WO2021023264A1 - 用于治疗非洲猪瘟的药物组合物及其用途 - Google Patents

用于治疗非洲猪瘟的药物组合物及其用途 Download PDF

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WO2021023264A1
WO2021023264A1 PCT/CN2020/107411 CN2020107411W WO2021023264A1 WO 2021023264 A1 WO2021023264 A1 WO 2021023264A1 CN 2020107411 W CN2020107411 W CN 2020107411W WO 2021023264 A1 WO2021023264 A1 WO 2021023264A1
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composition
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methyltetrahydrofolate
mice
virus
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成永之
连增林
刘康
顾睿
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连云港金康和信药业有限公司
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Priority to JP2022506894A priority Critical patent/JP2022545341A/ja
Priority to EP20850334.2A priority patent/EP4011372A4/en
Priority to BR112022002156A priority patent/BR112022002156A2/pt
Priority to US17/631,948 priority patent/US20220280508A1/en
Publication of WO2021023264A1 publication Critical patent/WO2021023264A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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

Definitions

  • the invention belongs to the field of medicine, and specifically relates to a pharmaceutical composition capable of producing nitric oxide in an animal body, which can provide a safe and sufficient amount of nitric oxide for the prevention and treatment of diseases.
  • African swine fever African swine fever
  • ASF African swine fever
  • the main hosts of ASFV are domestic pigs of various breeds, African and Eurasian wild boars, and Ornithological ticks. Warthogs and bush pigs can be infected, but they do not show clinical symptoms. African wild boars such as warthogs and Ornithological ticks are ASFV. Host. According to the virulence of ASFV, the clinical symptoms and infection process of ASF are significantly different. The mortality rate of the most acute and acute infections is 100%.
  • influenza viruses are developing rapidly.
  • the selective pressure of antibodies on seasonal influenza viruses promotes the emergence of escape mutants, which can be immune to early strains.
  • Epidemics are caused in the community, which is why seasonal flu vaccines need to be updated frequently.
  • seasonal flu vaccines need to be updated frequently.
  • the specificity of the antibody response also laid the foundation for the emergence of a pandemic.
  • influenza virus or coronavirus pandemics including, A(H1N1), A(H2N2), A(H3N2), A(H1N1), SARS new coronavirus and COVID-19, the aforementioned influenza Or coronavirus infections occurred in 1918, 1957, 1968, 2009, 2001 and 2020 respectively.
  • T cells can mediate cross-protective immunity.
  • T cells cannot prevent viral infections, but they can recognize viral proteins that are complexed with human leukocyte antigen (HLA) molecules on the surface of infected epithelial cells or antigen-presenting cells ( Epitope) fragments to sense infected cells.
  • HLA human leukocyte antigen
  • Epitope antigen-presenting cells
  • the African swine fever virus has unique properties that induce neutralizing antibodies in pigs that will not recover from the infection and further resist the recurrence of the virus.
  • the presence of non-neutralizing antibodies leads to the possible ADE (antibody-dependent enhancement) effect of African swine fever, that is, the presence of antibodies not only does not prevent the virus infection, but aggravates the virus infection and the pathological process of related diseases.
  • the presence of non-neutralizing antibodies leads to persistent antigen positive in pigs immunized with attenuated vaccines.
  • ADE antibody-dependent enhancement
  • SFV pA179 L Bcl-2 family proteins bind to and inhibit some BH3 domain-only pro-apoptotic proteins.
  • pA224 L IAP family proteins bind and inhibit caspase 3 and activate NF-kB signaling, thereby increasing the expression of anti-apoptotic genes (including cFLIP, cIAP2 and c-rel).
  • the existence of the above mechanism causes the African swine fever virus to inhibit the apoptosis of infected cells to ensure the continued replication of the virus, but it induces the apoptosis of uninfected lymphocytes through TNF- ⁇ .
  • the main characteristics of acute ASFV disease are lymphatic tissue and blood. A large number of apoptosis of B and T lymphocytes.
  • M2 ion channel blockers have an overall virus resistance effect and the side effects of the nervous system make clinical applications not ideal.
  • neuraminidase inhibitors can induce viruses, but the effect is relatively weak.
  • virus outbreaks such as avian influenza virus, African swine fever virus, and atypical pneumonia virus. The toxicological consequences of these viruses are very serious, and they cannot provide good treatment for patients or sick animal doctors.
  • Nitric oxide is colorless and tasteless, soluble in water, alcohols and fats.
  • nitric oxide was just an ordinary and useless chemical gas. People only knew that it existed in automobile exhaust and gas pollutants from certain chemical processes.
  • endothelial cells produced a substance (called "endothelial relaxing factor”).
  • endothelial relaxing factor (EDRF) was Nitric oxide.
  • Nitric oxide synthase can be divided into endothelial nitric oxide synthase (eNOS) and inducible nitric oxide Synthase (iNOS), neural nitric oxide synthase (nNOS). They are involved in the regulation of the heart and cerebrovascular system, immune regulation, and nervous system regulation in different tissue cells of the human body.
  • eNOS endothelial nitric oxide synthase
  • iNOS inducible nitric oxide Synthase
  • nNOS neural nitric oxide synthase
  • the NO involved in immunity can be produced by a variety of immune cells (dendritic cells, NK cells, macrophages, eosinophils and neutrophils).
  • iNOS a large amount of NO can be produced as an active defense of the body Mechanisms.
  • NO can inhibit virus replication.
  • Related mechanisms include reducing the palmitization of viral spike proteins, inhibiting viral proteases, and hindering viral protein and nucleic acid synthesis.
  • Nitric oxide synthase is a dimer, which will uncouple under oxidative conditions, resulting in the conversion of the original NO synthesis reaction pathway to the production of O2-, NO3- (PON) and other reactive oxygen radicals (ROS). NO itself can also react with reactive oxygen radicals (ROS) to generate reactive nitrogen (RNS).
  • ROS reactive oxygen radicals
  • Oxidation PON itself is a strong oxidant. Under acidic conditions, nitrogen dioxide and hydroxyl radicals are rapidly decomposed. Hydroxyl radicals are stronger oxidants and can oxidatively degrade almost all organic substances. In the living body, PON can react with many enzymes, proteins, cytokines, iron/sulfur centers, sulfhydryl groups, lipids, etc. to cause oxidative damage, causing cell function damage and apoptosis; it can also reduce the ability of glutathione to remove free radicals. The mechanism creates a vicious circle. PON oxidation may cause various diseases, such as acute chronic inflammation, sepsis, traumatic ischemia, arteriosclerosis, nerve regeneration disorders and so on.
  • Nitrolation PON can react with the tyrosine in the protein to generate nitrotyrosine, which affects the function of the protein, causing DNA breakage and other consequences.
  • Affect energy generation enzyme protein activity decreases under oxidation and nitration. Such as mitochondrial ATP synthase, aconitase activity is inhibited, resulting in a decrease in energy.
  • PON is a strong activator of poly ADP-ribose synthase. The activation of the enzyme initiates an ineffective repair cycle, causing the energy pool to quickly deplete. Cell metabolism and membrane integrity are destroyed, leading to cell death.
  • the tolerable dose of PON also shows a positive effect, such as resisting the harm of viruses, germs, pathogens, cancer cells, etc. to the human body.
  • NO the star molecule of 1992, is actually everywhere in life. It is the messenger of the immune system and plays an important role in regulating blood flow, nerve conduction, and brain development; it can kill germs, viruses, pathogens, and cancer cells, and is a very important part of non-specific immunity.
  • the foreign microorganisms or abnormal cells killed by NO can release a large amount of antigen material after autolysis, and initiate specific immunity.
  • NO also causes the body to release many cytokines such as interleukin, interferon, tumor necrosis factor TNF, colony stimulating factor CSF, etc., to regulate immune response.
  • PON peroxynitrite
  • NO is involved in immune regulation.
  • Acute inflammatory response is a complex but highly coordinated sequence of events involving molecular, cellular and physiological changes. If the host's response to infection is dysregulated, it will further produce abnormal immune response, resulting in a comprehensive organ dysfunction.
  • the sign is sepsis (Sepsis).
  • Sepsis research on the treatment of sepsis reflects the progress of our human understanding of pathophysiology and host-microbe interactions. In the early days, people mainly focused on microorganisms and their pathogenicity. In the 1980s, with the implementation of molecular cloning and sequencing of human inflammatory genes, the study of sepsis focused more on the host's response to invading pathogens.
  • sepsis is defined as an organ dysfunction that threatens the host’s life due to the imbalance of the host’s response to infection. Its clinical manifestations are fever and rapid breathing , Changes in the level of consciousness and low blood pressure, accompanied by symptoms related to the disease such as pneumonia caused by lung infection, kidney infection, urinary tract infection, etc.
  • Inflammation is the host's defense response to pathogen invasion. Therefore, the clinically preferred treatment for removing pathogens is to use antibiotics or antiviral drugs to reduce the external stimulation of pathogen antigens. Viral infectious diseases develop to the stage of immune disorders, and severe inflammation will occur. Some anti-inflammatory or anti-inflammatory treatments reduce the number of macrophages in the inflamed area, and the decision to improve immunity or reduce immune response is often difficult. Common anti-inflammatory drugs include non-steroidal anti-inflammatory drugs and glucocorticoids. When severe inflammation occurs, glucocorticoids are often used clinically, but for sepsis, the use of cortisol has no substantial benefit.
  • 5-Methyltetrahydrofolate is the active form of folic acid in the human body, and its direct antiviral effect has not been observed.
  • the direct link between folic acid and virus is mainly in the folate receptor alpha (FRalpha), which has been described as mediating Including Ebola and other factors that viruses enter cells.
  • FRalpha folate receptor alpha
  • 5-Methyltetrahydrofolate has a direct antioxidant effect. It promotes the conversion of BH2 to BH4 through the action of dihydrofolate reductase. It is well known that BH4 is an essential cofactor for eNOS.
  • 5-methyltetrahydrofolate is beneficial to the prevention and protection of cardiovascular diseases by promoting eNOS, but few studies have reported that 5-methyltetrahydrofolate can secrete iNOS and macrophages under conditions of innate immune activation. The influence of NO.
  • L-arginine is the precursor of the endogenous synthesis of NO. Under the action of nitric oxide synthase, it will react to produce NO and L-citrulline, although only a small part of L-arginine is used in this way. Metabolism in the body, but in the case of acute inflammation, the NO produced by the iNOS of macrophages will greatly exceed the normal dose of the human body.
  • L-arginine is a non-essential amino acid, it can be synthesized endogenously (through the process of systemic protein degradation) in the metabolic pathways of proline, glutamine or glutamate. In the kidneys, citrulline passes through arginine.
  • the present invention has discovered that 5-methyltetrahydrofolate has a physiological activity different from the low concentration of "nutrition support” at a "pharmacological" concentration, and a composition containing 5-methyltetrahydrofolate has the effect of treating viral infections. It was further discovered that it has therapeutic effects on different pathogens, including bacteria and fungi. The present invention also found that the activity of dehydrocorbic acid and NMN is similar to 5-methyltetrahydrofolate.
  • the pharmaceutical composition of the present invention includes a NO attenuator and an optional NO extender, and the NO attenuator is selected from antioxidant substances that scavenges peroxynitrite or its salt (PON) at a dose.
  • the attenuating agent does not inhibit the expression of inducible nitric oxide synthase (iNOS) at a concentration of not less than 10 ⁇ mol/L, for example, does not inhibit the expression of iNOS in macrophages induced by LSP.
  • iNOS inducible nitric oxide synthase
  • the NO attenuator of the present invention is selected from antioxidant substances, which do not affect the activation of iNOS synthase and selectively quench peroxynitrite.
  • antioxidant substances which do not affect the activation of iNOS synthase and selectively quench peroxynitrite.
  • it is selected from one or more of the following substances: 5-methyltetrahydrofolate or its salt, dehydroascorbic acid, and NMN.
  • the NO extender of the present invention is selected from enzyme-producing NO substrates, and the enzyme-producing NO substrate is selected from L-arginine or its salt, citrulline or its salt, or arginine.
  • the pharmaceutical composition of the present invention includes 5-methyltetrahydrofolate or its salt and arginine or its salt. Further, phytohemagglutinin may be included.
  • the single dose of 5-methyltetrahydrofolate is not less than 15 mg, and the single dose of arginine is not less than 50 mg.
  • the present invention also provides the use of the above-mentioned pharmaceutical composition for the preparation of drugs for preventing or treating diseases caused by pathogenic microbial infections.
  • the pathogenic microorganism infection is a viral infection.
  • the pharmaceutical composition of the present invention can increase the level of T cells of a virus-infected host, especially CD4 and CD8 T cells, and reduce the expression of inflammatory factors, thereby being used for anti-viral infection.
  • the virus is influenza virus, herpes virus, African swine fever virus, coronavirus such as COVID-19.
  • the present invention provides a pharmaceutical composition for the prevention and treatment of swine fever, which comprises a NO attenuator and a NO booster, and the NO attenuator is selected from 5-methyltetrahydrofolate or its salt.
  • Hydroascorbic acid, one or more of NMN, and the NO extender is selected from one or more of arginine, citrulline or arginine.
  • composition for treating swine fever includes 5-methyltetrahydrofolate and arginine. Further, it may include phytohemagglutinin, and the mass ratio of the three is 2:8:1.
  • the swine fever is African swine fever.
  • the composition for treating swine fever of the present invention, can increase the level of T cells of a virus-infected host, especially CD4 and CD8 T cells, reduce the expression of inflammatory factors, and resist viral infection.
  • the single dose of the active ingredient of the composition is at least 30 mg/kg, for example 50 mg/kg.
  • the composition is used to prepare a medicine for preventing and treating sepsis and systemic inflammatory response syndrome caused by infection.
  • the pharmaceutical composition of the present invention includes 5-methyltetrahydrofolate or its salt and vitamin C.
  • the mass ratio of 5-methyltetrahydrofolate calcium to vitamin C is 2:1 to 5:1, for example, 3:1, 4:1.
  • the present invention also provides the use of the above-mentioned pharmaceutical composition, which is used to prepare and treat systemic inflammatory response syndrome and sepsis caused by non-infectious factors.
  • the sepsis is caused by Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and influenza virus infection.
