition; (2) inhibiting the condition, disorder or condition (e.g. arresting, alleviating or delaying the progression of the disease or its recurrence (in maintenance); treatment) or at least one clinical or subclinical symptom thereof); and/or (3) alleviating the condition (ie causing regression of the condition, disorder or condition or at least one of its clinical or subclinical symptoms).
  • the benefit to the treated patient is statistically significant or at least perceptible by the patient or by the physician.
  • a combination comprising an interleukin-37 active and an interferon active is effective in reducing viral infection and its associated conditions.
  • Described virus is DNA virus and RNA virus, for example: DNA virus is selected from: adenovirus, herpes virus, pox virus, hepadnavirus, HBV, HPV etc.; Described RNA virus is selected from: coronavirus (such as SARS, SARS-CoV-2, MERS), fibroviruses (eg, Ebola), flaviviruses (eg, Zika, yellow fever, dengue, West Nile, JE, HCV), orthomyxoviruses (influenza viruses (eg H1N1, H3N2, H7N9, H5N1), avian influenza viruses), paramyxoviruses (measles virus, human respiratory syncytial virus), retroviruses (HIV) and picornaviruses (eg rhinovirus, enterovirus) Virus).
  • coronavirus such as
  • the viral infection is a respiratory viral infection, the symptoms of which include, but are not limited to: dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulopathy, Multiple organ failure, fever, dry cough, fatigue, nasal congestion, runny nose, sore throat, myalgia, and diarrhea.
  • the combination comprising interleukin 37 active substance and interferon active substance of the present disclosure can be used to treat viral infections of various severity, such as for the treatment of mild, common, severe, and critical viral infections .
  • a combination comprising a therapeutically effective amount of an interleukin 37 active and an interferon active, and optionally a pharmaceutically acceptable carrier.
  • the combination is used for the treatment of viral infections and related disorders.
  • the combination may be a pharmaceutical composition, a formulation combination, a kit, or a combination in use.
  • the term "pharmaceutical composition” refers to a pharmaceutical combination comprising both an interleukin-37 active and an interferon active.
  • the term “combination formulation” or “kit” means that the interleukin 37 active and the interferon active may be administered independently, in separate form or by using different fixed combinations with separate amounts of the active ingredients. In combinations, the ratio of the amounts of interleukin 37 active substance and interferon active substance to be administered may vary, for example, to meet the needs of a subgroup of subjects to be treated or the needs of an individual subject, which requirements vary by subject age, sex, weight and so on are different.
  • kits can be administered simultaneously or chronologically staggered, eg, at different time points and at the same or different time intervals for any part of the kit.
  • the present disclosure relates to combinations of interleukin 37 actives and interferon actives, eg, combined formulations or pharmaceutical compositions, for simultaneous, separate or sequential use.
  • boost or “boost therapy” refers to a set of agents used in a therapy that a subject receiving a first treatment regimen of one or more agents initiates after initiation of a first treatment regimen other than the first treatment regimen or A second treatment regimen of multiple different agents, so not all agents used in the therapy start at the same time. For example, add-on IFN therapy to patients already receiving IL-37 therapy, or vice versa.
  • the actives in the combinations or products of the present disclosure include interleukin-37 actives and interferon actives. In some embodiments, the actives in the combinations or products of the present disclosure consist essentially of, or consist of, an interleukin 37 active and an interferon active.
  • the term "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or animals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio.
  • the term “effective amount” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • the active substances in the combination or product of the present disclosure account for 0.01-100 wt % of the total weight of the drug or composition, and the balance is pharmaceutically acceptable carriers and other additives.
  • the active protein may account for 0.01-10 wt % of the total weight; when the drug or composition is a powder, it may be substantially or completely composed of active protein composition.
  • the active ingredients in the combinations or products of the present disclosure are present in amounts that produce superior or synergistic therapeutic effects.
  • the excellent therapeutic effect includes, but is not limited to, significantly shortening the time required for disease remission, such as significantly shortening the hospital stay of a patient, compared with conventional treatment.
  • the amount of IL-37 is 0.01-10 ⁇ g, 0.05-5 ⁇ g, 0.1-4 ⁇ g, 0.5-3 ⁇ g.
  • the amount of interferon IFN- ⁇ 2b or IFN- ⁇ is 1 ⁇ 10 4 to 1 ⁇ 10 8 active units, 5 ⁇ 10 4 to 5 ⁇ 10 7 active units, 1 ⁇ 10 5 to 1 ⁇ 10 7 active units, 5 ⁇ 10 5 to 5x10 6 active units
  • the mass ratio of interleukin-37 active substance and interferon active substance in the combination or product of the present disclosure is 1:100-100:1, 1:50-50:1, 1:10-10:1, 1:5 ⁇ 5:1, 1:2 ⁇ 2.5:1, 1:1 ⁇ 2:1.
  • the term "pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Pharmaceutically acceptable carriers in the formulation combination, pharmaceutical composition or kit may contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as fillers, disintegrating agents, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances and the like may also be present in these carriers.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably about a pH of about 6-8.
  • buffers that can be used in the combinations herein include, but are not limited to: phosphate buffer, 5% dextrose injection, 9% sodium chloride injection, acetate buffer, sodium bicarbonate buffer, citrate buffer solution, etc., preferably phosphate buffered saline.
  • unit dosage form refers to the preparation of compositions of the present disclosure into dosage forms required for a single administration for convenience of administration, including but not limited to various solids (eg, powders), liquids, aerosols tablets, capsules, sustained-release formulations.
  • the composition is in a unit dosage form or a multi-dosage form, and wherein the amount of IL-37 active substance can range from, for example, 0.1-100 mg/dose, 0.5-50 mg/dose, 1-40 mg /dose, 5-30 mg/dose; the amount of IFN active substance can be, for example, 0.01-100 mg/dose, 0.05-80 mg/dose, 0.1-70 mg/dose, 0.5-50 mg/dose.
  • the amount of the IFN active substance is 1 ⁇ 10 4 to 1 ⁇ 10 8 active units/dose, 5 ⁇ 10 4 to 5 ⁇ 10 7 active units/dose, 1 ⁇ 10 5 to 1 ⁇ 10 7 active units/dose, and 5 ⁇ 10 5 to 5 ⁇ 10 6 active units unit/dose.
  • a subject may be administered an active of the present disclosure as needed, eg, 1-6 doses, 1-3 doses, or 1 dose of a product of the present disclosure daily, every 2 days, every 3 days, weekly.
  • the effective dose of the active agent employed may vary depending on the severity of the subject to be administered or treated. The specific situation is determined according to the individual conditions of the subject (eg, the subject's weight, age, physical condition, and desired effect), which is within the judgment of a skilled physician.
  • the routes of administration of the medicaments or pharmaceutical compositions or kits of the present disclosure may include, but are not limited to, one or more of the following: inhalation, intranasal, topical administration, targeted administration to target tissues, injection, oral administration, and the like.
  • the active substances or formulations or compositions containing the active substances may be administered simultaneously or separately in the same or different ways.