  • the pharmaceutical composition according to the present invention can be made of active ingredients and pharmaceutically acceptable auxiliary materials.
  • the pharmaceutical preparation is selected from tablets, capsules, granules, injections, topical ointments or sprays.
  • the pharmaceutical composition according to the present invention is an immune adjuvant.
  • a safe amount of nitric oxide means that the ratio of nitric oxide converted to toxic free radicals and reactive nitrogen represented by peroxynitrous acid is controllable, which can meet the safety of using nitric oxide to prevent and treat diseases. demand.
  • These free radicals severely affect the metabolism of substances and energy in the body, affect or even destroy the functions of cells and tissues, and significantly increase the chance of gene mutations. They are also the cause of many diseases.
  • composition of the present invention controls toxic free radicals and effectively increases the production of nitric oxide to meet the needs of preventing and treating diseases.
  • the pharmaceutical composition that produces a safe amount of nitric oxide has application potential in the treatment of various diseases.
  • the composition of the present invention can promote the proliferation and activation of T cells, and increase the CD4 and CD8 of the host during infection. At the cell level, it blocks the apoptosis of CD4 and CD8 T cells, significantly improves the survival rate of the host, and improves the inflammatory response during infection.
  • the invention uses a composition containing 5-methyltetrahydrofolate and arginine to mice infected with influenza virus, and obtains a large-scale recovery result, and the disease course is significantly shortened.
  • the main function of folic acid is a carbon transmitter, participates in DNA methylation, participates in the synthesis of purine and thymine, and further synthesizes DNA and RNA.
  • the virus uses DNA or RNA structure to replicate in host cells in large quantities. Adequate supply of folic acid should be conducive to the replication and spread of the virus. The experimental results are surprising. 5-Methyltetrahydrofolate combined with nitric oxide extender , But suppressed the virus.
  • the present invention first proposes the application of 5-methyltetrahydrofolate and arginine in microbial infections, especially viral infections.
  • iNOS is a key enzyme for generating NO in the immune system. It is known in the prior art that oxidation of this enzyme will cause the uncoupling of dimers, and the reaction path for generating NO is transformed into a reaction path for generating free radicals and reactive nitrogen.
  • 5-Methyltetrahydrofolate is an endogenous antioxidant, which can activate NADPH to achieve a good antioxidant effect and has a direct antioxidant effect.
  • the composition of the present invention can make the body produce NO under pathogen infection while avoiding the generation of free radicals that are unfavorable to the body, including reactive oxygen species (ROS) and reactive nitrogen (RNS).
  • ROS reactive oxygen species
  • RNS reactive nitrogen
  • the present invention provides a method for producing sufficient nitric oxide in the body.
  • the composition of the present invention inhibits the generation of peroxynitrite, does not inhibit and induce the increase of the activity of nitric oxide synthase, and further increases the way the enzyme produces arginine and its precursor, which is a substrate for nitric oxide, so that Enough nitric oxide is produced in the body.
  • the concept of a sufficient amount refers to reaching or exceeding the minimum dose of nitric oxide required to prevent and treat diseases.
  • the invention provides a systematic scheme for providing sufficient nitric oxide, which can be selected and optimized according to needs.
  • a nitric oxide synthase inducer such as lectin
  • Phytohemagglutinin PHA is a kind of mitogen, an efficient and safe inducer of nitric oxide synthase, and the technology extracted from legumes can already be produced on a large scale.
  • Another object of the present invention is to provide a variety of uses of the above-mentioned safe nitric oxide compositions.
  • the active ingredient in the composition of the present invention includes 5-methyltetrahydrofolate or its salt.
  • the salt is selected from but not limited to calcium salt, arginine salt, glucosamine salt, sodium salt.
  • the amount of 5-methyltetrahydrofolate or its salt in each single-dose composition of the present invention is 15 mg or more, preferably 25 mg or more, more preferably 50-1000 mg.
  • the composition contains 5-methyltetrahydrofolate or its salt or dehydroascorbic acid or NMN, and arginine; each single-dose composition contains the amount of 5-methyltetrahydrofolate or its salt It is 15 mg or more, preferably 25 mg or more; preferably 50-1000 mg; more preferably 50-500 mg.
  • the amount of arginine is 50-5000 mg, preferably 100-1000 mg.
  • the composition includes 5-methyltetrahydrofolate or its salt and arginine and phytohemagglutinin PHA.
  • the amount of 5-methyltetrahydrofolate or its salt contained in each unit dose of the composition is 15 mg or more (equivalent to the amount of 5-methyl tetrahydrofolate), preferably 25 mg or more; preferably 50-1000 mg; more preferably 50-500 Milligrams; the amount of arginine in each unit dose composition is 50-5000 mg, preferably 100-1000 mg; the amount of phytohemagglutinin per unit dose composition is 10-500 mg, preferably 20-100 mg.
  • the pharmaceutical preparations can be selected from tablets, capsules, granules, injections, topical ointments or gas preparations.
  • Cigarette-smoke-induced oxidative/nitrosative stress impairs VEGF-and fluid-shear -stress-mediated signaling in endothelial cells[retracted in:Rahman I.Antioxid Redox Signal.2013 Apr 2018(12):1535].
  • Antioxid RedoxSignal.2010;12(12):1355–1369.] so the NO level and The subsequent downstream signal transduction is regulated by reactive oxygen species, which is also a factor in regulating redox signals.
  • NF- ⁇ B acts as the main switch of inflammation and is related to the production of H 2 O 2
  • NF- ⁇ B is regulated by redox.
  • Most reducing agents or antioxidants have anti-inflammatory effects to some extent. They inhibit the NF- ⁇ B pathway and can inhibit the expression of iNOS.
  • the macrophages under the influence of iNOS expression show that 5-methyltetrahydrofolate, dehydroascorbic acid, BH 4 , glutathione, and nicotinamide mononucleotide can almost express iNOS at a concentration of 10 ⁇ mol/L Does not affect.
  • the reactivity of the above antioxidants to peroxynitrite was investigated, and the results showed that 5-methyltetrahydrofolate, dehydroascorbic acid, and NMN all have higher ability to eliminate peroxynitrite. It has been proved that under hypoxic conditions, the immune function of lymphocytes is inhibited and the apoptotic rate increases.
  • antioxidants Due to the lack of active oxygen, it hinders the synthesis of iNOS, destroys the combination of iNOS and a-actinin4, and prevents iNOS from attaching to the muscles. Protein on the cytoskeleton. Therefore, the presence of antioxidants may cause the down regulation of iNOS.
  • the present invention found that the following antioxidants: 5-methyltetrahydrofolate, dehydroascorbic acid, and NMN have unique properties. They will not reduce the expression of iNOS at a certain concentration, but they have a better removal of peroxynitrite. The ability to salt.
  • the above-mentioned antioxidants all have the ability to not reduce the immune response after the antigen activates the immunity, especially not to negatively affect the expression of iNOS during infection, and also reduce the production of peroxynitrite.
  • NO has the effect of inhibiting cell apoptosis. S-nitrosylation inhibits caspases-8, caspases-9 or caspases-3, while peroxynitrite promotes cell apoptosis through DNA damage and upregulation of p53.
  • NO has direct and indirect effects on infecting microorganisms. NO can directly destroy the enzyme structure of pathogenic microorganisms, especially [Fe-S] clusters. In virus infection, expressing NO can inhibit the enzyme activity of the virus and inhibit the replication of the virus. The direct toxicity of NO, especially the extracellular antiviral activity, has been fully proved, but the indirect effect of NO on the regulation of immune function is much more complicated. Studies have shown that iNOS-deficient mice infected with influenza virus have almost no histopathological evidence of pneumonia. Therefore, the scholar believes that host iNOS may contribute more to pneumonia than viral replication [Karupiah G, Chen JH, Mahalingam S, Nathan CF, MacMicking JD.
  • exogenous NO inhibits the proliferation of T lymphocytes, and exogenous NO (that is, NO is not produced by T cells itself) inhibits proliferation or even causes the death of T cells [Bogdan C. Regulation of lymphocytes by nitric oxide.[J].Methods Mol Biol,2011,677:375-393.].
  • Mice lacking an important antioxidant mechanism ie, GSNOR
  • GSNOR a small amount of NO branch T cell subsets, especially the negative regulatory T cell populations of Th1 cells and FoxP3, can effectively inhibit Th17 cell differentiation.
  • recent studies have shown that exogenous NO also regulates Th9 and Th17 cells.
  • 5-methyltetrahydrofolate in the cell culture medium at a concentration of 15.625 ⁇ m hardly affects the secretion of NO by macrophages. More interestingly, when LPS stimulation is not added , 5-Methyltetrahydrofolate is found to promote the secretion of NO at low concentrations.
  • the combined use of the NO attenuating agent selected in the present invention and the NO increasing agent shows that it can significantly increase the activity of CD4+ T cell proliferation after antigen stimulation.
  • Existing studies have shown that virus clearance is mediated by antigen-specific CD8+ effector T cells, and memory CD4+ T cells play an important role in maintaining the memory response of CD8+ T and B cells [Stambas J, Guillonneau C, Kedzierska K,et al.Killer T cells in influenza[J].Pharmacology&therapeutics,2008,120(2):186-196.].
  • both CD4+ and CD8+ T cells are involved in the control of pneumonia, and through the production of interleukin-10 to limit excessive tissue damage. Therefore, the pharmaceutical composition of the present invention containing the above-mentioned NO attenuating agent and NO increasing agent can be used in virus elimination and anti-inflammatory treatment.
  • arginine is used as an increaser of NO, and when used in combination with 5-methyltetrahydrofolate, it produces unexpected antiviral and sepsis effects.
  • the composition of the present invention can significantly stimulate the proliferation of T cells in the thymus and spleen of mice.
  • the combination of arginine and 5-methyltetrahydrofolate is administered.
  • CD4 The proliferation of the cells was significantly increased, indicating that the composition can increase the proliferation ability of effector CD4+ T cells.
  • the number of virus-specific memory CD4 + T cells can predict the severity of human infection with influenza virus, and the number of virus-specific T cells is inversely proportional to the severity of the disease.
  • the composition of the present invention has the potential to treat influenza virus infection and can reduce the severity of illness. It has been known before that peroxynitrite affects the immune response of cells. Studies have supported that peroxynitrite prevents the feedback ability of inhibiting inflammation and repairing, and it is easy to make the host immune disorder during infection.
  • the used composition can not only improve the host's immunity but also maintain the negative feedback mechanism of inflammation, so that the host can resist infection, especially viral infection.
  • the above-mentioned pharmaceutical composition of the present invention has a subversive effect on the treatment of colds: the onset of action is fast. According to the results of more than 40 trial users, the cold symptoms disappeared within 48 hours after taking the composition, although no double-blind control was performed. Clinical trials, but the feedback results of the trial composition also exceeded expectations.
  • the present invention verifies the antiviral infection effect of the composition in an animal model, and the result shows that the composition can protect the immune function of mice, reduce the pathological state of influenza virus infection of the lung, and reduce lung tissue damage.
  • the combination of 5-methyltetrahydrofolate and arginine can significantly reduce the level of inflammatory factors caused by infection, and significantly reduce the lung virus titer 5 days after infection, indicating that the composition has a certain antiviral effect.
  • the use of the composition can significantly increase the levels of CD4 + and CD8 + T cells infecting the spleen and thymus of mice, suggesting that although the composition reduces inflammatory factors, it does not reduce the immunity of the host.
  • the results of lung tissue sections show that the composition can reduce lung tissue damage and inflammation, and shows very good efficacy in a host model for cold viruses.
  • the composition is applied to the treatment of African swine fever virus infected pigs, and a very good effect is obtained, which significantly improves the survival rate of pigs infected with African swine fever virus, and further proves that the composition is resistant to The potential of the virus.
  • the present invention found that the above-mentioned composition of the present invention can significantly protect the survival of the host in a high-dose virus challenge experiment, showing a certain potential for treating sepsis.
  • the present invention found that 5-methyltetrahydrofolate can significantly reduce the mortality of LPS-induced sepsis mice, suggesting that 5-methyltetrahydrofolate may be beneficial for the treatment of sepsis caused by severe allergies.
  • the present invention found that the combination of 5-methyltetrahydrofolate and arginine can significantly reduce the mortality of septic mice caused by infection with microorganisms (for example, Staphylococcus aureus). Sepsis is a highly fatal disease characterized by extensive apoptosis-induced depletion of immune cells and subsequent immunosuppression.
  • the present invention finds that the combination of 5-methyltetrahydrofolate and arginine can significantly increase the survival rate of the host, block the apoptosis of CD4 and CD8 T cells, and the combination is proven in a variety of sepsis models caused by bacteria and viruses The curative effect of the thing.
  • the antioxidant selected by the composition of the present invention has unique properties, which can alleviate the symptoms caused by pathogenic microorganism infection, and at the same time increase the level of NO, so that the pathogen killing effect mediated by NO is better than the oxidative stress mechanism triggered by NO.
  • T cells do play an important role in cross-protection.
  • inactivated virus vaccines often protect against certain viruses by inducing specific antibodies, but have little effect on enhancing the cross-protective T cell response.
  • the composition of the present invention has the effect of enhancing the immune response.
  • the use of the composition significantly increases the antibody level of the immunized animal after inoculation with rabies vaccine.
  • the present invention utilizes the immune system of human and animal body, which is a natural and powerful antiviral tool. African swine fever, a severe infectious disease, is completely powerless in the prior art, with a fatality rate of 95%-100%. However, the use of the present invention shows a clear curative effect, showing that the composition has an ability to resist viral infections beyond expectations. [Oura,CA L.
  • the present invention proposes for the first time the concept of safe and sufficient amount of nitric oxide to be applied to anti-viral infections, and the used compositions are synergistic with each other to show good effects.
  • There are complex regulatory mechanisms in the immune system multiple signaling pathways or target targets have beneficial or harmful effects, and there are tens of thousands of research papers on nitric oxide, but the conclusions of the research are inconsistent, contradictory and difficult. Sort out.
  • Some researchers even introduced the concept of “yin and yang balance” in Eastern civilization, which has expressed the role of a double-edged sword of various target mechanisms [Burke A J, Sullivan F J, Giles F J, et al.
  • the composition of the present invention from the overall perspective of immunity constitutes a very significant technical effect: 1. It can significantly increase the level of immune cells in the host, or prevent the apoptosis of immune cells; 2. Effectively reduce inflammatory factors and reduce inflammatory damage; 3 . Using the immune function to regulate the virus can make the host better resist secondary infection; 4. The safety of each component in the composition is better.
  • NOS nitric oxide synthase
  • SNO safe nitric oxide
  • nitric oxide with a controllable content of toxic free radicals such as nitrous acid peroxide which can meet the safety requirements when using nitric oxide to treat and prevent diseases
  • NO attenuator It is used to reduce the reducing substances generated by toxic nitrogen-containing free radicals such as peroxide and nitrous acid.
  • NO extender a precursor substance that produces NO. It is divided into chemical substances that can release NO in the body, arginine that produces enzymes to produce NO, and arginine biogenic substances.
  • NO synthase inducer a substance used to induce the production of NO synthase
  • Folic acid calcium 6S-5-methyltetrahydrofolate.
  • the salt in the present invention refers to a pharmaceutically acceptable salt.
  • Figure 1 The body weight change curve of each group in Example 7;
  • Figure 8 The effect of the composition in Example 10 on stimulating the proliferation of CD4 T cells for three days;
  • Figure 9 The results of the quantitative composition in Example 10 on stimulating the proliferation of CD4 T cells for three days;
  • Fig. 13 The changes in the index of the middle spleen and the total number of immune cells in the spleen in Example 13;
  • Figure 14 The changes in the index of the middle spleen and the total number of immune cells in the spleen in Example 13;
  • Figure 15 The changes in the index of the middle spleen and the total number of immune cells in the spleen in Example 13;
  • Figure 16 The changes in the middle thymus index and the total number of immune cells in the thymus in Example 13;
  • Figure 17 is a section of lung tissue in Example 13;
  • Figure 18 Changes in the secretion of inflammatory factors in peripheral blood in Example 13;
  • Figure 21 is a graph of changes in LYMP/NEUP of the composition in Example 24 on the blood routine of pig ear veins.
  • mice Balb/c mice, female, 6 weeks old, 15-17g, 25 mice.
  • A administration group, infection administration, 10 animals;
  • Infection method 150 ⁇ l 5% chloral hydrate was injected intraperitoneally to anesthetize mice, and PR8 influenza virus (1 ⁇ 106pfu/mouse) was used to infect the mice through the nose.
  • mice start weighing, body temperature, water intake, and food intake on the day of infection.
  • the body weight, food and water intake of mice were measured once a day at a fixed time; body temperature measurement: measured twice a day within 3 days after infection, with an interval of 12 hours, starting from 4 days after infection, and measured once a day at a fixed time. In the experiment until the 15th day after infection, the weight of the mice basically recovered.
  • Cell line Macrophage RAW264.7.
  • Reagents LPS (Sigma); iNOS detection kit (Stressgen); MTT (Biotopped).
  • Mouse macrophages RAW264.7 were cultured in DMEM high glucose medium containing 10% FBS in a 37°C, 5% CO 2 incubator.
  • LPS induction add 40 ⁇ L LPS to all wells (to a final concentration of 0.1 ⁇ g/mL);
  • Vitamin C group Vitamin C and LPS per well (to a final concentration of 10 ⁇ mol/L Vitamin C, 0.1 ⁇ g/mL LPS);
  • Vitamin E group Vitamin E, LPS per well (to a final concentration of 10 ⁇ mol/L Vitamin E, 0.1 ⁇ g/mL LPS);
  • Glutathione group glutathione per well, LPS (to a final concentration of 10 ⁇ mol/L glutathione, 0.1 ⁇ g/mL LPS);
  • 5-Methyltetrahydrofolate group each well of 5-methyltetrahydrofolate calcium, LPS, 5-methyltetrahydrofolate calcium to a final concentration of 10 ⁇ mol/L, and LPS to a final concentration of 0.1 ⁇ g/mL LPS);
  • Dehydroascorbic acid group Dehydroascorbic acid per well, LPS (Dehydroascorbic acid to a final concentration of 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL LPS);
  • Anthocyanin group anthocyanin per well, LPS (the final concentration of anthocyanin is 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL);
  • Curcumin group curcumin per well, LPS (curcumin to a final concentration of 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL LPS);
  • Resveratrol group Resveratrol per hole, LPS (resveratrol to a final concentration of 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL);
  • Andrographolide group andrographolide per hole, LPS (andrographolide to a final concentration of 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL);
  • Baicalin group Astragaloside per hole, LPS (astragaloside to a final concentration of 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL);
  • NMN group NMN, LPS per well (NMN final concentration is 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL);
  • Tetrahydrobiopterin group Tetrahydrobiopterin, tetrahydrobiopterin, LPS (tetrahydrobiopterin, to a final concentration of 10 ⁇ mol/L, LPS 0.1 ⁇ g/mL) per well; normal group: add 50 ⁇ L of complete medium to each well.
  • the level of iNOS protein in macrophages was determined by ELISA using polyclonal antibody anti-human iNOS (Stressgen). Use an automated flow cytometer to determine the number of macrophages.
  • mice were sacrificed by cervical dislocation.
  • the mouse spleen and lymph nodes were aseptically separated and placed in Hank's solution.
  • CD4 T cells were purified from the mouse spleen and lymph nodes using immune beads (CD4+ cell extraction kit; Miltenyi Biotec, USA).
  • CD4+ cell extraction kit Miltenyi Biotec, USA.
  • Composition group A add 5-methyltetrahydrofolate calcium to the cell culture solution to a final concentration of 10 ⁇ mol/L, and arginine to a final concentration of 40 ⁇ mol/L;
  • composition group B add dehydroascorbic acid to the cell culture solution to the final concentration Concentration 10 ⁇ mol/L, arginine to a final concentration of 40 ⁇ mol/L;
  • composition group C add NMN to the cell culture solution to a final concentration of 10 ⁇ mol/L, arginine to a final concentration of 40 ⁇ mol/L;
  • arginine group cell culture Add arginine to the final concentration of 40 ⁇ mol/L; blank group: use the initial cell culture medium (without L-arginine).
  • the purified T cells were stained with Cell Violet Trace Proliferation kit (Invitrogen) and cultured for three days, and analyzed by flow cytometry to determine the proliferation.
  • Cell Violet Trace Proliferation kit Invitrogen
  • the results are shown in Figures 8, 9.
  • the results show that the selected compositions can improve the proliferation of CD4 cells after stimulation to some extent, and show the ability to improve the cellular immunity of the infected host.
  • Tonsillitis and other symptoms other than sore throat are defined as basic rehabilitation; including tonsillitis and sore throat are defined as complete recovery.
  • Example 12 The effect of 5-methyltetrahydrofolate on some inflammatory factors and the effect of NO secretion
  • Cell line Macrophage RAW264.7.
  • Reagents LPS (Sigma); MTT (Biotopped); Folic acid is calcium 5-methyltetrahydrofolate (Lianyungang Jinkang Hexin Pharmaceutical Co., Ltd.); NO detection kit (Biyuntian)
  • LPS group 50 ⁇ L LPS (to a final concentration of 0.1 ⁇ g/mL);
  • Example 13 Investigating the early protective effect of the composition with different doses of one-time administration on influenza virus infection mice
  • mice 20 (5 in each group), female, 6 weeks old, 15-17g, purchased from Weitong Lihua Experimental Animal Company;
  • G1 group blank control group
  • Group G2 Model Group
  • Model group only modeled without administration, given equal volume of deionized water.
  • Blank control group give equal volume of deionized water.
  • mice were anesthetized by intraperitoneal injection of 150 ⁇ l of 5% chloral hydrate, and PR8 influenza virus (1 ⁇ 10 6 pfu/mouse) was injected intranasally.
  • mice were sacrificed 5 days after infection, and lungs, thymus, spleen and peripheral blood were collected.
  • Serum Serum is prepared, part of which is tested for cytokines (external test), and the rest is frozen.
  • the lung tissue is divided into 2 parts, one part (the right lobe) is used to determine the virus titer; the other part (the upper tip of the left lung lobe) is fixed, embedded in paraffin, sectioned and HE stained.
  • Spleen and thymus weighing, photographing, cell counting and immune cell staining.
  • Weight changes weigh your body every day. Keep the sample: the serum sample on day 5 is used to detect inflammatory factors (use Biolegend's LEGENDplex Mouse Inflammation Panel detection, external inspection).
  • Lungs measure virus titer; make pathological sections of lung tissue; detect changes in inflammatory factors in lung tissue.
  • Thymus weighing, photographing, total thymus cell count, lymphocyte staining (CD4+, CD8+ T cells).
  • Spleen weighing, photographing, total spleen cell count, splenic lymphocyte staining analysis (surface staining, B cells, CD4+T cells, CD8+T cells, NK cells, NKT cells, monocytes, macrophages, dendrites Like cells, neutrophils).
  • mice 60 Kunming mice, male, weighing 14-18g, HSV-1 attacked Hela cells, HSV-1 was cultured in Hela cells for 48 hours, the virus was collected to determine the virus titer, and the mass score was 100TCID 50 10 -5 for virus inoculation Mice.
  • mice were divided into control group, model group, saline treatment group, acyclovir treatment group (10mg/kg), composition group (5-methyltetrahydrofolate 14mg/kg, arginine 50mg/kg, plant Hemagglutinin 7mg/kg), the control group was injected with 0.03ml sterile normal saline, the model group and each treatment group were injected with 0.03ml HSV-1 virus solution, and then administered by gavage for 4 consecutive days. Observe the death and Other changes. After 7 days, 0.5ml of blood was taken from the eyeball, stored in a 35°C incubator for 2 hours, centrifuged at 1000r/min for 5min, and the results of detecting NO and 1L-1 ⁇ were as follows:
  • composition can significantly reduce the mortality of mice under herpes virus infection, increase the release of NO in mice during the infection process, and reduce the level of inflammatory factors.
  • mice Male, 6-8 weeks old, weighing 18-22g, observe the animal's general physiological indicators, body weight and eating status.
  • LPS purchased from sigma company, article number: L2880.
  • mice 49 male C57 mice were divided into 7 groups, 6 administration groups, 1 model group, 7 mice in each group, intraperitoneal injection of 13mg/kg LPS (dose determined by pre-experiment, because 20mg LPS dose cannot be observed for 120 hours In order to prolong the survival time of the model group, the pre-experiment confirmed that the dose was 13 mg/kg).
  • Vitamin C 1:3 (equivalent to human body dosage is 100mg 5-methyltetrahydrofolate calcium, 300mg vitamin C);
  • Vitamin C 1:12 (equivalent to human body dosage is 5-methyltetrahydrofolate calcium 50mg, vitamin C 600mg);
  • Vitamin C 12:1 (equivalent to human body dosage is 600mg 5-methyltetrahydrofolate calcium, 50mg of vitamin C);
  • Vitamin C 3:1 (equivalent to human body dosage is 1200mg 5-methyltetrahydrofolate calcium, 400mg vitamin C
  • mice in groups C and D showed signs of trembling and obvious listlessness.
  • Groups E and F were in the best condition, followed by groups A and B. No animals died in each group after 144h.
  • mice were randomly divided into 5 groups, half male and half male, and grouped as follows:
  • the administration was started. According to the above dose, the administration was divided into three doses (0, 2, and 4 days), once every other day.
  • mice weighing 18-24g, half male and half male, Staphylococcus aureus and Streptococcus pneumoniae were selected as test bacteria, and randomly divided into normal group, model group, and low-dose composition group (40mg/ kg), the composition dose group (80mg/kg), the composition high dose group (160mg/kg), the amoxicillin group (120mg/kg).
  • the above groups were administered intraperitoneally at 20ml/kg once a day. Except for the normal group, mice in each group were intraperitoneally injected with 0.5ml/mouse of Staphylococcus aureus solution (5 ⁇ 10 9 CFU/ml).
  • mice pneumoniae infection methods and grouping conditions are the same as those of Staphylococcus aureus. Observe the death of mice in each group within 4 days after the injection of the bacterial solution, compare the differences between the groups, and calculate the survival rate. The experimental results are as follows.
  • the composition can significantly improve the survival rate of the host.
  • an independent experiment was carried out. ICR mice were taken and injected with 0.5ml of Staphylococcus aureus solution (5 ⁇ 10 9 CFU/ml ), among which 10 model mice were injected intraperitoneally with 20 mg/kg of the composition solution of 5-methyltetrahydrofolate and arginine at the same time, 24 hours after modeling, the mice were sacrificed and the spleens were collected. Spleen total cell count, splenic lymphocyte staining analysis (surface staining, B cells, CD4+T cells, CD8+T cells, NK cells.
  • the results are shown in Figure 20.
  • the results show that the composition can prevent the apoptosis of CD4 and CD8 T cells in sepsis. After 24 hours of model building in septic mice, sepsis induces apoptosis of all types of immune effector cells.
  • BAL B/C male rats Take 50 BAL B/C male rats, weighing 18-22g, and divide them into 6 groups, each with 8 rats, of which the experimental group is 2 groups, and the rest are various control groups, which are the composition low-dose group (40mg/kg ), composition high-dose group (80mg/kg), normal group, model group, penicillin group (450mg/kg), meropenem group (75mg/kg).
  • the composition low-dose group 40mg/kg
  • composition high-dose group 80mg/kg
  • normal group normal group
  • model group penicillin group
  • meropenem group 75mg/kg
  • a lethal dose of Pseudomonas aeruginosa was intraperitoneally injected into BAL B/C male mice in each experimental group and three control groups at a dose of 500 ⁇ L/head. Thirty minutes after the BAL B/C male mice in the experimental group were infected with the bacteria, the dosage group and the high-dose composition group were administered intragastrically, the penicillin group and the meropenem group were also administered intragastrically, and the normal group was intragastrically administered purified water.
  • BAL B/C male mice of each experimental group and control group were given a second administration, and the type and dosage of administration were the same as the first administration.
  • BAL B/C male rats were observed every 24h after administration, and their survival was recorded. All animals were killed on the 15th day.
  • the composition of the present invention can maintain 100% survival of mice infected with a lethal dose of Pseudomonas aeruginosa for 14 days.
  • the survival rate of mice in the meropenem group was 100% after 14 days, while the penicillin group all died, and the model group also all died.