  • a combination comprising an interleukin 37 active and an interferon active produces a beneficial, eg, synergistic, therapeutic effect, or other unexpected beneficial effect, such as a significantly better efficacy than existing drugs, compared to monotherapy using only a single active , fewer and/or milder side effects.
  • interleukin 37 active and interferon active may be used in combination, or a synergistic effect may be produced, compared to monotherapy with interleukin 37 active or interferon active alone. For example, not only can the dose used be smaller, but it can also be used less frequently. Furthermore, the occurrence of side effects can be reduced and/or the response rate to therapy based on interleukin 37 actives and interferon actives can be increased.
  • mice The survival status of mice was observed and recorded at fixed time points every day until the end point of observation on the 14th day. As shown in Figure 1C, it can be seen that when the TCID 50 is 250, the survival rate of mice is the highest, and 70% of the mice still survive until the end of the 14-day observation, indicating that this challenge dose is low. When the TCID 50 was 500, the survival rate of mice decreased significantly, and 10% of the mice survived at the end of the 14-day observation. When the TCID 50 reached 1000, the survival rate of the mice was the lowest, and the 14-day end point could not be observed, and all the mice died on the tenth day, indicating that the challenge dose was too high.
  • the optimal challenge dose for H1N1-PR8 mouse influenza virus infection model is 500 TCID 50 .
  • Example 2 The combination of IL-37 and IFN ⁇ 2b protein has a synergistic protective effect on influenza virus infection in mice
  • IL-37 and IFN ⁇ 2b proteins were purchased from Shanghai Nearshore Technology Co., Ltd., and the model construction conditions in Example 1 were explored. On the 0th day, 500 TCID 50 was used for H1N1-PR8 challenge.
  • the group, the interferon alone nebulization group and the combination of IL-37 and IFN ⁇ 2b protein were treated by nebulization inhalation, wherein the administration concentration of interferon ⁇ 2b was 5 ⁇ 10 4 IU/only, and the administration concentration of interleukin 37 was 12.5 ⁇ g/kg , the equipment for atomization and inhalation is the British EMMS atomization support system, the body weight of each group of mice is recorded every day, and the survival situation is observed until the end point of observation on the 14th day.
  • a schematic diagram of the model design is shown in Figure 2A.
  • mice in the IL-37 combined with IFN ⁇ 2b group were the lowest, and the phenomenon of weight recovery was observed from the 9th day, and the weight recovery was very obvious when the observation end point was reached on the 14th day. It is proved that the combined drug has a better therapeutic effect on H1N1 influenza virus infection.
  • the survival status of mice in different groups is shown in Figure 2C. It can be seen that the survival rate of mice in different groups is significantly different. The survival rate of mice in the interferon alone group is the lowest, and all of them died on the 11th day. Infection with H1N1 influenza virus has a certain protective effect (the weight loss rate is lower than that of the PBS control group), but it also has greater toxic and side effects (the survival rate is lower than that of the PBS control group); followed by the PBS control group (without any therapeutic intervention) group), only 10% of the mice survived on day 11.
  • mice in the IL-37 treatment group was higher, and 50% of the mice survived on the 11th day, indicating that the IL-37 treatment group had a more obvious protective effect on mice infected with H1N1 influenza virus.
  • the survival rate of mice in the combined treatment group of IL-37 and IFN ⁇ 2b was the highest, 70% of the mice survived on the 11th day, and 50% of the mice were still alive at the end of the 14th day of observation. It shows that the combination has very obvious therapeutic and protective effects on mice infected with H1N1 influenza virus.
  • the antiviral effect of interferon and the anti-inflammatory effect of interleukin 37 act synergistically, which not only effectively inhibits virus replication, but also avoids excessive activation of inflammation in the body. It can recover quickly and greatly reduce the mortality rate.
  • the weight change rate of the mice in the interleukin-37 alone group was significantly lower, P ⁇ 0.01, and the survival rate was significantly higher than that in the control group, P ⁇ 0.05.
  • the weight change rate of mice in the combined treatment group of interleukin 37 and IFN ⁇ 2b was significantly reduced, P ⁇ 0.001, and the survival rate was significantly higher than that of the control group, P ⁇ 0.05.
  • the synergistic effect of treatment with interleukin 37, interferon ⁇ 2b, and interleukin 37 combined with interferon ⁇ 2b was verified as follows: the survival rate of the three groups of mice on the 8th day and the 11th day or more was evaluated. Statistical analysis was calculated, where the mean survival rate of mice treated with interleukin 37 alone was X, the mean survival rate of mice treated with interferon ⁇ 2b alone was Y, and the survival rate of mice treated with interleukin 37 combined with interferon ⁇ 2b was R. .
  • Z is the theoretical expected survival rate, when the actual survival rate R of the combined treatment group is greater than the theoretical expected survival rate Z, that is, interleukin 37 and interferon ⁇ 2b
  • Z is the theoretical expected survival rate, when the actual survival rate R of the combined treatment group is greater than the theoretical expected survival rate Z, that is, interleukin 37 and interferon ⁇ 2b
  • Z is the theoretical expected survival rate, when the actual survival rate R of the combined treatment group is greater than the theoretical expected survival rate Z, that is, interleukin 37 and interferon ⁇ 2b
  • Z is the theoretical expected survival rate
  • the plasma interleukin-37 levels of 254 patients were quantitatively determined by using the IL-37 enzyme-linked immunosorbent assay kit (purchased from Beijing Sizhengbai Biotechnology Co., Ltd.) protocol.
  • IL-37 enzyme-linked immunosorbent assay kit purchased from Beijing Sizhengbai Biotechnology Co., Ltd.
  • IL-6 level (Fig. 3A), plasma IL-8 level (Fig. 3B) and plasma HS-CRP level (Fig. 3C) of the IL-37 high secretion group were significantly lower, further confirming that the IL-6 secreted by the patient's body was significantly lower.
  • -37 can effectively antagonize the hyperactivation of inflammation, and the patients with high secretion of IL-37 have higher hospitalization time (Fig. 3D), improvement time of lung CT (Fig. 3E), and cough elimination time (Fig. 3F) than those with low secretion significantly shortened, suggesting that IL-37 has a strong anti-inflammatory ability during COVID-19 infection.
  • Example 4 Patients with COVID-19 who maintain high levels of IL-37 and IFN ⁇ have better clinical prognosis
  • Patient plasma sample IFN ⁇ data were derived from the patient's clinical immunization report. According to the median levels of IL-37 and IFN ⁇ secretion in the plasma samples of COVID-19 patients, we divided the patients into two groups, the co-high level group and co-low level group of IL-37 and IFN ⁇ , respectively, and compared the two groups respectively. Differences in clinical outcomes among patients.
  • mice with ICR background Six 5- to 6-week-old female angiotensin-converting enzyme transgenic mice with ICR background were purchased from Beijing Huafukang Company, and were divided into control group and interleukin-37 treatment group, with 3 mice in each group.
  • Each mouse was intranasally challenged with 10 5 TCID 50 of SARS-CoV-2 virus (from Wuhan Institute of Virology, Chinese Academy of Sciences), 12h and 48h after the challenge, the interleukin 37 treatment group was injected with 12.5 ⁇ g of PBS as solvent via tail vein /kg recombinant human interleukin 37 (purchased from R&D company), the control group was injected with the same amount of PBS through the tail vein, and the mice were raised in a biosafety tertiary laboratory after challenge.