  • Example 19 The death protection effect of the composition treatment and preventive administration on H1N1 (FM1) influenza virus infection mice
  • ICR mice SPF grade, weighing 13-15g, half male and half female. Provided by Beijing Weitonglihua Experimental Animal Co., Ltd., license number: SCXK ( ⁇ )2016-0006, animal certificate: 1100111911082385.
  • test substances were all used 1000 times dilution of FM1 strain to carry out nasal infection in mice, and the composition was divided into three dosage groups of high, medium and low.
  • a preventive administration group was set up, and a low-dose one-time administration was used, and the vitamin group was set to compare with the middle-dose group.
  • the official test concentration is 1000 times FM1 diluent, 45 ⁇ l/head, nasal infection.
  • a total of 130 ICR mice were taken, and the animals were randomly divided into 7 groups according to their weight levels, which were a normal control group, a model control group, a high, medium, and low-dose composition group, a composition prevention group, and a composition post-treatment group. Take 10 animals as the normal control group, and the other 20 animals in each group were infected with H1N1 influenza virus at a rate of 45 ul/each. After infection, each administration group was given by gavage, 0.1ml/10g.
  • Model control group give the same amount of saline
  • the composition (5-methyltetrahydrofolate:arginine:vitamin C 3:12:1) 0.173g/kg body weight, and the two administration times are respectively on the 3rd day and the 6th day after infection.
  • the results show that the high-dose composition group has a protective effect on animal death after the first infection and repeated infections.
  • the preventive administration group also has a certain protective effect. It is suggested that in addition to reducing the mortality of mice caused by influenza virus, the composition has better therapeutic and death protection effects on animals, and its preventive administration also shows a certain protective effect, which can prolong the survival time of mice.
  • Example 20 The inhibitory effect of the composition of formula A on the fever caused by endotoxin
  • composition preparation 5-methyltetrahydrofolate calcium and vitamin C are mixed at a mass ratio of 1:1, and after three-dimensional mixing and total mixing, a composition of formula A is prepared.
  • Endotoxin preparation According to previous reports, this experiment was reported in the literature. After pre-experiment, the calorific value of endotoxin was determined to be 250ng/ml/kg, and it was prepared with normal saline before the experiment.
  • Each rabbit was injected endotoxin from the ear vein.
  • the rectal temperature was measured, and the groups were divided into groups according to the changes in body temperature. They were the model group, the positive drug group, and the pharmaceutical composition A of the invention with a large formula (40mg/kg), Medium (20mg/kg) and small (10mg/kg) dose groups.
  • Each administration group was given once by gavage, 2ml/kg, and the model group was given distilled water under the same conditions.
  • the rectal temperature was measured 0.5h, 1h, 1.5h, 2h after administration.
  • the experimental results are shown in Table 13.
  • Example 21C formula in vivo anti-Mycoplasma pneumonia test
  • the international standard strain of Mycoplasma pneumoniae (ATCCFH15531) was purchased from the American Culture Collection.
  • Formula C composition self-made in the laboratory, 5-methyltetrahydrofolate calcium, arginine, and phytohemagglutinin were mixed in a ratio of 2:8:1, and after total mixing, the composition of formula C was obtained.
  • mice Fifty BALB/C mice, half male and half male, weighing 16-20 g, were purchased from Guangdong Medical Experimental Animal Center.
  • mice were randomly divided into 5 groups, each with half male and female. They were normal control group, model control group, positive drug control group (40mg/kg), and C composition high-dose group ( 80mg/kg), C (40mg/kg) composition low dose group. Except for the normal control group, the mice in the other groups were anesthetized with ether, and infected with 50 ⁇ L of Mycoplasma pneumoniae (MP) bacterial solution with a concentration of 10 6 CCU/ml through the nose for 3 consecutive days. After that, intragastric administration was started, once a day for 10 consecutive days. 4 hours after the last administration, the mice’s eyeballs were taken blood and sacrificed. The lungs, spleen, and thymus were weighed for pathological observation. Another small piece of lung tissue was taken, and the content of MP was quantitatively detected by PCR after grinding. The results are as follows.
  • the spleen index of the mice in the blank control group is significantly different, and the spleen index of the mice in each administration group is significantly different, which suggests that MP is killed in the body after administration.
  • Example 22 The effect of nitric oxide composition as an immune adjuvant on the effect of rabies virus vaccine
  • mice 30 adult Kunming mice, weighing 20-28g, male and female, were obtained from Experimental Animal Center of Xinjiang Medical University, rabies rSRV 9 live attenuated oral freeze-dried vaccine (Beijing Zhongliankang Biotechnology Co., Ltd.), nitric oxide composition B formula preparation: 5-methyltetrahydrofolate calcium and arginine are mixed at a mass ratio of 1:4, which is worthy of the B formula composition.
  • the 30 mice were divided into 3 groups, 10 mice in each group. They are the blank control group, the virus oral immunization group, and the formula B + virus oral immunization group.
  • the corresponding vaccines were taken orally on the first, 7th, and 14th days of the test, and blood was collected from the orbit at 0, 14, 21, 35, 42, and 70 days after immunization. 300 ⁇ L/mouse was collected from the orbit. After standing for 1 hour, centrifuged at 5000r/min for 5min, and the serum was drawn ; Simultaneously collect mouse feces about 0.05g, put in 500 ⁇ L of PBS (pH value about 7.4), crush to form a turbid liquid, centrifuge to extract the supernatant, and store in the refrigerator at -20°C. Serum IgG antibody is detected by ELISA detection kit, stool IgA The antibody was detected by the mouse serum rabies specific IgA antibody ELISA detection kit. The results are as follows.
  • composition B can increase the level of IgG antibodies in the serum.
  • composition C After the oral vaccine is combined with composition C, there is a considerable degree of antibodies on the 14th day. After 21 days of immunization, there are significant differences in antibodies between different groups.
  • the formula B composition can improve the immune activity of the vaccine.
  • the rSRV 9 virus oral attenuated vaccine can significantly increase the expression of antibodies in mice and reduce the immunity The number of vaccinations can improve the immune effect.
  • a case of African swine fever was confirmed by the China Center for Animal Health and Epidemiology.
  • the positive sample came from a farmer in Ganyu District, Lianyungang, Jiangsu province. This household had 300 live pigs and 130 cases of disease. More than 120 heads died.
  • Anatomy of the dead pigs, pathological examination found: pulmonary hemorrhage, interstitial pneumonia and other symptoms, anatomy of the spleen revealed severe spleen enlargement, severe splenomegaly 7 times; stomach anatomy revealed diffuse gastric serosal surface Bleeding; kidney swelling is obvious, in line with the symptoms of African swine fever.
  • the blood of 3 pigs with diseased hair and 10 healthy pigs from the household was centrifuged at 3000 r/min, and the serum was taken into Roche with ceramic beads, and PBS buffer was added.
  • the DNA was extracted using a viral DNA kit. The detection was determined to be ASFV African pigs. Plague Genotype II, a genus of viruses broadcast in the Russian Far East and Eastern Europe in 2017. Use the formula C composition to intervene in the treatment of African swine fever.
  • composition injection 5-methyltetrahydrofolate calcium, L-arginine and phytohemagglutinin are mixed in a ratio of 2:8:1, and after total mixing, a composition of formula C is obtained. After sterilization, the composition of formula C is dissolved in physiological saline, filtered through a microfiltration membrane, activated carbon absorbs the heat source, and then prepared into an injection.
  • results show that: it has an unexpected effect on African swine fever pigs, suggesting that the formula C composition has a very good antiviral effect.
  • the results showed that only one pig died within 7 days. Afterwards, due to policy requirements, all infected pigs were put to death, and other infected pigs of the farmer died 3 to 4 days after the onset of illness.
  • the blood was collected, and after standing for 1 hour, centrifuged at 5000 r/min for 5 min, and the serum was drawn. Detect IgG antibody levels. Add Krebs-HEPES buffer to the blood, keep it at 37°C, let it stand for 30 minutes, add L-NAME (100 ⁇ M), and detect the content of superoxide, nitrite and NO by electrochemical methods.
  • the cured pigs were sacrificed and dissected for pathological observation. It was found that the cured pigs had local bleeding in the lungs except for the slightly larger spleen.
  • the blood was collected, and after standing for 1 hour, centrifuged at 5000 r/min for 5 min, and the serum was drawn. Detect IgG antibody levels. Krebs-HEPES buffer was added to the blood, maintained at 37°C, and allowed to stand for 30 minutes, then L-NAME (100 ⁇ M) was added, and the content of nitrite and NO was detected by photochemical method.
  • composition of the present invention can reduce the death of malignant virus due to immune overexpression by reducing RNS, and maintain the normal operation of the immune system to achieve the purpose of eliminating the virus.
  • the implementation effect of the composition of the present invention greatly exceeds expectations.
  • Example 24 Effect of composition on pig immune cells
  • compositions containing 5-methyltetrahydrofolate Three common domestic pigs, about 3 months old and weighing about 25 kg, were given a composition containing 5-methyltetrahydrofolate.
  • the preparation process of the composition is as follows: 5-methyltetrahydrofolate calcium and L- Arginine and phytohemagglutinin are mixed in a ratio of 1:4:0.1 to obtain a pharmaceutical composition.
  • the composition Before taking the composition, take blood from the ears and test blood routinely. After that, the composition was taken orally at 30 mg per kilogram of pig body weight every day, and ear blood was taken in the first week and the second week, and the blood was measured routinely.
  • the main indicators of routine blood testing are LYMP (lymphocytes) and NEUP (neutrophils).
  • the blood routine test indicators are as follows:

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Abstract

一种在体内产生安全量的一氧化氮的药物组合物及其用途。其包括以下组分：NO减毒剂、NO增量剂和一氧化氮合酶诱导剂。提供了通用性强而且又极为有效治疗致病微生物感染的药物组合物。其中NO减毒剂选自5—甲基四氢叶酸，NMN以及去氢抗坏血酸，NO增量剂选自精氨酸，一氧化氮合酶诱导剂选自植物血凝素。提供了5—甲基四氢叶酸的新药用活性，其对因病原体感染导致的免疫系统具有多种活性作用，可用于治疗或预防病毒感染以及其他病原体感染所带来的疾病，特别的，组合物用于预防或治疗非洲猪瘟。

Description

用于治疗非洲猪瘟的药物组合物及其用途
本申请要求2019年08月06日向中国国家知识产权局提交的专利申请号为201910719544.6，发明名称为“安全的一氧化氮组合物及其用途”的在先申请的优先权，上述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于医药领域，具体涉及一种可在动物体内产生一氧化氮的药物组合物，它能提供安全足量的一氧化氮，用于疾病的预防和治疗。
背景技术
非洲猪瘟(African swine fever，ASF)是由非洲猪瘟病毒(African swine fevervirus，ASFV)引起的一种急性、热性、高度接触性、致死性动物传染病。ASFV的主要宿主为各品种的家猪、非洲和欧亚野猪、钝缘蜱等，其中疣猪和灌木猪均可被感染，但不表现临床症状，疣猪等非洲野猪和钝缘蜱为ASFV的宿主。根据ASFV的毒力，ASF的临床症状和感染过程有显著差异，最急性和急性型感染死亡率为100％。
非洲猪瘟无有效的预防疫苗，无特效治疗药物。各阶段家猪、野猪和软蜱是非洲猪瘟自然宿主，可在家猪和野猪之间直接传播，也可通过蜱虫叮咬传播，还可以通过污染了病毒的泔水、饲料以及腌制的干火腿等猪肉产品跨国家和地区传播。发现疫情，必须进行扑杀，是全球防范内危害猪群最严重的传染病之一，是我国重点防范的头号外来动物疫病。
人类的历史中新的病毒不断出现，已知的病毒不断变异，面对新的传染性病毒时，由于人体内没有相对应的特异性抗体，导致类周期性的出现大规模的感染。人类的几次流感病毒大流行，带走了很多人的生命。2009年美国和墨西哥爆发了H1N1流感病毒，2020年全球爆发COVID-19病毒，不同个体感染相同的流感病毒，结果却不相同，有些病人会失去生命，而有些却几乎没有症状，尽管病毒的毒力可能会有所不同，但宿主的免疫状态也很重要。
当需要预防和治疗流感病毒感染时，抗体是最好的手段，但是流感病毒发展迅速，抗体对季节性流感病毒的选择性压力推动了逃逸突变体的出现，这些突变体可以在早期菌株免疫的社区中引起流行病，这就是为什么季节性流感疫苗需要经常更新的原因。不幸的是抗体反应的特异性也为流感大流行的出现奠定了基础。上个世纪，已经发生了多次流感病毒或冠状病毒大流行，包括，A(H1N1)，A(H2N2)，A(H3N2)，A(H1N1)，非典新型冠状病毒和COVID-19，上述流感或冠状病毒感染分别发生在1918、1957、1968、2009、2001和2020年。有趣的是上述的流感病毒感染流行发展中，宿主患病的严重程度均差异巨大。有学者证明免疫细胞中，特别是T细胞感染与激活的差异，导致抵抗流感病毒的能力差异[Kelso,Anne.CD4+T cells limit the damage in influenza[J].Nature Medicine,2012,18(2):200-202.]。
有证据表明，T细胞可以介导交叉保护免疫，T细胞无法预防病毒感染，但它们可以通过识别与感染的上皮细胞或抗原递呈细胞表面上的人类白细胞抗原(HLA)分子复合的病毒蛋白(表位)片段来感知被感染的细胞。由于T细胞优先看到源自病毒保守内部蛋白的表位，因此交叉保护性免疫已被归因于预先存在的细胞毒性CD8+T细胞，这些细胞会杀死递呈这些保守表位的病毒感染细胞，减少因缺乏抗体保护的大流行病毒感染的时间和严重程度。
非洲猪瘟病毒具有独特的性质，导致不会从感染中恢复过来的猪中诱导中和抗体，进一步的抵抗病毒的复发。未中和抗体的存在导致了非洲猪瘟可能存在ADE(抗体依赖性增强)效应，即抗体的存在不仅不阻止病毒的感染，反而加剧的病毒的感染以及有关疾病的病理进程。同时未中和抗体 的存在导致，受到减毒疫苗免疫保护猪中持续的抗原阳性。在综述[Dixon L K,Islam M,Nash R,et al.African swine fever virus evasion of host defences[J].Virus research,2019.]中，作者描述分析了非洲猪瘟病毒的免疫逃脱策略，SFV pA179 L Bcl-2家族蛋白结合并抑制一些仅BH3的结构域促凋亡蛋白。pA224 L IAP家族蛋白结合并抑制caspase 3并激活NF-kB信号传导，从而增加抗凋亡基因(包括cFLIP，cIAP2和c-rel)的表达。上述机制的存在导致非洲猪瘟病毒能够抑制感染细胞的凋亡从而保证病毒的继续复制，而却通过TNF-α诱导未感染的淋巴细胞的凋亡，急性ASFV疾病的主要特征就是淋巴组织和血液中B和T淋巴细胞的大量凋亡。
由于抗生素的发现，人们对于细菌感染已经具有了非常好的临床治疗手段，而对于病毒，目前还没有很好的手段去治疗。目前人类对于病毒的治疗药物主要分为两类，M2离子通道阻断剂以及神经氨酸酶抑制剂，M2离子通道阻断剂存在整体的病毒抵抗效应以及神经系统的副作用导致临床应用不是很理想，而神经氨酸酶抑制剂虽然能诱导病毒，但是效果比较弱。近年来出现了大量的病毒爆发，禽流感病毒、非洲猪瘟病毒、非典型肺炎病毒，这些病毒的毒理学后果非常严重，对于患者或患病动物医生也无法拿出良好的治疗手段。不仅新病毒无良好的治疗手段，很多长期存在的病毒人们也束手无策，包括登革热病毒、艾滋病病毒等等。治疗病毒最佳方案就是预防，也就是疫苗，通过人体的免疫系统达到预防病毒的效果，以上事实也说明了对于病毒的治疗，开发以抗生素那种直接杀死或抑制病原体的药物的思路其实是事倍功半。
人们需要以新的思路来开发抗病毒药物，利用人体免疫系统达到治疗病毒感染就是其中重要的方向，特别是针对NO和免疫的有关药物，以实现通用性强的抗病毒效果。
一氧化氮气体无色无味，能溶于水，醇类，脂肪。上世纪80年代以前,一氧化氮只是一种普通而无用的化学气体,人们只知道它存在于汽车尾气和某些化学过程的气体污染物中。在1980年之前的27中，人们发现了内皮细胞产生一种物质(称为“内皮源性舒张因子”)，1986年Ignarro提交的第一篇实验论文，声称内皮源性舒张因子(EDRF)为一氧化氮。这些成果激起了人们极大的热情来关注和研究NO。它快速的进出细胞，传导信号调节血管扩张，神经传导，大脑发育甚至学习与记忆，它能够强化免疫，杀死部分外来微生物，它能够降低血压，预防中风，心脏病，肿瘤，老年痴呆。
NO是由L-精氨酸在一氧化氮合酶(NOS)催化下，经过NADPH还原而成，一氧化氮合酶可以分为内皮型一氧化氮合酶(eNOS)、诱导性一氧化氮合酶(iNOS)、神经型一氧化氮合酶(nNOS)。它们分别在人体不同的组织细胞中，分别参与了心、脑血管系统的调节、免疫调节、神经系统的调节。