  • mice On the 5th day after challenge, mice were euthanized, and lung tissue sections were taken for hematoxylin-eosin staining, and pathological score calculation and inflammatory cell quantification were performed using Tissue FAXS Confocal Plus 200 software.
  • the left picture of Fig. 5A is the staining of the whole lung lobe tissue section in the control group
  • Fig. 5B is the staining of the whole lung lobe tissue section of the treatment group
  • the two right pictures are enlarged display of the left picture. It can be seen that compared with the control group, the inflammatory cell infiltration, the thickening of the alveolar wall and the degree of congestion were significantly lighter in the treatment group.
  • Figure 5C is the result of scoring by pathological analysis software, showing that the pathological score of the interleukin 37 treatment group is significantly lower;
  • Figure 5D is the number of inflammatory cell infiltration per unit area of lung tissue calculated by the pathological analysis software, showing that the inflammatory cells in the interleukin 37 treatment group The number of infiltrates was significantly less.

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Abstract

本发明提供用于治疗病毒感染性疾病或症状的产品，其包含：(a)白介素37 (IL-37)活性物质；(b)一种或多种干扰素(IFN)活性物质；以及(c)可任选的，药学上可接受的载剂。本发明还提供白介素37活性物质和干扰素活性物质在制备所述产品中的应用。

Description

白细胞介素37与干扰素联用在治疗病毒感染中的应用 技术领域
本申请属于生物技术和医学领域。具体而言，本申请涉及白细胞介素37与干扰素的联合用药、及其相关产品和在治疗病毒感染中的应用。
背景技术
病毒是只有一种核酸，且十分微小的具有感染性的致病实体，但其对人类社会产生的影响是十分深远且显著的。1918年的西班牙大流感，感染了全球近四分之一人口；1976年非洲爆发的埃博拉疫情，死亡率极高；2003年突然出现的SARS，2013年出现的H7N9禽流感以及目前肆虐全球的2019年新型冠状病毒疾病，无疑给人们的生命和生活带来巨大影响。
由于病毒的种类奇多，按照基因结构划分，可分为DNA病毒(其中包括腺病毒科，疱疹病毒科，小DNA病毒科，痘病毒科以及嗜肝DNA病毒科等)以及RNA病毒(齐总包括冠状病毒科，纤维病毒科，正黏液病毒科，副黏液病毒科，反转录病毒科等)。按照传播途径可分为呼吸道传播，消化道传播，血液传播等。据国际病毒分类委员会统计，截止2018年，已分类的病毒共有4958种，并仍有大量新的病毒由于现有分类方法不完善而无法被分类。有研究提出，目前在人类中传播的263种已知病毒只占所有潜在病毒的0.1％，而那些未知的病毒都是有可能传染人类的。尤其是对于新发现的病毒，人类毫无治疗经验和特定有效的治疗药物。
临床上的广谱抗病毒治疗通常为抗病毒药物如利巴韦林、阿糖腺苷等，此类药物可以通过抑制病毒的代谢类似物而起选择性的抑制作用，但长期使用会对造血系统，消化系统带来严重副作用，并且有很大的肝脏毒性。近年来干扰素作为广谱抗病毒分子，开始应用于临床用于治疗病毒感染，其主要是通过与细胞表面受体的作用激活下游的干扰素刺激基因，从而抑制病毒在细胞中的复制，总体的抗病毒效果由于起效快，作用强等优势优于广谱抗病毒药物。
然而，干扰素也有一定的毒副作用，可介导肝、肾毒性，尤其是在清除病毒的过程中，很可能引起机体炎症的过度活化，产生大量炎症因子，甚至发生炎症因子风暴，极易导致感染性休克以及多器官衰竭，增加死亡率。
白介素37是IL-1家族的第7号成员，也是IL-1家族唯一一个抑炎细胞因子。由于其强大的抑炎能力使之在多种炎症性疾病中发挥重要作用。早期研究者发现IL-37可以入核与TGF-β信号通路的关键转录调节因子Smad3结合形成功能复合物，从而通过影响基因转录来实现抑炎功能。新近发现的IL-37的另一个抑炎机制，IL-37可以与IL-18Ra结合，从而招募孤儿受体IL-1R8(SIGIRR或TIR8)，利用IL-1R8抑制下游促炎信号激酶mTOR、MAPK等以及促炎转录因子NF-kB，髓样分化因子MyD88；激活抑炎信号分子Mer、PTEN、STAT3和p62(dok)，从而实现抑炎效果。然而，如何更好地发挥IL-37的抗炎效果，以更有效地治疗呼吸道病毒感染尚需进一步研究开发。
综上，本领域中迫切需要开发出可有效治疗呼吸道病毒感染的产品。
发明内容
本申请中正是提供了一种基于白介素37的呼吸道病毒感染治疗应用、产品以及方法。
在本申请的第一方面中，提供了一种用于治疗病毒感染性疾病或症状的产品，其包含：
(a)白介素37(IL-37)活性物质；
(b)一种或多种干扰素(IFN)活性物质；以及
(c)可任选的，药学上可接受的载剂。
在一些实施方式中，所述产品为药物组合物、制剂组合或药盒。
在一些实施方式中，所述IL-37活性物质选自：天然IL-37蛋白、重组IL-37蛋白、编码前述IL-37蛋白的核酸分子、包含所述核酸分子的表达载体或细胞、包含所述IL-37蛋白的细胞膜组分、合成的IL-37蛋白。