参与免疫的NO可以由多种免疫细胞产生(树突状细胞，NK细胞，巨噬细胞，嗜酸粒细胞和中性粒细胞)，在iNOS表达时，可产生大量的NO，作为人体主动防御的机制。有证据表明NO能够抑制病毒的复制，相关机制包括导致病毒刺突蛋白棕榈化减少，抑制病毒蛋白酶，阻碍病毒蛋白和核酸合成。
一氧化氮合酶是一种二聚体，其在氧化条件下会解偶联，从而导致原本合成NO的反应路径转变为产生O2-，NO3-(PON)等活性氧自由基(ROS)。而NO本身也可以与活性氧自由基(ROS)反应生成活性氮(RNS)。
NO在体内跟超氧阴离子迅速反应生产过氧亚硝酸。在酸性条件下，又会迅速分解产生羟基自由基。过氧化亚硝酸是氧化性极强的物质，能够导致蛋白质硝基化、导致DNA链断裂。种种原因导致生物体内产生很多氧化性的自由基，既包括活性氧也包括活性氮。这些自由基的存在相当程度的破坏了过往的平衡。这些自由基中，影响最大的当属过氧亚硝基阴离子(PON)。其生产途径主要是一氧化氮跟超氧阴离子反应获得。
PON在人体内的作用大部分都是负面的。包括而不限于：
1.氧化作用：PON本身是一种强氧化剂。在酸性条件下迅速分解出二氧化氮和羟基自由基。羟 基自由基是更强的氧化剂，几乎可以氧化降解所有有机物。在生命体内，PON能够跟很多酶、蛋白质、细胞因子等的铁/硫中心，巯基，脂质等反应造成氧化损害，造成细胞功能损伤和凋亡；还能够降低谷胱甘肽清除自由基的机制，造成恶性循环。PON氧化作用可能引起各种疾病发生，如急性慢性炎症，败血症，外伤性局部缺血，动脉硬化，神经再生性失调等。
2.硝基化作用：PON能够跟蛋白质中的酪氨酸反应生成硝基酪氨酸而影响蛋白质的功能，造成DNA断裂等后果。
3.影响能量代：酶蛋白在氧化作用和硝基化作用下活性降低。如线粒体的ATP合成酶，乌头酶活性受到抑制，导致能量减少。PON是多聚ADP-核糖合成酶的强烈激活物。该酶激活后会启动无效的修复循环，导致能量池迅速耗尽。细胞代谢和膜的完整性被破坏，导致细胞死亡。
4.干扰钙的运转：Na+/Ca2+交换蛋白的巯基被氧化，发生功能障碍，导致细胞内钙超载，造成功能障碍。
当然，可耐受剂量的PON也表现出正面的作用，比如抵抗病毒，病菌，病原虫，癌细胞等对人体的危害。
NO这个1992年的明星分子，实际上在生命体内无处不在。它是免疫系统的信使，调节血流，神经传导，大脑发育等方面起到重要的作用；它可以杀死病菌，病毒，病原虫，癌细胞，是非特异性免疫非常重要的组成部分。被NO杀死的外来微生物或者异常细胞自溶后可以释放大量的抗原物质，启动特异性免疫。NO还会使机体释放出很多细胞因子比如白细胞介素，干扰素，肿瘤坏死因子TNF，集落刺激因子CSF等，调节免疫应答。
NO跟超氧阴离子等自由基反应生成过氧化亚硝酸盐(PON)，氧化性极强，具有特别的硝基化能力，积累到一定程度就会造成炎症，释放出影响病理进程的细胞因子。PON通过蛋白质硝基化破坏蛋白质功能，使DNA断裂，促使病毒变异，破坏免疫平衡，唤醒原癌基因，促发癌症。
NO参与免疫调控，急性炎症反应是一个复杂但高度协调的事件序列，涉及分子，细胞和生理学改变，其中如果宿主对感染的反应失调，进一步的产生异常免疫反应，从而产生以器官功能障碍的综合征即为脓毒症(Sepsis)。脓毒症的治疗的研究反映了我们人类对病理生理学和宿主-微生物相互作用的理解的进步。早期人们主要关注在微生物及其致病性上，在20世纪80年代，随着分子克隆的实施和人类炎症基因的测序，研究脓毒症更专注于宿主对入侵病原体的反应。
根据2016年脓毒症3的国际会议(The Third International Consensus Definitions for Sepsis)将脓毒症定义为由于宿主对感染的反应失调，而威胁宿主生命的器官功能障碍，其临床表现为发烧，呼吸加快，意识水平改变和血压低，并且伴随该病相关的症状比如肺部感染导致的肺炎、肾脏感染、尿路感染等。
尽管人类对脓毒症起源，进展的认识有了极大的提高，但是脓毒症的死亡率依然十分高，根据文章[Hotchkiss R S,Moldawer L L,Opal S M,et al.Sepsis and septic shock[J].Nature reviews Disease primers,2016,2(1):1-21.]所述，对高收入国家的数据进行的初步推断表明，全球每年发生3150万例脓毒症和1940万例严重脓毒症，每年可能有530万人死亡。在许多情况下，尤其是在患有慢性疾病(例如癌症，充血性心力衰竭和慢性阻塞性肺疾病)的患者中，官方的死亡记录通常报告的是潜在疾病，而不是直接的死亡原因(脓毒症)，这可能使得脓毒症死亡率明显被低估。这些数字只是估计，因为有关的发病率和低收入、中等收入国家的脓毒症死亡率记录仍然匮乏。
炎症是宿主对病原体入侵的防御反应，因此临床上优先采用的清除病原体的治疗方案是，使用抗生素药物或抗病毒药物来减少病原体抗原的外来刺激。病毒感染性疾病发展到免疫失调阶段，就会发生严重的炎症。一些终止炎症或者抗炎的治疗会减少炎症区域的巨噬细胞数量，提高免疫或是降低免疫反应的决策往往是困难的。常见的抗炎药物有非甾体抗炎药，糖皮质激素等。在严重炎症发生时，临床上往往使用糖皮质激素，然而对脓毒症来说，皮质醇的使用却没有实质益处，根据一 项随机对照试验[Annane D,Cariou A,Maxime V,et al.Corticosteroid treatment and intensive insulin therapy for septic shock in adults:a randomized controlled trial[J].Jama,2010,303(4):341-348.]表明氟氢可的松并没有降低脓毒症患者的死亡率。而之后的萃取分析[Wang C,Sun J,Zheng J,et al.Low-dose hydrocortisone therapy attenuates septic shock in adult patients but does not reduce 28-day mortality:a meta-analysis of randomized controlled trials[J].Anesthesia&Analgesia,2014,118(2):346-357.]也说明氢化可的松并不能降低严重感染患者或脓毒症的死亡率，目前临床上对严重感染患者，使用类固醇是有争议的。
过去20年内，人们一直尝试弄清维生素C与脓毒症之间的关系，脓毒症患者一般具有非常低的血清维生素C水平，人们认为危重病人低维生素C水平与血管加压，肾损伤，多器官功能障碍和死亡率增加相关。通过研究维生素C的作用机制，发现多种可能对脓毒症有作用的机制，包括抗氧化、抗炎、微循环、抗血栓、增加肾上腺敏感性、促进伤口愈合等。然而和预期不同的是，临床上使用维生素C未见具有显著的效果，根据[常雪妮,李敏,张正馨,et al.维生素C在脓毒症及脓毒性休克患者治疗中疗效的Meta分析[J].中华危重症医学杂志(电子版),2019,012(001):37-41.]统计，静脉输注维生素C不能改善脓毒症及脓毒性休克患者的病死率。
5-甲基四氢叶酸是叶酸在人体的活性形式，其并未观察到直接的抗病毒作用，目前叶酸与病毒的直接联系主要在叶酸受体α(FRalpha)，其已被描述为介导包括埃博拉等病毒进入细胞的因子。5-甲基四氢叶酸具有直接的抗氧化作用，通过二氢叶酸还原酶的作用促进BH2转化为BH4，众所周知BH4是eNOS必要的辅助因子。已经有人证明了5-甲基四氢叶酸通过促进eNOS,有利于预防和保护心血管疾病,但几乎没有研究报道5-甲基四氢叶酸在先天免疫激活的条件下对iNOS及巨噬细胞分泌NO的影响。
L-精氨酸是NO内源性合成的前体，在一氧化氮合成酶作用下，会反应产生NO和L-瓜氨酸，尽管L-精氨酸只有一小部分通过这种途径在体内代谢，但是急性炎症的状况下，由巨噬细胞的iNOS所产生的NO会大大超过人体的正常剂量。L-精氨酸虽然是非必需氨基酸，可以在在脯氨酸，谷氨酰胺或谷氨酸的代谢途径中内源性合成(通过全身蛋白质降解过程)，在肾脏中，瓜氨酸通过精氨酸琥珀酸合酶和精氨酸琥珀酸裂合酶的作用而转化为精氨酸，然而当精氨酸内源性合成不足以满足生物体的代谢需求时，其在不同的病理生理条件下非常重要。
发明内容
本发明发现，5-甲基四氢叶酸在“药理”浓度下，具有与“营养支持”的低浓度不同的生理活性，含有5-甲基四氢叶酸的组合物具有治疗病毒感染的作用，进一步发现其对不同病原体，包括细菌、真菌等均具有治疗效果。本发明还发现，去氢坏血酸以及NMN的活性与5-甲基四氢叶酸类似。
基于以上发现，本发明提供如下技术方案：
一种能在动物体内产生安全量的一氧化氮，即控制或降低体内活性氮比例的药物组合物，该组合物使得体内产生的一氧化氮能够达到预防和治疗疾病所需的剂量。
本发明的药物组合物，包括NO减毒剂，和任选的NO增量剂，所述NO减毒剂选自在用药剂量下清除过氧化亚硝酸或其盐(PON)的抗氧化物质。优选的，所述减毒剂在不低于10μmol/L浓度下不抑制诱导性一氧化氮合酶(iNOS)的表达，例如不抑制LSP诱导下巨噬细胞的iNOS的表达。
本发明的NO减毒剂选自抗氧化物质，该物质不影响iNOS合酶的激活，且对过氧化亚硝酸盐选择性猝灭。示例性的，其选自下列物质中的一种或者几种：5-甲基四氢叶酸或其盐，去氢抗坏血酸，NMN。
本发明的NO增量剂选自酶生NO底物，所述酶生NO底物选自L-精氨酸或其盐、瓜氨酸或其盐或精氨酸生素。
本发明的药物组合物，包括5-甲基四氢叶酸或其盐和精氨酸或其盐。进一步的，可包括植物血 凝素。
本发明的药物组合物，所述5-甲基四氢叶酸单剂量不小于15mg,所述精氨酸单剂量不小于50mg。
本发明还提供上述药物组合物的用途，用于制备预防或治疗致病微生物感染导致的疾病的药物方面的用途。优选的，所述致病微生物感染为病毒感染。
根据本发明药物组合物的用途，其能够提高病毒感染宿主的T细胞水平，特别是CD4以及CD8T细胞，降低炎症因子的表达，从而用于抗病毒感染。
根据本发明药物组合物的用途，所述病毒为流感病毒、疱疹病毒、非洲猪瘟病毒、冠状病毒例如COVID-19。
特别的，本发明提供一种用于预防和治疗猪瘟的药物组合物，其包括NO减毒剂和NO增量剂，所述NO减毒剂选自5-甲基四氢叶酸或其盐，去氢抗坏血酸，NMN中的一种或者几种，所述NO增量剂选自精氨酸、瓜氨酸或精氨酸生素中的一种或几种。
根据本发明上述治疗猪瘟的组合物，其包括5-甲基四氢叶酸和精氨酸。进一步的，其可以包括植物血凝素，三者的质量比为2:8:1。
根据本发明上述治疗猪瘟的组合物，所述猪瘟为非洲猪瘟。
根据本发明上述治疗猪瘟的组合物，所述组合物能够提高病毒感染宿主的T细胞水平，特别是CD4以及CD8T细胞，降低炎症因子的表达，抗病毒感染。
根据本发明上述治疗猪瘟的组合物，所述组合物单剂量的活性成分为至少30mg/公斤，例如50mg/公斤。根据本发明药物组合物的用途，所述组合物用于制备预防和治疗感染导致的脓毒症、全身性炎症反应综合征的药物用途。
根据本发明药物的组合物，其包括包含5-甲基四氢叶酸或其盐和维生素C。优选的，所述5-甲基四氢叶酸钙与维生素C的质量比为2：1至5：1，例如3：1，4：1。
本发明还提供上述药物组合物的用途，所述组合物用于制备治疗由非感染因素引起的全身性炎症反应综合征、脓毒症的用途。
根据本发明药物组合物的用途，所述脓毒症由金黄色葡萄球菌、肺炎链球菌、绿脓杆菌、流感病毒感染所引起的。
根据本发明的药物组合物，可以由活性成分和药学上可接受的辅料制成，例如，所述药物制选自片剂、胶囊剂、冲剂、注射剂、外用膏剂或喷剂。
根据本发明的药物组合物，其是一种免疫佐剂。
本发明中，安全量的一氧化氮是指，一氧化氮中转变为以过氧化亚硝酸为代表的毒性自由基、活性氮的比例可控，能够满足使用一氧化氮预防和治疗疾病的安全需求。这些自由基在体内严重影响物质和能量的代谢，影响甚至破坏细胞和组织的功能，显著增加基因突变的几率，还是很多疾病发生的原因。
本发明的组合物通过控制毒性自由基，并有效地提升一氧化氮的产生量到能够满足预防和治疗疾病的需求。
本发明中，产生安全量一氧化氮的药物组合物，在多种疾病的治疗中具有应用潜力，本发明的组合物能够促进T细胞的增殖与活化，提高宿主在感染过程中的CD4和CD8细胞的水平，阻断CD4和CD8T细胞的凋亡，显著提高宿主的生存率，并且改善感染过程中的炎症反应。
本发明给感染流感病毒的小鼠使用含有5-甲基四氢叶酸、精氨酸组合物，得到了大比例痊愈的结果，而且病程显著缩短。
叶酸的主要功能为碳的传递者，参与DNA的甲基化，参与嘌呤和胸腺嘧啶的合成，进一步合成DNA和RNA。而病毒以DNA或RNA的结构，在宿主细胞中大量复制，充足的叶酸供给应该有利于病毒的复制与传播，而实验结果让人意外，5-甲基四氢叶酸联合一氧化氮增量剂，反而抑制了病 毒。本发明首次提出5-甲基四氢叶酸和精氨酸等组合物在微生物感染，特别是病毒感染的应用。
iNOS是免疫系统中生成NO的关键酶，现有技术已知，该酶氧化会导致二聚体解偶联，使生成NO的反应路径转变为生成自由基，活性氮的反应路径。5-甲基四氢叶酸是内源性的抗氧化剂，能够激活NADPH，从而达到很好的抗氧化效果，具有直接的抗氧化作用。本发明组合物能够使得机体在病原体感染下，产生NO的同时避免产生不利于机体的自由基，包括活性氧(ROS)以及活性氮(RNS)。本发明已经验证了，包括5-甲基四氢叶酸或其盐，去氢抗坏血酸，NMN在内的抗氧化剂能够消除过氧化亚硝酸盐，并不影响iNOS表达的功能。
本发明提供一种在体内产生足量一氧化氮的方法。本发明的组合物通过抑制过氧化亚硝酸盐的生成，且不抑制并诱导提升一氧化氮合酶的活性，并进一步增加酶生一氧化氮底物精氨酸及其前体的方式，使得体内产生足量一氧化氮。
本发明中，足量的概念是指在达到或者超过预防和治疗疾病所需要的一氧化氮的最低剂量。
本发明为提供足量的一氧化氮提供了系统的方案，可以根据需要选择和优化。为提高一氧化氮产量，组合物中还可以使用一氧化氮合酶诱导剂，比如植物血凝素。植物血凝素PHA是一种有丝分裂原，是一种高效安全的一氧化氮合酶诱导剂，提取于豆类植物的技术已经可以规模化生产。本发明的另一目的是提供多种上述安全的一氧化氮组合物的用途。
本发明组合物中的活性成份包括5-甲基四氢叶酸或其盐。所述盐选自但并不限于钙盐，精氨酸盐，氨基葡萄糖盐,钠盐。
在一种优选方案中，本发明每单剂量组合物中5-甲基四氢叶酸或其盐的用量为15mg以上，优选25mg以上，更优选50-1000毫克。
在一种方案中，组合物包含5-甲基四氢叶酸或其盐或去氢抗坏血酸或NMN，和精氨酸；每单剂量组合物中包含5-甲基四氢叶酸或其盐的用量为15mg以上，优选25mg以上；优选50-1000毫克；更优选50-500毫克。示例性的，精氨酸的用量为50-5000毫克，优选100-1000毫克。
在一种方案中，组合物包括5-甲基四氢叶酸或其盐和精氨酸以及植物血凝素PHA。每单位剂量组合物中包含5-甲基四氢叶酸或其盐的用量为15mg以上(折合为5-甲基四氢叶酸用量)，优选25mg以上；优选50-1000毫克；更优选50-500毫克；每单位剂量组合物中精氨酸的用量为50-5000毫克，优选100-1000毫克；每单位剂量组合物中植物血凝素用量为10-500毫克，优选20-100毫克。
所述药物制剂可以选自片剂、胶囊剂、冲剂、注射剂、外用膏剂或气体制剂。
发明详述
NO诱导的HIF-1α的稳定化和磷酸化的p53水平被活性氧减少[Thomas DD,Ridnour LA,Espey MG,et al.Superoxide fluxes limit nitric oxide-induced signaling.J Biol Chem.2006；281(36):25984–25993.]，事实上，添加抗氧化剂对亚硝化信号具有保护作用[Edirisinghe I,Arunachalam G,Wong C,et al.Cigarette-smoke-induced oxidative/nitrosative stress impairs VEGF-and fluid-shear-stress-mediated signaling in endothelial cells[retracted in:Rahman I.Antioxid Redox Signal.2013 Apr 2018(12):1535].Antioxid Redox Signal.2010；12(12):1355–1369.]，因此NO水平和随后的下游信号传导被活性氧调节，也是调节氧化还原信号的一个因素。