在一些实施方式中，编码所述IL-37蛋白的核酸分子选自：(i)具有Gene ID：27178或SEQ ID NO：6～10中任一项所示的核苷酸序列的核酸分子或其简并序列或其剪接体核酸分子；(ii)在(i)的核酸分子中包含一个或多个核苷酸取代、缺失和/或添加且与(i)中的核酸分子具有能够编码活性IL-37的核酸分子；(iii)与(i)中核酸分子具有至少80％同源性(例如至少85％、85％、90％、95％、96％、97％、98％、99％同源性)且能够编码IL-37蛋白的核酸分子。
在一些实施方式中，所述IL-37蛋白选自：(a)具有SEQ ID NO：1～5中任一项所示的氨基酸序列的IL-37蛋白、或其剪接体分子；(b)在(a)的IL-37蛋白或其剪接体分子中包含一个或多个氨基酸取代和/或缺失和/或添加且具有抑制炎症功能的蛋白质；和/或(c)与前述多肽具有至少80％同源性(例如至少85％、85％、90％、95％、96％、97％、98％、99％同源性)且具有抑制炎症功能的IL-37蛋白；(d)由(i)、(ii)或(iii)中所述核酸分子表达的IL-37蛋白或多肽。
在一些实施方式中，所述IFN活性物质选自：天然IFN蛋白或重组IFN蛋白(例如重组人IFN)、前述IFN蛋白的编码分子、包含所述编码分子的表达载体或细胞、包含所述IFN蛋白的细胞膜组分。
在一些实施方式中，所述IFN为选自下组中的一种或多种：IFN-α、IFN-β、IFN-γ、IFN-λ家族中的一种或多种，优选地，所述IFN为IFN-α1b、IFN-α2a、IFN-α2b、IFN-κ、IFN-ω、IFN-λ1、IFN-λ2中的一种或多种，更优选地，所述干扰素为IFN-α(如IFN-α2b)或IFN-κ。
在一些实施方式中，所述表达载体为选自下组中的一种或多种：腺病毒载体、腺相关病毒载体、痘病毒载体、逆转录病毒载体、疱疹病毒载体、RNA病毒载体，EB病毒载体、杆状病毒载体、噬菌体载体、动物病毒载体、植物病毒载体、DNA质粒载体、RNA载体。
在一些实施方式中，所述IL-37活性物质的量为0.01～10μg，0.05～5μg，0.1～4μg，0.5～3μg；和/或，所述IFN活性物质的量为1x10 4～1x10 8个活性单位，5x10 4～5x10 7个活性单位，1x10 5～1x10 7个活性单位，5x10 5～5x10 6个活性单位。
在一些实施方式中，所述产品的形式适于通过选自下组相同或不同途径给予IL-37活性物质和IFN活性物质：雾化吸入、滴鼻、喷雾、静脉给药、靶组织内给药或口服给药。
在一些实施方式中，所述产品的形式适于同时、先后或间隔给予IL-37活性物质和IFN活性物质。
在一些实施方式中，所述产品还包括：一个或多个容纳IL-37活性物质和IFN活性物质的容器，例如容纳单位剂量IL-37活性物质和IFN活性物质的一个或多个容器；用于给药的器具；指导使用的说明书等。
在一些实施方式中，所述IL-37活性物质和干扰素活性物质同时或分别以如 下形式提供：活性蛋白形式(例如单独的活性蛋白和/或两种或多种活性蛋白的融合蛋白形式)、编码活性蛋白或其融合蛋白的核酸分子形式、包含所述核酸分子的表达载体(例如单独的表达载体或同时包含两种核酸分子的表达载体)或细胞形式、包含所述活性蛋白的细胞膜组分。
在一些实施方式中，所述IL-37活性物质与所述干扰素活性物质：分别存在或表达，并联合使用；融合表达形成多功能分子；或所述IL-37活性物质与一种干扰素融合后，再与另一种或多种干扰素联合使用。
在一些实施方式中，所述IL-37活性物质与一种或多种IFN活性物质联合使用，或所述IL-37活性物质与一种或多种IFN活性物质由相同或不同载体表达，或分别表达在不同细胞膜上后联合应用细胞膜的方式，或所述IL-37活性物质与一种或多种IFN活性物质表达在同一细胞膜上后应用细胞膜的方式。
在一些实施方式中，所述IL-37活性物质与一种或多种IFN活性物质融合表达，包含通过基因重组技术构建融合基因进行蛋白表达的方式，通过载体表达融合基因的方式，通过细胞质膜上表达融合重组蛋白并利用细胞膜的方式，通过化学方法连接的方式。优选地，IL-37与IFN-α或IFN-κ融合表达的方式。
在一些实施方式中，所述产品还包含用于治疗病毒感染的其他药物，例如中药、化学药物、生物药物，所述药物包括但不限于抗病毒药物、抗病毒制剂、抗病毒血清、抑炎药物、激素、细胞因子、抗体等。
在一些实施方式中，所述病毒为DNA病毒和RNA病毒。
在一些实施方式中，所述的DNA病毒选自：腺病毒、疱疹病毒、痘病毒、嗜肝DNA病毒、HBV、HPV等。在一些实施方式中，所述的RNA病毒选自：冠状病毒(如SARS、SARS-CoV-2、MERS)、纤维病毒(如埃博拉病毒)、黄病毒(如寨卡病毒、黄热病毒、登革病毒、西尼罗病毒、乙脑病毒、HCV)、正黏液病毒(流感病毒(如H1N1、H3N2、H7N9、H5N1)、禽流感病毒)、副黏液病毒(麻疹病毒、人类呼吸道融合病毒)、逆转录病毒(HIV)以及小核糖核酸病毒(如鼻病毒、肠道病毒)。
在一些实施方式中，所述病毒为新型冠状病毒，其病毒感染性疾病或症状包括：新型冠状病毒肺炎；选自下组的一种或多种与新型冠状病毒感染相关的病症：呼吸困难、低氧血症、急性呼吸窘迫综合征、脓毒症休克、代谢性酸中毒、凝血 功能障碍、多器官功能衰竭、肺纤维化、迁延形成的慢性炎症、发热、干咳、乏力、鼻塞、流涕、咽痛、肌痛和腹泻。
在一些实施方式中，所述病毒感染的对象为哺乳动物，优选人。
本申请的一些方面中，提供了IL-37活性物质和IFN活性物质在制备如本申请所述的用于治疗病毒感染的产品中的应用。
在本公开的另一个方面中，提供了一种治疗对象中病毒感染性疾病或症状的方法，所述方法包括给予有需要的对象治疗有效量的IL-37活性物质和IFN活性物质的组合或如本文所述的产品或通过本文所述方法制得的产品。该方面所涉及的特征可如前文中所述。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本发明的发明构思和保护范围。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
下面结合附图对本发明作进一步说明，其中这些显示仅为了图示说明本发明的实施方案，而不是为了局限本发明的范围。
图1：H1N1-PR8攻毒小鼠流感病毒感染模型的建立
图1A：小鼠生存情况和体重变化监测流程示意图：三组小鼠，每组8只，分别滴鼻攻毒剂量为250、500和1000 TCID50的H1N1-PR8流感病毒，连续称重14天，并监测小鼠生存情况和体重变化；
图1B：14天内小鼠体重随时间的变化率，其中纵坐标表示小鼠体重的下降率；
图1C：14天观测终点时各组小鼠的生存情况。
图2：H1N1-PR8小鼠模型测试IL-37与IFNα2b组合物的保护作用
图2A：实验流程示意图：选择实施例1中500TCID50 H1N1-PR8攻毒剂量，攻毒后，每组分别雾化PBS、IL-37、IFNα2b以及IL-37与IFNα2b的组合，连续记录体重14天，并同时监测生存情况；
图2B：14天内小鼠的体重随时间的变化率，其中纵坐标表示的为小鼠体重的下降率；
图2C：14天观测终点时各组小鼠的生存情况如图2C所示。
图3：COVID-19患者血浆IL-37水平与体内炎症因子的关系
图3A：COVID-19患者血浆IL-37水平与炎症因子IL-6水平的相关性；
图3B：COVID-19患者血浆IL-37水平与炎症因子IL-8水平的相关性；
图3C：COVID-19患者血浆IL-37水平与炎症相关指标超敏C反应蛋白(HS-CRP)水平的相关性。
图3D：血浆IL-37高分泌水平的COVID-19患者住院天数显著更短；
图3E：血浆IL-37高分泌水平的COVID-19患者肺CT改善天数显著更短；
图3F：血浆IL-37高分泌水平的COVID-19患者咳嗽消除天数显著更短。
通过血浆IL-37水平的中位数将患者分为IL-37低分泌和高分泌两组，其中IL-37高分泌的患者相较于IL-37低分泌的患者，其炎症因子IL-6水平(图3A)、炎症因子IL-8水平(图3B)和炎症相关指标超敏C反应蛋白(HS-CRP)的水平(图3C)均显著更低。并且，还发现血浆IL-37高分泌水平的COVID-19患者住院天数显著更短(图3D)，肺CT改善天数显著更短(图3E)，咳嗽消除天数也显著更短(图3F)。
图4：IL-37和IFNα均维持高水平的COVID-19患者的临床预后
图4A：高IL-37且高IFNα与低IL-37且低IFNα的COVID-19患者的住院天数比较；
图4B：高IL-37且高IFNα与低IL-37且低IFNα的COVID-19患者的病毒核酸转阴天数比较；
图4C：高IL-37且高IFNα与低IL-37且低IFNα的COVID-19患者的肺CT改善天数比较；
图4D：高IL-37且高IFNα与低IL-37且低IFNα的COVID-19患者的咳嗽好转天数比较。
将COVID-19患者分为高IL-37且高IFNα以及低IL-37且低IFNα两组，统计发现其中高IL-37且高IFNα组的患者住院天数(图4A)、病毒核酸转阴天数(图4B)、肺CT改善天数(图4C)以及咳嗽好转天数(图4D)显著更短。
图5：白介素37治疗显著减轻SARS-CoV-2病毒感染的ACE2转基因小鼠的肺部炎症
图5A：左图为PBS对照组整个肺叶组织切片染色，右图均为左图截取区域 的放大显示；
图5B：左图为治疗组整个肺叶组织切片染色，右图为左图截取区域的放大显示；
图5C：病理分析软件进行的病理评分数据，显示白介素37治疗组病理评分显著更低；
图5D：病理分析软件计算的单位面积肺组织的炎症细胞浸润数量，显示白介素37治疗组炎症细胞浸润数量显著更少。
以上统计学分析方法均采用非配对Mann-Whitney检验，显著性表示方法：“*”，P＜0.05；“**”，P＜0.01；“***”，P＜0.001；“****”，P＜0.0001。
具体实施方式
本发明人在研究中出乎意料地发现将白介素37与干扰素(例如干扰素α2b)的组合应用于小鼠感染H1N1流感模型中，发现该组合对小鼠有明显的保护作用，包括小鼠的体重减轻率降低，生存率显著提高。由于组合物中的白介素37能够拮抗炎症的过度活化，结合干扰素对病毒复制的抑制作用，二者联合将对病毒感染具有明显的治疗和保护作用，极具临床应用价值。
由此，本申请中提供了白介素37与干扰素的联合应用，其用于治疗病毒感染，尤其是呼吸道病毒感染。该组合提供一种更加安全且有效的病毒感染治疗药物和/或方案。
不希望受限于理论，干扰素能够广谱抑制病毒复制，而IL-37可抑制病毒感染以及干扰素使用所引发的炎症致病作用。本申请中至少包含白介素37以及一种干扰素的组合，具有重要的抗病毒优势，可同时作用于病毒复制与炎症致病两个靶点、协同起效；在发挥迅速、广谱的抗病毒效果的同时，又能拮抗干扰素在抗病毒过程中可能引起的炎症因子过度应答，达到安全有效地治疗效果，对于病毒感染，尤其是新发的，难治的病毒感染具有重要的治疗价值和广阔的应用前景。
如本文所用，“单位剂量”和“单位剂量形式”表示单一药物给予实体。
如本文所用，在数值或范围上下文中的“约”表示所引用或要求保护的数值或范围的±10％。
应理解，当提供参数范围时，本发明同样提供了在该范围内的所有整数及其十分位小数。例如，“0.1-2.5毫克/天”包括0.1毫克/天、0.2毫克/天、0.3毫克/天 等直至2.5毫克/天。比值范围亦然。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由......构成”、“基本上由......构成”、和“由......构成”；“主要由......构成”、“基本上由......构成”和“由......构成”属于“含有”、“具有”或“包括”的下位概念。
白介素37活性物质和干扰素活性物质
如本文所用，术语“白介素37活性物质”或“IL-37活性物质”是指白介素37蛋白或可用于产生或补充白介素37蛋白的物质。所述IL-37活性物质包括但不限于：天然IL-37蛋白、重组IL-37蛋白、编码前述IL-37蛋白的核酸分子、包含所述核酸分子的表达载体或细胞、包含所述IL-37蛋白的细胞膜组分、合成的IL-37蛋白等。
如本文所用，术语“白介素37(IL-37)蛋白或多肽”是指由IL-37基因编码的蛋白质或多肽、它们的保守性变异多肽、或其同源蛋白或多肽、或其活性片段。白介素37蛋白在本领域中是已知白介素1家族成员，例如人白介素37的序列可如Gene ID：27178所示。
如本文所用，术语“干扰素活性物质”或“IFN活性物质”是指干扰素蛋白或可用于产生或补充干扰素的物质。所述IFN活性物质包括但不限于：天然IFN蛋白、重组IFN蛋白、编码前述IFN蛋白的核酸分子、包含所述核酸分子的表达载体或细胞、包含所述IFN蛋白的细胞膜组分、合成的IFN蛋白等。
如本文所用，术语“IFN蛋白或多肽”是指由IFN基因编码的蛋白质或多肽、它们的保守性变异多肽、或其同源蛋白或多肽、或其活性片段。可用的干扰素包括但不限于：IFN-α、IFN-β、IFN-γ、IFN-λ家族中的一种或多种；优选IFN-α1b、IFN-α2a、IFN-α2b、IFN-κ、IFN-ω、IFN-λ1、IFN-λ2中的一种或多种；更优选，IFN-α2b或IFN-κ。
进一步地，所述的干扰素IFN-α2b或IFN-κ为重组人干扰素，可以通过商业渠道获得。
可用于本申请中的白介素37活性物质和干扰素活性物质可如前文中所详细描述。
本公开活性物质的全长序列或其片段通常可以用PCR扩增法、重组法或人工 合成的方法获得。例如，对于PCR扩增法，可例如根据本公开所公开的或其他数据库中的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
根据重组生产方案所用的宿主，本公开的蛋白质或多肽可以是糖基化的，或可以是非糖基化的。
抗病毒感染的应用