iNOS的表达需要同时激活STAT和NF-κB，NF-κB充当炎症的主要开关，与H 2O 2产生有关，NF-κB受到氧化还原调节。绝大部分的还原剂或抗氧化剂都会某种程度上上具有抗炎的作用，抑制NF-κB通路，能够抑制iNOS的表达，在我们的一个实施例中，我们比较了不同抗氧化剂对LPS诱导下的巨噬细胞表达iNOS的影响，结果表明5-甲基四氢叶酸、去氢抗坏血酸、BH 4、谷胱甘肽、烟酰胺单核苷酸在10μmol/L的浓度下对iNOS的表达几乎不影响。考察上述抗氧化剂对过氧化亚硝酸盐的反应性，结果表明5-甲基四氢叶酸、去氢抗坏血酸、NMN均具有较高的消除过氧化亚硝酸盐的能力。已经证明在低氧条件下，淋巴细胞的免疫功能被抑制并且凋亡率增加，由于缺乏活性氧，它阻碍了iNOS的合成，破坏了iNOS和a-actinin4的结合，阻止了iNOS附着在肌动蛋白细胞骨架 上。因此，抗氧化剂的存在可能会导致iNOS的下调。而本发明却发现，如下抗氧化剂：5-甲基四氢叶酸、去氢抗坏血酸、NMN具有独特的性质，它们在一定浓度下不会降低iNOS的表达，却具有较好的清除过氧化亚硝酸盐的能力。上述抗氧化剂均具有在抗原激活免疫后，不降低免疫应答能力，特别是不对感染过程中iNOS表达产生负面影响，且还会降低过氧化亚硝酸盐的产生，NO具有抑制细胞凋亡的作用，通过S-亚硝基化作用抑制caspases-8，caspases-9或caspases-3，而过氧化亚硝酸盐则会通过DNA损伤以及p53的上调促进细胞的凋亡。
NO对感染微生物具有直接和间接的作用，NO能够直接破坏病原微生物的酶结构，特别是[Fe-S]簇，在病毒感染中，表达NO能够抑制病毒的酶活性，抑制病毒的复制。NO的直接毒性尤其是细胞外抗病毒活性已经得到充分的证明，但是NO对免疫功能的调节的间接作用则复杂很多。有研究证明流感病毒感染的iNOS缺陷小鼠几乎没有肺炎的组织病理学证据，因此该学者认为宿主的iNOS可能比病毒复制对肺炎的贡献更大[Karupiah G,Chen JH,Mahalingam S,Nathan CF,MacMicking JD.Rapid interferon gamma-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice.J Exp Med.1998；188(8):1541–1546.]。在内毒素血症中，早期使用的iNOS抑制剂治疗的临床前模型结果令人失望[Hauser B,Bracht H,Matejovic M,et al.Nitric oxide synthase inhibition in sepsis？Lessons learned from large-animal studies[J].Anesthesia&Analgesia,2005,101(2):488-498.]迄今为止已经描述了有益的和有害的效果，人们疑惑NO是感染的积极因素还是消极因素。
人们观察到外源性的NO会抑制T淋巴细胞的增殖，外源性NO(即，NO不是由T细胞产生的本身)抑制增殖或甚至引起T细胞的死亡[Bogdan C.Regulation of lymphocytes by nitric oxide.[J].Methods Mol Biol,2011,677:375-393.]。缺乏重要抗氧化剂机制的小鼠(即GSNOR)由于过度的S-亚硝基化和淋巴细胞凋亡而在外周显示出T和B细胞的明显缺乏。在另一方面，少量的NO支T细胞亚群，尤其是Th1细胞和FoxP3的阴性调节性T细胞群，可有效抑制Th17细胞分化。此外，最近的研究表明，外源NO也调节Th9和Th17细胞。
在本发明的一个实施例中发现，细胞培养液中5-甲基四氢叶酸在15.625μm的浓度下，几乎不影响巨噬细胞NO的分泌，更为有趣的是，在未加LPS刺激时，5-甲基四氢叶酸在低浓度时，发现可促进NO的分泌。
本发明中筛选的NO减毒剂与NO增量剂的联合使用显示出能够显著增加抗原刺激后CD4+T细胞增殖的活性。现有研究以表明，病毒清除是由抗原特异性CD8+效应T细胞介导的，而记忆CD4+T细胞在维持CD8+T和B细胞记忆反应中起到重要作用[Stambas J,Guillonneau C,Kedzierska K,et al.Killer T cells in influenza[J].Pharmacology&therapeutics,2008,120(2):186-196.]。另外，近期还有研究表明，CD4+和CD8+T细胞均与肺炎的控制有关，并通过产生白介素10来限制过度的组织损伤。因此本发明的含有上述NO减毒剂与NO增量剂的药物组合物能够用于病毒清除和抗炎治疗中。
本发明中精氨酸作为NO的增量物，与5-甲基四氢叶酸联合使用时，产生了意想不到的抗病毒、治疗脓毒症的效果。在一个实施例中，本发明的组合物能够显著的刺激小鼠胸腺和脾脏的T细胞的增殖，施用精氨酸和5-甲基四氢叶酸的组合，相对于仅添加精氨酸，CD4细胞的增殖明显提高，说明组合物能够提高效应CD4+T细胞的增殖能力。正如背景技术中所讲，病毒特异性记忆CD4 +T细胞的数量可以预测人类感染流感病毒的严重程度，病毒特异性T细胞数量与疾病的严重程度成反比。因此本发明的组合物具有治疗流感病毒感染的潜力，可降低患病的严重程度。前已经知道，过氧化亚硝酸盐影响细胞的免疫反应，有研究支持过氧化亚硝酸盐阻止了抑制炎症反应和修复的反馈能力，容易使得宿主在感染中免疫失调。使用的组合物既能提高宿主免疫能力而且维持了炎症的负反馈机制，能够使得宿主能够抵御感染，特别是病毒感染。
现有的病毒感冒药，多数用于缓解症状、减轻感冒的痛苦，但是不能可靠且明显的缩短病程。而本发明的上述药物组合物对感冒治疗产生了颠覆性的效果：起效快，针对40多名试用者的回访结 果，服用组合物之后一般48小时内感冒症状消失，虽然没有进行双盲对照的临床实验，但是有关的试用组合物的回馈结果也超乎预期。
进一步的，本发明在动物模型中验证了组合物的抗病毒感染效果，结果表明组合物能够保护小鼠的免疫功能，减轻流感病毒感染肺部的病理状态，减轻肺组织损伤。5-甲基四氢叶酸与精氨酸的组合物能够明显降低感染所致的炎性因子的水平，显著降低感染后5天肺部病毒滴度，提示组合物具有一定的抗病毒作用。此外，组合物的使用能够显著提高感染小鼠脾脏及胸腺的CD4 +和CD8 +T细胞的水平，提示虽然组合物降低炎性因子但是未降低宿主的免疫力。肺组织切片的结果表明组合物能够减少肺组织损伤和炎症状态，在对于感冒病毒的宿主模型中显示了非常好的疗效。
最近的研究表明，NO能够促进T细胞受体(TCR)介导的免疫突触(IS)信号[Garcia-Ortiz A,Martin-Cofreces N B,Ibiza S,et al.eNOS S-nitrosylatesβ-actin on Cys374 and regulates PKC-θat the immune synapse by impairing actin binding to profilin-1[J].PLoS biology,2017,15(4):e2000653.]，IS对于调节T细胞活化、分泌和细胞间免疫信号通讯非常重要，这也是组合物能够显著提高T细胞数量的可能原因。
在本发明的一个实施例中，将组合物应用在非洲猪瘟病毒感染猪的治疗，获得了非常好的效果，显著提高了感染非洲猪瘟病毒的猪的存活率，进一步证明了组合物抗病毒的潜力。
此外，本发明发现，本发明上述组合物能够显著保护高剂量病毒的攻毒实验中宿主的生存，显示一定的治疗脓毒症的潜力。
在过去的三十年中，已经进行了100多次Ⅱ期和Ⅲ期临床试验，以测试各种新型药物和治疗干预措施，以期改善严重脓毒症和败血性休克患者的预后。所有这些努力最终都未能产出能够减少器官衰竭并改善脓毒症患者生存率的新型药物[Artenstein AW,Higgins TL,Opal SM.Sepsis and scientific revolutions.Crit Care Med.2013；41(12):2770–2772.]。所有这些研究都使用特定分子或途径的单一药物，由于涉及到非常复杂的免疫代谢途径，以及上千个可能靶点，以此思路筛选药物并不容易。
外源性精氨酸的补充在脓毒症的治疗中是存在争议的，由于之前人们认为NO介导的过氧化在脓毒症的病理发展起重要作用。有人假设，NO产生过程中的药学阻断剂是一种治疗脓毒症的可行策略，因此开发了NOS合酶抑制剂，但是回顾临床结果，有关抑制NOS的疗法总体上无益处。另一方面脓毒症患者精氨酸水平降低，但是增加NO的内源性供体可能导致氧化应激的增强的有害影响。本发明的组合物中5-甲基四氢叶酸与精氨酸的联合使用在临床前动物模型中取得意想不到的非常好的疗效。
本发明发现5-甲基四氢叶酸能够显著降低LPS诱导下的脓毒症小鼠的死亡率，提示5-甲基四氢叶酸对由于严重过敏导致的脓毒症的治疗可能有益。
本发明发现5-甲基四氢叶酸与精氨酸组合物能够显著降低由于感染微生物(例如，金黄色葡萄球菌)所导致的脓毒症小鼠的死亡率。脓毒症是一种高度致死性疾病，其特征在于广泛的细胞凋亡诱导的免疫细胞耗竭和之后的免疫抑制。本发明发现5-甲基四氢叶酸和精氨酸的组合可以显著提供宿主存活率，阻断CD4和CD8T细胞的凋亡，在多种细菌和病毒导致的脓毒症模型中均证明了组合物具有的疗效。
应该认识到，人体中的某些氧化性信号对感染性疾病是有益的，在保护性氧化信号和活性氧的有害作用之间存在微妙的平衡。本发明的组合物选用的抗氧化剂具有独特的性质，可以缓解病原微生物感染导致症状，且同时提高NO的水平，使得NO介导的病原体杀伤作用胜过NO触发的氧化应激机制。
T细胞确实在交叉保护中起着重要作用，在疫苗领域灭活的病毒疫苗往往针对某些病毒通透诱导特异性抗体来进行保护，而对增强交叉保护性T细胞反应的作用却很小，本发明组合物具有增强免疫反应的作用，在一个实施例中组合物的使用显著提高了接种狂犬疫苗后免疫动物的抗体水平。
本发明利用了人和动物体的免疫系统，这一天然而强大的抗病毒工具。非洲猪瘟这一烈性传染病，现有技术完全无能为力，致死率95％-100％，而使用本发明显示明确的疗效，显示组合物抗病毒感染的能力超乎预期。[Oura,C.A L.In vivo depletion of CD8+T lymphocytes abrogates protective immunity to African swine fever virus[J].Journal of General Virology,2005,86(9):2445-2450.]中报道了，减毒病毒分离株OUR/T88/3，使用该病毒作为疫苗后发现，无毒ASFV分离株OUR/T88/3的感染可以保护远亲猪免受葡萄牙ASFV强毒分离株OUR/T88/1的攻击。但是暴露于OUR/T88/3然后耗尽了CD8(+)淋巴细胞的猪不再能完全免受OUR/T88/1攻击。这表明CD8+淋巴细胞对ASFV感染的保护性免疫应答中起到了重要作用。
近年来，人们对NO在免疫力的作用认识已发生巨大的进步，除了直接对病原微生物的抑制外，还广泛调节免疫功能，除iNOS外，还必须将eNOS作为免疫条件NO的来源。因此对NO的多种功能和目标靶点的认识的补充使我们认识到针对NOS亚型的抑制或激活难以应用到临床实践，但是，本发明的组合物的临床前动物模型的结果令人鼓舞，显示NO在抗病毒和脓毒症治疗潜力。
本发明首次提出安全和足量一氧化氮应用于抗病毒感染疾病的概念，使用的组合物相互协同体现了很好的效果。在免疫系统中存在复杂的调控机制，多个信号通路或目标靶点存在有益或有害的作用，有关一氧化氮的研究论文也多达数万篇，但是研究的结论却不一致，相互矛盾，难以理清。有研究人员甚至将东方文明的“阴与阳平衡”的概念引入，已表达各种靶点机制的双刃剑的作用[Burke A J,Sullivan F J,Giles F J,et al.The yin and yang of nitric oxide in cancer progression[J].Carcinogenesis,2013,34(3):503-512.]。本发明从免疫的整体角度发明的组合物，构成十分显著的技术效果：1.可以显著提升宿主的免疫细胞水平，或者阻止免疫细胞的凋亡；2.有效降低炎症因子，降低炎症损伤；3.利用调节免疫功能来抗病毒，能够使宿主较好的抵抗二次感染；4.组合物中各个成分安全性较好。
名词解释：
NO，一氧化氮；
NOS，一氧化氮合酶；
SNO，安全的一氧化氮，指过氧化亚硝酸等毒性自由基含量可控的一氧化氮，能够满足使用一氧化氮治疗和预防疾病时的安全需求
PON，过氧化亚硝酸及其盐；
NO减毒剂:用以减少过氧化亚硝酸等毒性含氮自由基生成的还原性物质。
NO增量剂:产生NO的前体物质。分为能够在体内释放NO的化学物质以及产生酶生NO的精氨酸，精氨酸生素等物质。
NO合酶诱导剂:用以诱导产生NO合酶的物质
叶源酸：6S-5-甲基四氢叶酸钙。
本发明所述的盐，是指药学上可接受的盐。
附图说明
图1实施例7中各组体重变化曲线；
图2实施例7各组体温变化曲线；
图3实施例7各组采食量变化曲线；
图4实施例7各组饮水量变化曲线；
图5实施例7各组生存曲线；
图6实施例8中各种抗氧化剂在LPS诱导下对巨噬细胞表达iNOS的影响；
图7实施例9中各种抗氧化剂对过氧化亚硝酸盐清除作用；
图8实施例10中组合物对刺激CD4T细胞三天的增殖的影响；
图9实施例10中定量组合物对刺激CD4T细胞三天的增殖的结果；
图10实施例12中炎性因子检测结果；
图11实施例12中炎性因子检测结果；
图12实施例12中炎性因子检测结果；
图13实施例13中中脾脏指数及脾脏各免疫细胞总数变化；
图14实施例13中中脾脏指数及脾脏各免疫细胞总数变化；
图15实施例13中中脾脏指数及脾脏各免疫细胞总数变化；
图16实施例13中中胸腺指数及胸腺各免疫细胞总数变化；
图17实施例13中肺组织切片图；
图18实施例13中外周血炎性因子分泌变化；
图19实施例13中各组小鼠病毒滴度；
图20实施例17中脓毒症模型小鼠脾脏淋巴细胞计数图；
图21实施例24中组合物对家猪耳静脉血常规的LYMP/NEUP变化图。
具体实施方式
下文通过对本发明实施例的描述，更加详细地对本发明的上述及其他特性和优势进行解释和说明。应当理解，下列实施例旨在对本发明的技术方案进行示例性的说明，而并非旨在对由权利要求及其等价方案所限定的本发明保护范围进行任何限制。
除非另有说明，本文中的材料和试剂均为市售商品，或可由本领域技术人员根据现有技术制备。
实施例1胶囊
6s-甲基四氢叶酸钙       200mg
维生素c                 200mg
填充剂适量
粘合剂适量
崩解剂适量
实施例2胶囊
6s-甲基四氢叶酸钙      100mg
精氨酸                 400mg
填充剂适量
粘合剂适量
崩解剂适量
实施例3片剂
6S-5-甲基四氢叶酸钙   100毫克
植物血凝素            50毫克
精氨酸                400毫克
填充剂适量
粘合剂适量
崩解剂适量
实施例4片剂
6S-5-甲基四氢叶酸钙   200毫克
维生素C               600毫克
填充剂适量
粘合剂适量
崩解剂适量
实施例5片剂
6S-5-甲基四氢叶酸钙       400毫克
维生素C钠                 100毫克
1，6-二磷酸果糖           2毫克
填充剂适量
粘合剂适量
崩解剂适量
实施例6冻干粉针
6S-5-甲基四氢叶酸钙      800毫克
精氨酸                   3克
溶解，过滤，冻干即可
实施例7小鼠抗感冒试验
小鼠：Balb/c小鼠，雌性，6周龄，15-17g，25只。
A,给药组，感染给药，10只；
B,模型组，感染不给药，10只；
C,正常组，不感染不给药，5只。
感染方法：使用150μl 5％水合氯醛腹腔注射麻醉小鼠，PR8流感病毒(1×106pfu/mouse)滴鼻感染。
给药方法：5-甲基四氢叶酸钙蒸馏水配制浓度6mg/ml，每只小鼠200μl，1.2mg/每只，灌胃，本次实验感染32小时后给药。
感染当天开始称体重、体温、饮水量、食量。小鼠体重、食物和水摄取量每日固定时间测定一次；体温测定：感染后3天内每日测定2次，间隔12小时，从感染4天起，每日固定时间测定一次。实验直至感染后第15天，小鼠体重基本恢复。