如本文所用，术语“治疗”包括：(1)在可能罹患或易患病况、病症或病状但尚未经历或呈现该病况、病症或病状的临床或亚临床症状的动物(尤其为哺乳动物且尤其为人类)中，预防或延迟所述病况、病症或病状的临床症状的出现；(2)抑制该病况、病症或病状(例如阻滞、减轻或延迟该疾病的发展或其复发(在维持治疗的情况下)或其至少一种临床或亚临床症状)；和/或(3)减轻病状(即导致病况、病症或病状或其临床或亚临床症状中的至少一者的消退)。对所治疗的患者的益处在统计上显著或至少可被患者或被医师察觉。
在一些实施方式中，包含白介素37活性物质和干扰素活性物质的组合能有效减轻病毒感染及其相关病症。所述病毒为DNA病毒和RNA病毒，例如：DNA病毒选自：腺病毒、疱疹病毒、痘病毒、嗜肝DNA病毒、HBV、HPV等；所述的RNA病毒选自：冠状病毒(如SARS、SARS-CoV-2、MERS)、纤维病毒(如埃博拉病毒)、黄病毒(如寨卡病毒、黄热病毒、登革病毒、西尼罗病毒、乙脑病毒、HCV)、正黏液病毒(流感病毒(如H1N1、H3N2、H7N9、H5N1)、禽流感病毒)、副黏液病毒(麻疹病毒、人类呼吸道融合病毒)、逆转录病毒(HIV)以及小核糖核酸病毒(如鼻病毒、肠道病毒)。
在一些实施方式中，所述病毒感染是呼吸道病毒感染，其症状包括但不限于：呼吸困难、低氧血症、急性呼吸窘迫综合征、脓毒症休克、代谢性酸中毒、凝血功能障碍、多器官功能衰竭、发热、干咳、乏力、鼻塞、流涕、咽痛、肌痛和腹泻等症状。
在一些实施方式中，可采用本公开的包含白介素37活性物质和干扰素活性物质的组合对各种严重程度的病毒感染进行治疗，例如用于轻型、普通型、重型、危重型病毒感染的治疗。
产品及其应用
本公开中还提供了一种组合，其中包括治疗有效量的白介素37活性物质和干扰素活性物质，以及可任选的药学上可接受的载剂。在本公开的一些实施方式中，所述组合用于毒感染及其相关病症的治疗。在本公开的一些实施方式中，所述组合可为药物组合物、制剂组合、药盒或使用上的组合。
如本文所用，术语“药物组合物”是指同时包含白介素37活性物质和干扰素活性物质的药物组合。如本文所用，术语“组合制剂”或“药盒”，意指白介素37活性物质和干扰素活性物质可独立给药，以分开的形式或通过使用有独立量活性成分的不同固定组合给药。在组合中，待给药的白介素37活性物质和干扰素活性物质的量的比值可以变化，例如以满足待治疗的对象亚群的需求或单独对象的需求，这些需求因对象年龄、性别、体重等而各不相同。药盒的各部分可同时或按时间顺序交错给予，例如对于药盒的任意部分，在不同的时间点以及以相同或不同的时间间隔给予。由此，本公开涉及供同时、分开或顺序使用的白介素37活性物质和干扰素活性物质的组合，例如组合的制剂或药物组合物。
该组合还可用作追加疗法。如本文所用，“追加”或“追加疗法”表示疗法中所用的试剂集合，接受该疗法的对象在开始一种或多种试剂的第一治疗方案之后开始该第一治疗方案以外的一种或多种不同试剂的第二治疗方案，因此该疗法中所用的所有试剂并非都在相同时间开始。例如，向已接受IL-37疗法的患者追加IFN疗法，或反之。
在一些实施方式中，本公开的组合或产品中的活性物质包括白介素37活性物质和干扰素活性物质。在一些实施方式中，本公开的组合或产品中的活性物质基本上由白介素37活性物质和干扰素活性物质组成，或由白介素37活性物质和干扰素活性物质组成。
如本文所用，术语“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
本公开的组合或产品中的活性物质占药物或组合物总重量的0.01～100wt％，余量为药学上可接受的载剂以及其它添加剂等物质。例如，当所述药物或组合物 为包含活性蛋白的溶液时，活性蛋白可占总重量的0.01～10wt％；当所述药物或组合物为粉末剂时，其可基本上或完全由活性蛋白组成。
在一些实施方式中，本公开的组合或产品中活性成分以产生优异治疗效果或协同治疗效果的量存在。所述优异治疗效果包括但不限于：相对于采用常规治疗而言，显著缩短病情缓解所需的时间，例如显著缩短患者住院时间。
在一些实施方式中，IL-37的量为0.01～10μg，0.05～5μg，0.1～4μg，0.5～3μg。在一些实施方案中，所述干扰素IFN-α2b或IFN-κ的量为1x10 4～1x10 8个活性单位，5x10 4～5x10 7个活性单位，1x10 5～1x10 7个活性单位，5x10 5～5x10 6个活性单位
在一些实施方式中，本公开的组合或产品中白介素37活性物质和干扰素活性物质的质量比为1∶100～100∶1，1∶50～50∶1，1∶10～10∶1，1∶5～5∶1，1∶2～2.5∶1，1∶1～2∶1。
如本文所用，术语“药学上可接受的载剂”指用于治疗剂给药的载剂，包括各种赋形剂和稀释剂。该术语指这样一些药剂载剂：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载剂是本领域普通技术人员所熟知的。在《雷明顿药物科学》(Remington’s Pharmaceutical Sciences，Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。
在制剂组合、药物组合物或药盒中药学上可接受的载剂可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如填充剂、崩解剂、润滑剂、助流剂、泡腾剂、润湿剂或乳化剂、矫味剂、pH缓冲物质等。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。例如，可用于本申请组合的缓冲液包括但不限于：磷酸盐缓冲液、5％葡萄糖注射液、9％氯化钠注射液、醋酸盐缓冲液、碳酸氢钠缓冲液、柠檬酸盐缓冲液等，优选地磷酸盐缓冲液。
如本文所用，术语“单位剂型”是指为了施用方便，将本公开的组合物制备成单次施用所需的剂型，包括但不限于各种固体剂(如粉末剂)、液体剂、气雾剂、胶囊剂、缓释剂。
在本公开的另一优选实施方式中，所述组合物为单位剂型或多剂型，且其中IL-37活性物质量的范围可为例如0.1～100mg/剂，0.5～50mg/剂，1～40mg/剂，5～30mg/剂；IFN活性物质的量可为例如0.01～100mg/剂，0.05～80mg/剂，0.1～70mg/剂，0.5～50mg/剂。或者，所述IFN活性物质的量为1x10 4～1x10 8个活性单位/剂，5x10 4～5x10 7个活性单位/剂，1x10 5～1x10 7个活性单位/剂，5x10 5～5x10 6个活性单 位/剂。
在本公开的一些实施方式中，可根据需要给予对象本公开活性物质，例如每天、每2天、每3天、每周施用1～6剂，1～3剂或1剂本公开的产品。
应理解，所用活性物质的有效剂量可随待施用或治疗的对象的严重程度而变化。具体情况根据对象的个体情况(例如对象体重、年龄、身体状况、所需达到的效果)来决定，这在熟练医师可以判断的范围内。
本公开的药物或药物组合物或药盒的给药途径可包括但不限于如下一种或多种：雾化吸入、滴鼻、局部给药、靶组织定向给药、注射、口服等。可采用相同或不同的方式同时或分别给予所述活性物质或包含所述活性物质的制剂或组合物。
与仅使用单一活性物质的单一疗法相比，给予包含白介素37活性物质和干扰素活性物质的组合产生有益的例如协同的治疗效果，或者其他出人意料的有益效果，例如显著优于现有药物的疗效、更少和/或更轻的副作用。
与仅使用白介素37活性物质或干扰素活性物质的单一疗法相比，在联用时可使用更低剂量的白介素37活性物质和干扰素活性物质，或可产生协同效果。例如，所用剂量可不仅更小，使用频率还可更低。而且，副作用发生可降低，和/或对于基于白介素37活性物质和干扰素活性物质的疗法的响应率可提高。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验室指南》或按照供应商所建议的条件。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1、H1N1-PR8攻毒小鼠流感病毒感染模型的构建
采用上海西格玛-必凯生物公司购入8～10周龄，平均体重20g的雌性C57 小鼠，第0天分别采用250、500、1000 TCID 50剂量的PR8滴鼻攻毒小鼠，攻毒后统一饲养于生物安全二级实验室，每个独立分组共8只小鼠，每天观测记录小鼠体重变化以及生存情况，直至第14天观测终点。模型构建的示意图如图1A所示。
每天固定时间点对每组小鼠的体重进行测量和记录，直至第14天观测终点。观测到不同TCID 50PR8攻毒剂量的组体重变化率不同，如图1B所示。结果显示：TCID 50为250时，小鼠体重下降率最低，且第10天后有所回升，说明此攻毒剂量下，在未经治疗干预的情况下，小鼠可以自行清除部分病毒，并逐渐康复。而在TCID 50为500时，小鼠的体重下降率明显，14天观察终点结束之前体重仅有轻微回升现象。当TCID 50达到1000时，小鼠的体重下降率最快，且无体重回升现象，说明此攻毒剂量较高。
每天固定时间点观察小鼠的生存状况并记录，直至第14天观测终点。如图1C所示，可见TCID 50为250时，小鼠生存率最高，直至14天观测终点，仍有70％的小鼠存活，说明此攻毒剂量较低。而在TCID 50为500时，小鼠生存率下降明显，至14天观测终点时有10％的小鼠存活。当TCID 50达到1000时，小鼠的生存率最低，且无法观测到14天终点，第十天时小鼠则全部死亡，说明此攻毒剂量过高。
综上，H1N1-PR8小鼠流感病毒感染模型的攻毒最适剂量为500 TCID 50。
实施例2、IL-37与IFNα2b蛋白联用对小鼠感染流感病毒具有协同保护作用
IL-37与IFNα2b蛋白购自上海近岸科技有限公司，通过实施例1中的模型构建条件摸索，第0天采用500 TCID 50进行H1N1-PR8攻毒，设置PBS对照组、白介素37单独雾化组、干扰素单独雾化组以及IL-37与IFNα2b蛋白的组合物进行雾化吸入治疗，其中干扰素α2b的施用浓度为5×10 4IU/只，白介素37的施用浓度为12.5μg/kg，雾化吸入的仪器设备为英国EMMS雾化支持系统，每天对各组小鼠进行体重记录，生存情况观测，直至第14天观测终点。模型设计示意图如图2A所示。
观测结果显示，如图2B所示，各组小鼠的体重变化率差异明显，其中PBS对照组(未经任何治疗干预组)的体重下降率最高，其次是IFNα2b单独使用组，说明单独使用干扰素进行治疗对小鼠有一定的保护作用，但效果并不明显。白介素 37组单独使用组的效果优于干扰素IFNα2b单独使用组，体重下降率较干扰素IFNα2b单独使用组低，说明白介素37单独使用对小鼠感染H1N1流感病毒有一定的治疗效果。而IL-37与IFNα2b联用组小鼠的体重下降率最低，且从第9天开始，即观测到了体重回升的现象，且达到第14天观测终点时体重回升非常明显。证明了该联合用药对H1N1流感病毒感染具有较佳的治疗效果。
不同组小鼠的生存状况如图2C所示，可见不同组小鼠的生存率差异明显，其中干扰素单独使用组小鼠的生存率最低，第11天则全部死亡，说明虽然IFNα2b对小鼠感染H1N1流感病毒感染后有一定的保护作用(体重下降率较PBS对照组低)，但也有较大的毒副作用(生存率较PBS对照组低)；其次为PBS对照组(未经任何治疗干预组)，第11天时仅有10％小鼠存活。而白介素37单独治疗组的小鼠生存率较高，第11天时还有50％小鼠存活，说明单独IL-37治疗组对小鼠感染H1N1流感病毒后有较为明显的保护作用。而IL-37与IFNα2b的联合治疗组小鼠的生存率最高，第11天时有70％的小鼠存活，且至第14天观测终点时仍有50％的小鼠存活。说明该组合对小鼠感染H1N1流感病毒后有十分明显的治疗和保护作用。
上述试验结果证明了，单独给予白介素37对于H1N1流感病毒感染具有一定的保护和治疗作用；单独给予IFNα2b效果不佳，甚至于会降低存活率，具有一定毒性；出乎意料的是，IFNα2b与IL-37效果好极佳，使小鼠感染H1N1流感病毒后能迅速恢复，大大降低了死亡率。此模型的结果很好地证明了白介素37单独或与干扰素联合用于病毒干扰的显著保护作用和治疗效果。
不限于理论，联合治疗后，干扰素的抗病毒作用于白介素37的抗炎作用协同起效，既有效抑制了病毒复制，又避免了体内发生炎症的过度活化，使小鼠感染H1N1流感病毒后能迅速恢复，大大降低了死亡率。
与PBS对照组相比，白介素37单独作用组的小鼠体重变化率显著降低，P＜0.01，而生存率显著高于对照组P＜0.05。白介素37与IFNα2b联合治疗组的小鼠体重变化率显著降低，P＜0.001，生存率显著高于对照组P＜0.05。
在以上小鼠H1N1病毒感染模型中，分别使用白介素37、干扰素α2b以及白介素37联合干扰素α2b进行治疗的协同作用验证如下：以第8天和第11天以上三组小鼠的生存率进行统计分析计算，其中单独使用白介素37进行治疗的小鼠平 均生存率为X，单独使用干扰素α2b进行治疗的小鼠平均生存率为Y，白介素37联合干扰素α2b的治疗组的生存率为R。
根据Colby法协同作用计算公式：Z＝X+Y-X*Y/100，Z值为理论预期生存率，当联合治疗组的实际生存率R＞理论预期生存率Z时，即白介素37与干扰素α2b存在协同治疗作用，当联合治疗组的实际生存率R≤理论预期生存率Z时，即白介素37与干扰素α2b不存在协同作用。计算结果如下表所示：
Figure PCTCN2020116399-appb-000001
根据表中的计算结果，可得出结论，白介素37与干扰素α2b存在协同治疗作用。
实施例3、COVID-19患者血浆中高的IL-37水平伴随低的炎症因子水平
我们收集了上海市公共卫生临床中心2020年2月～2020年5月通过病毒核酸检测阳性确诊为COVID-19的254例患者血浆样本。患者年龄为42.76±0.95，其中女性114例，男性140例。此研究获伦理委员会的批准，所有入组的患者均签署了知情同意书。
通过白介素37酶联免疫吸附测定试剂盒(购自北京四正柏生物科技有限公司)，对254例患者的血浆白介素37水平进行了定量测定，操作步骤按照白介素37酶连免疫吸附测定试剂盒操作规程。我们进一步按照血浆IL-37分泌量的中位数将254位患者分为IL-37高分泌组和IL-37低分泌组两组，观测两组患者血浆中炎症相关因子的分泌差异。
结果显示IL-37高分泌组的血浆IL-6水平(图3A)、血浆IL-8水平(图3B)和血浆HS-CRP水平(图3C)显著偏低，进一步证实了患者体内分泌的IL-37可有效地拮抗炎症的过度活化，同时白介素37高分泌的患者相较于低分泌的患者其住院时间(图3D)、肺CT改善时间(图3E)以及咳嗽消除时间(图3F)均显著缩短，提示IL-37在COVID-19感染过程中具有强大的抑炎能力。
实施例4、IL-37和IFNα维持高水平的COVID-19患者有更好临床预后
患者血浆样本IFNα数据来源于患者的临床免疫报告。根据COVID-19患者血浆样本中IL-37与IFNα分泌量的中位数水平，我们将患者分为两组，分别为IL-37和IFNα共同高水平组与共同低水平组，分别比较两组患者临床预后之间的差异性。
结果显示共同高水平组的患者住院天数(图4A)、核酸转阴的天数(图4B)、肺CT改善的天数(图4C)以及咳嗽消除天数(图4D)均显著低于共同低水平组。
以上的数据更直观地揭示出体内IL-37与IFNα共同维持高水平有利于SARS-CoV-2病毒感染后的良性临床预后，为IL-37与IFNα在SARS-CoV-2病毒感染中的治疗价值提供了进一步的临床数据支持。
实施例5、白介素37单独治疗显著减轻SARS-CoV-2病毒感染的血管紧张素 转化酶转基因小鼠的肺部炎症
从北京华富康公司购入6只5～6周龄ICR背景雌性血管紧张素转化酶转基因小鼠，并分为对照组和白介素37治疗组，每组3只。每只小鼠滴鼻攻毒10 5TCID 50的SARS-CoV-2病毒(来源于中国科学院武汉病毒所)，攻毒后12h和48h，白介素37治疗组尾静脉注射以PBS为溶剂的12.5μg/kg重组人白介素37(购自R&D公司)，对照组尾静脉注射等量的PBS，攻毒后小鼠均饲养于生物安全三级实验室。攻毒后第5天，对小鼠进行安乐死，取肺组织切片进行苏木素-伊红染色，并使用Tissue FAXS Confocal Plus 200软件进行病理评分计算以及炎症细胞定量。
如图5所示，其中图5A左图为对照组整个肺叶组织切片染色，图5B为治疗组整个肺叶组织切片染色，其中的两幅右图均为左图的放大显示。可见治疗组相较于对照组，其炎性细胞浸润，肺泡壁增厚、充血程度均明显更轻。图5C为经过病理分析软件进行评分的结果，显示白介素37治疗组病理评分显著更低；图5D为经过病理分析软件计算后的单位面积肺组织的炎症细胞浸润数量，显示白介素37治疗组炎症细胞浸润数量显著更少。
本实施例说明白介素37单独治疗SARS-CoV-2病毒感染的血管紧张素转化酶转基因小鼠，可以看到显著的抑制炎症的效果，提示可能对疾病的转归具有重要 的治疗价值。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于治疗病毒感染性疾病或症状的产品，其包含：
    (a)白介素37(IL-37)活性物质；
    (b)一种或多种干扰素(IFN)活性物质；以及
    (c)可任选的，药学上可接受的载剂。
  2. 如权利要求1所述的产品，其中，所述产品为药物组合物、制剂组合或药盒。
  3. 如权利要求1所述的产品，其中，所述IL-37活性物质选自：天然IL-37蛋白、重组IL-37蛋白、编码前述IL-37蛋白的核酸分子、包含所述核酸分子的表达载体或细胞、包含所述IL-37蛋白的细胞膜组分、合成的IL-37蛋白。
  4. 如权利要求1所述的产品，其中，所述IFN活性物质选自：天然IFN蛋白或重组IFN蛋白(例如重组人IFN)、前述IFN蛋白的编码分子、包含所述编码分子的表达载体或细胞、包含所述IFN蛋白的细胞膜组分；例如：
    所述IFN为选自下组中的一种或多种：IFN-α、IFN-β、IFN-γ、IFN-λ家族中的一种或多种，优选地，所述IFN为IFN-α1b、IFN-α2a、IFN-α2b、IFN-κ、IFN-ω、IFN-λ1、IFN-λ2中的一种或多种，更优选地，所述干扰素为IFN-α(如IFN-α2b)或IFN-κ。
  5. 如权利要求3或4所述的产品，其中，所述表达载体为选自下组中的一种或多种：腺病毒载体、腺相关病毒载体、痘病毒载体、逆转录病毒载体、疱疹病毒载体、RNA病毒载体，EB病毒载体、杆状病毒载体、噬菌体载体、动物病毒载体、植物病毒载体、DNA质粒载体、RNA载体。
  6. 如权利要求1所述的产品，其中，所述IL-37活性物质的量为0.01～10μg，0.05～5μg，0.1～4μg，0.5～3μg；和/或，所述IFN活性物质的量为1x10 4～1x10 8个活性单位，5x10 4～5x10 7个活性单位，1x10 5～1x10 7个活性单位，5x10 5～5x10 6个活性 单位。
  7. 如权利要求1所述的产品，其中，所述产品的形式适于通过选自下组相同或不同途径给予IL-37活性物质和IFN活性物质：雾化吸入、滴鼻、喷雾、静脉给药、靶组织内给药或口服给药。
  8. 如权利要求1所述的产品，其中，所述IL-37活性物质和干扰素活性物质同时或分别以如下形式提供：活性蛋白形式(例如单独的活性蛋白和/或两种或多种活性蛋白的融合蛋白形式)、编码活性蛋白或其融合蛋白的核酸分子形式、包含所述核酸分子的表达载体(例如单独的表达载体或同时包含两种核酸分子的表达载体)或细胞形式、包含所述活性蛋白的细胞膜组分。
  9. 如权利要求1所述的产品，其中，所述病毒为DNA病毒和RNA病毒，例如：
    所述的DNA病毒选自：腺病毒、疱疹病毒、痘病毒、嗜肝DNA病毒、HBV、HPV等；
    所述的RNA病毒选自：冠状病毒(如SARS、SARS-CoV-2、MERS)、纤维病毒(如埃博拉病毒)、黄病毒(如寨卡病毒、黄热病毒、登革病毒、西尼罗病毒、乙脑病毒、HCV)、正黏液病毒(流感病毒(如H1N1、H3N2、H7N9、H5N1)、禽流感病毒)、副黏液病毒(麻疹病毒、人类呼吸道融合病毒)、逆转录病毒(HIV)以及小核糖核酸病毒(如鼻病毒、肠道病毒)。
  10. IL-37活性物质和IFN活性物质在制备如权利要求1-9中任一项所述的用于治疗病毒感染的产品中的应用。
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CN115531315A (zh) * 2022-12-05 2022-12-30 上海惠盾因泰生物科技有限公司 重组人干扰素λ1鼻喷雾剂及其用途
CN117919385A (zh) * 2024-02-05 2024-04-26 中国医学科学院医学实验动物研究所 IL-37在MERS-CoV感染疾病的治疗中的应用

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