结果：各组体重变化曲线见图1，体温变化曲线见图2，采食量变化曲线见图3，饮水量变化曲线见图4，生存曲线见图5。从各图可以看出，给药组的状况大为改善。其中附图1-4的纵坐标以第一天数值为1的相对比值。
实施例8组合物中减毒剂的筛选
一、实验材料
1、细胞株：巨噬细胞RAW264.7。
2、试剂：LPS(Sigma)；iNOS检测试剂盒(Stressgen)；MTT(Biotopped)。
二、实验方案
1、细胞培养：
小鼠巨噬细胞RAW264.7在37℃、5％CO 2培养箱用含10％FBS的DMEM高糖培养基培养。
2、加药处理：
调整细胞密度为5×10 4cell/mL，于96孔板中每孔加入100μL细胞悬液，置于CO 2培养箱中培养24h。
基于LPS诱导炎症模型：
LPS诱导：所有孔加入40μL LPS(至终浓度为0.1μg/mL)；
维生素C组：每孔维生素C，LPS(至终浓度为10μmol/L维生素C，0.1μg/mL LPS)；
维生素E组：每孔维生素E，LPS(至终浓度为10μmol/L维生素E，0.1μg/mL LPS)；
谷胱甘肽组：每孔谷胱甘肽，LPS(至终浓度为10μmol/L谷胱甘肽，0.1μg/mL LPS)；
5-甲基四氢叶酸组：每孔5-甲基四氢叶酸钙，LPS，5-甲基四氢叶酸钙至终浓度为10μmol/L，LPS至终浓度为0.1μg/mL LPS)；
去氢抗坏血酸组：每孔去氢抗坏血酸，LPS(去氢抗坏血酸至终浓度为10μmol/L，LPS 0.1μg/mL LPS)；
花青素组：每孔花青素，LPS(花青素至终浓度为10μmol/L，LPS 0.1μg/mL)；
姜黄素组：每孔姜黄素，LPS(姜黄素至终浓度为10μmol/L，LPS 0.1μg/mL LPS)；
白藜芦醇组：每孔白藜芦醇，LPS(白藜芦醇至终浓度为10μmol/L，LPS 0.1μg/mL)；
穿心莲内酯组：每孔穿心莲内酯，LPS(穿心莲内酯至终浓度为10μmol/L，LPS 0.1μg/mL)；
黄芩苷组：每孔黄芪苷，LPS(黄芪苷至终浓度为10μmol/L，LPS 0.1μg/mL)；
NMN组，每孔NMN，LPS(NMN至终浓度为10μmol/L，LPS 0.1μg/mL)；
四氢生物蝶呤组，每孔四氢生物蝶呤，LPS(四氢生物蝶呤，至终浓度为10μmol/L，LPS 0.1μg/mL)；正常组：每孔加入50μL完全培养基。
混匀，于CO 2培养箱中继续培养24h。
3、iNOS的检测
使用多克隆抗体抗人iNOS(Stressgen)通过ELISA测定巨噬细胞中的iNOS蛋白水平。使用自动流式细胞分析仪确定巨噬细胞的数量。
4、结果
结果见图6，结果表明5-甲基四氢叶酸、谷胱甘肽、NMN、四氢生物蝶呤在10μmol/L浓度下不影响iNOS的表达。
实施例9不同抗氧化剂清除过氧化亚硝酸盐的比较
取过氧化亚硝酸盐供体3-morpholino-sydnonimine(SIN-1)分别置于15只试管中，浓度为1μmol/L。分别向含有SIN-1溶液的试管中加入5-甲基四氢叶酸钙至终浓度为1μmol/L、10μmol/L、100μmol/L，分别向含有SIN-1溶液的试管中加入去氢抗坏血酸至终浓度为1μmol/L、10μmol/L、100μmol/L，分别向含有SIN-1溶液的试管中加入谷胱甘肽至终浓度为1μmol/L、10μmol/L、100μmol/L，分别向含有SIN-1溶液的试管中加入NMN至终浓度为1μmol/L、10μmol/L、100μmol/L，分别向含有SIN-1溶液的试管中加入四氢生物蝶呤至终浓度为1μmol/L、10μmol/L、100μmol/L。过氧化亚硝酸盐浓度用分光光度法测定，检测波长为302nm。
结果见图7，结果表明去氢抗坏血酸、5-甲基四氢叶酸、NMN均具有非常好的过氧化亚硝酸盐消除效果。
实施例10组合物介入下T细胞的增殖分化实验
颈椎脱臼法处死小鼠，无菌分离小鼠脾脏及淋巴结放于Hank’s液中，使用免疫微珠(CD4+细胞提取试剂盒；美国Miltenyi Biotec)从小鼠的脾脏和淋巴结中纯化CD4T细胞。在96多孔板上分别添加4μg/mL的小鼠CD3单抗以及2μg/mL的抗CD28(Biolegend公司)，并且添加Dulbecco的改良Eagle培养基(不含L-精氨酸)，微量青霉素，甘氨酸，10％的胎牛血清中。
组合物A组：细胞培养液中添加5-甲基四氢叶酸钙至终浓度10μmol/L，精氨酸至终浓度40μmol/L；组合物B组：细胞培养液中添加去氢抗坏血酸至终浓度10μmol/L，精氨酸至终浓度40μmol/L；组合物C组：细胞培养液中添加NMN至终浓度10μmol/L，精氨酸至终浓度40μmol/L；精氨酸组：细胞培养液中添加精氨酸至终浓度40μmol/L；空白组：使用初始细胞培养液(不含L-精氨酸)。
纯化的T细胞使用Cell Violet Trace Proliferation kit(Invitrogen)染色并培养三天，使用流式细胞仪分析测定增殖。
结果如图8，9。结果表明，经过筛选的组合物某种程度均能提高刺激后CD4细胞的增殖，显示 出能够提高感染中宿主的细胞免疫的能力。
实施例11感冒患者试用组合物临床预实验
制备400毫克/粒叶源酸(6S-5-甲基四氢叶酸钙)胶囊，给予感冒患者试用，表1中记录用药后各症状的消失时间(小时)。扁桃体炎，咽喉疼痛之外的其他症状都消失的，定义为基本康复；包括扁桃体炎，咽喉疼痛在内的全部消失的定义为完全康复。
表1感冒患者临床统计(症状消失时间，小时)
Figure PCTCN2020107411-appb-000001
Figure PCTCN2020107411-appb-000002
感冒患者试食处方GK301(叶源酸300毫克，L-精氨酸100毫克)胶囊，结果在表2中统计。
表2感冒患者临床分析(症状消失小时数)
Figure PCTCN2020107411-appb-000003
Figure PCTCN2020107411-appb-000004
对照表1可以看出，加入精氨酸缩短了病程。服用组合物的抗感冒效果优于单独的5-甲基四氢叶酸。但不论是表1还是表2数据均显示，服用5-甲基四氢叶酸后，患者的感冒几乎都在2天内痊愈。
实施例12 5-甲基四氢叶酸对部分炎性因子的影响及NO分泌的影响
一、实验材料
1.细胞株：巨噬细胞RAW264.7。
2.试剂：LPS(Sigma)；MTT(Biotopped)；叶源酸即为5-甲基四氢叶酸钙(连云港金康和信药业有限公司)；NO检测试剂盒(碧云天)
二、实验方案
1.细胞培养：小鼠巨噬细胞RAW264.7在37℃、5％CO 2培养箱用含10％FBS的DMEM高糖培养基培养。采用ELISA法检测上清液炎性因子(TNF-α、IL-1α、IL-6)。
2.加药处理：
1)调整细胞密度为2×10 5cell/mL，于96孔板中每孔加入100μL细胞悬液，置于CO2培养箱中培养24h。
2)叶源酸组每孔加入50μL叶源酸(至终浓度为15.625，62.5，250μmol/L)；
LPS+叶源酸组，每孔加入50μL LPS(至终浓度为0.1μg/mL)，培养箱中培养6h后，每孔加入10μL叶源酸(至终浓度为15.625，62.5，250μmol/L)；
LPS组，50μL LPS(至终浓度为0.1μg/mL)；
正常组每孔加入50μL完全培养基。
3)混匀，于CO2培养箱中培养24h。
在520nm的吸光度值用均数±标准差表示。计算NO分泌率＝(OD样品孔-OD空白孔)/(OD正常孔-OD空白孔)×100％，根据标准曲线计算NO分泌量。
三、实验结果
表3 NO检测试剂盒在520nm测定OD值，以均值表示(n＝6)
Figure PCTCN2020107411-appb-000005
Figure PCTCN2020107411-appb-000006
结果表明5-甲基四氢叶酸在15.625μmol/L的浓度下，并不会抑制LPS诱导的巨噬细胞表达NO。炎症因子结果见说明书附图10，说明书附图11，说明书附图12。
上述结果表明5-甲基四氢叶酸钙对巨噬细胞及LPS诱导下的炎性因子表达未有显著影响。
实施例13考察组合物不同剂量一次性给药对流感病毒感染小鼠的早期保护作用
1.1材料与方法
1.1.1小鼠
Balb/c小鼠，20只(每组5只)，雌性，6周龄，15-17g，购自维通利华实验动物公司；
1.1.2药物配制
使用去离子水溶解药物，现用现配，30分钟内使用。
1.1.3给药方法
G1组：空白对照组
G2组：模型组
G3组：低剂量给药组(5-甲基四氢叶酸钙：精氨酸＝1：4，0.173g/kg)
G4组：高剂量给药组(5-甲基四氢叶酸钙：精氨酸＝1：4，0.346g/kg)
低剂量组、高剂量组灌胃给药，模型组：只造模不给药，给与等体积去离子水。空白对照组：给与等体积的去离子水。一次性给药。
1.1.4感染方法
使用150μl 5％水合氯醛腹腔注射麻醉小鼠，PR8流感病毒(1×10 6pfu/mouse)滴鼻感染。
1.1.5小鼠处理方法
感染当天开始称体重，小鼠体重每天固定时间测定一次。感染后3天，眼眶取静脉血100ul，制备血清、冻存。感染后5天处死小鼠，取肺、胸腺、脾脏及外周血。
血：制备血清，一部分检测细胞因子(外检)，其余冻存。
肺脏：肺组织分为2份，一份(右侧肺叶)测定病毒滴度；一份(左侧肺叶的上尖端)固定，做石蜡包埋、切片并进行HE染色。
脾、胸腺：称重、拍照、细胞计数及免疫细胞染色。
1.1.6观察指标
体重变化，每天称量体重。留取样本：第5天血清样品用于检测炎性因子(使用Biolegend的LEGENDplex Mouse Inflammation Panel检测，外检)。肺脏：测量病毒滴度；做肺组织病理切片；检测肺组织中炎性因子的变化。胸腺：称重、拍照、胸腺总细胞计数、淋巴细胞染色(CD4+、CD8+T细胞)。脾脏：称重、拍照、脾脏总细胞计数、脾淋巴细胞染色分析(表面染色,B细胞、CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞、单核细胞、巨噬细胞、树突状细胞、中性粒细胞)。
1.2实验结果
1.2.1脾脏指数及脾脏各免疫细胞总数变化
见说明书附图13、14、15。
1.2.3胸腺指数及各免疫细胞总数变化
见说明书附图16。
1.2.2肺部病理变化
见说明书附图17。
1.2.6外周血炎性因子分泌变化
见说明书附图18。
1.2.7肺部病毒滴度(5dpi)的变化
见说明书附图19。
结果表明，在感染流感病毒后，除正常对照组外，其他各组小鼠脾脏淋巴细胞都减少、胸腺都缩小，高剂量组胸腺变化较小。脾脏和胸腺均属于免疫器官，与小鼠免疫功能有关，胸腺缩小是导致免疫功能低下的原因之一，提示组合物可能有助于保护免疫器官，对抵抗病毒感染导致得免疫低下有一定的保护作用。肺部病理切片经HE染色后，模型组及治疗各组均呈现程度相似的淋巴细胞浸润及肺部组织结构的改变，高剂量治疗组较模型组的肺组织损伤程度略轻。提示组合物有助于减轻流感病毒感染早期肺部的病理状态，减轻肺组织损伤。
感染流感病毒后，感染后5天模型组外周血的多种细胞因子显著升高，给药组可明显降低感染所致的炎性因子的分泌。说明药物干预能够有效减低炎性因子水平，可能有助于减弱炎性因子风暴，防治炎性因子风暴所致的肺损伤。感染后5天的肺部病毒滴度，治疗组与模型组相比均呈现不同程度的下降趋势。特别是高剂量治疗组，与模型组相比病毒滴度的下降已经接近统计学的显著性差异的临界值。提示组合物降低病毒滴度能够抑制病毒在体内的复制，有一定的抗病毒作用。
实施例14组合物对疱疹病毒I型脑炎小鼠的治疗
昆明小鼠60只，雄性，体重14～18g，HSV-1攻击Hela细胞，HSV-1在Hela细胞中培养48小时，收集病毒测定该病毒滴度，选择质量分数为100TCID 5010 -5病毒接种小鼠。小鼠分为对照组、模型组、生理盐水治疗组、阿昔洛伟治疗组(10mg/kg)、组合物组(5-甲基四氢叶酸14mg/kg、精氨酸50mg/kg、植物血凝素7m g/kg),对照组注射0.03ml无菌生理盐水，模型组和各治疗组注射HSV-1病毒液0.03ml后，灌胃给药，连用4天，观察各组死亡情况及其他变化。7天后，眼球取血0.5ml,35℃温箱保存2h，1000r/min离心5min,检测NO与1L-1β结果如下：
表4不同组死亡数及死亡率
Figure PCTCN2020107411-appb-000007
表5对照组、模型组、阿昔洛伟和一氧化氮组合物组小鼠血清NO、1L-1β水平
Figure PCTCN2020107411-appb-000008
Figure PCTCN2020107411-appb-000009
本实验表明组合物能够显著降低疱疹病毒感染下的小鼠死亡率，提高感染过程中小鼠NO的释放和降低炎症因子的水平。
实施例15 5-甲基四氢叶酸组合物抗脓毒症配方筛选
C57小鼠，雄性，6～8周龄，体重18～22g，观察动物的一般生理指标、体重和进食情况。适应性喂养一周。标准颗粒饲料饲养，自由饮水。自然昼夜光线照明，室温18～26℃，相对湿度40％～70％。LPS,购买于sigma公司,货号:L2880。
将49只雄性C57小鼠分为7组，6组给药组，1组模型组，每组7只，腹腔注射LPS13mg/kg(经预实验确定的剂量，由于20mgLPS剂量下无法观察120小时的情况，为了延长模型组存活时间，经预实验确认剂量为13mg/kg)。
A组，5-甲基四氢叶酸钙：维生素C＝3：1(折合人体用量为5-甲基四氢叶酸钙300mg，维生素C100mg)；
B组，5-甲基四氢叶酸钙：维生素C＝1：3(折合人体用量为5-甲基四氢叶酸钙100mg，维生素C300mg)；
C组，5-甲基四氢叶酸钙：维生素C＝1:12(折合人体用量为5-甲基四氢叶酸钙50mg，维生素C 600mg)；
D组，5-甲基四氢叶酸钙：维生素C＝12:1(折合人体用量为5-甲基四氢叶酸钙600mg，维生素C 50mg)；
E组，5-甲基四氢叶酸钙：维生素C＝4:1(折合人体用量为5-甲基四氢叶酸钙1200mg，维生素C 300mg)
F组，5-甲基四氢叶酸钙：维生素C＝3:1(折合人体用量为5-甲基四氢叶酸钙1200mg，维生素C 400mg
H组，模型组。
所有给药组均分3次：分别于造模后9小时给药一次(第一天晚上9点)，次日早晨再给药一次，第三日早晨再给药一次，共3次，给药体积一致。结果如下。
表6腹腔注射LPS后各时间点动物表现及死亡情况
Figure PCTCN2020107411-appb-000010
C组、D组在给药过程中，部分小鼠出现浑身发抖，明显精神萎靡等症状，E、F组状态最好，A、B组次之，144h后各组没有死亡动物。
结果表明不同剂量，不同配比的5-甲基四氢叶酸与维生素C组合物可不同程度抑制LPS诱导的小鼠死亡率，提高小鼠的存活率，口服灌胃给药即可达到良好的降低脓毒症模型小鼠死亡率的作用，其最佳比例为3：1。效果随剂量的加大明显改善，在折合人体用量1200mg的5-甲基四氢叶酸剂量下，与400mg维生素C作用能够对LPS诱导的脓毒症模型小鼠100％存活，具有极大的临床价值。
实施例16 5-甲基四氢叶酸组合物对金黄色葡萄球菌脓毒症模型小鼠的保护尝试
SPF级昆明种小鼠，体重20克左右，金黄色葡萄球菌取单个菌落接种于培养液中，37℃振荡培养过夜，收集菌液4000rpm离心3min，收集沉淀，用无菌生理盐水洗涤2次。菌液约(5×10 9CFU/ml，预实验表明腹腔注射菌液2ml，7天死亡率在90％以上)。
小鼠随机分为5组，雌雄各半，分组如下：
A组，高剂量组，5-甲基四氢叶酸钙：维生素C＝3：1(折合人体用量为1200mg/日即192mg/日)；
B组，中剂量组，5-甲基四氢叶酸钙：维生素C＝3：1(折合人体用量为600mg/日即96mg/kg/日)；
C组，低剂量组，5-甲基四氢叶酸钙：维生素C＝3：1(折合人体用量为300mg/日即48mg/kg/日)；
D组，联合治疗，5-甲基四氢叶酸钙：维生素C＝3：1(折合人体用量为600mg/日即96mg/kg/日)+ 苯唑西林30mg/kg/d模型组。
腹腔注射菌液后4h，开始给药，按上述剂量，分三次给药(0、2、4天)，隔一天施用一次。
表7金黄色葡萄球菌脓毒症模型的治疗
Figure PCTCN2020107411-appb-000011
实验结果令人惊奇，在LPS模型组小鼠上切实具有保护性的组合物，在葡萄球菌模型小鼠上不能减缓疾病，二者没有显著差异。
实施例17组合物C配方治疗脓毒症及抑菌试验
5-甲基四氢叶酸钙、精氨酸、植物血凝素，按:2:8:1比例混合，得到配方C的组合物。
健康ICR小鼠120只，体重18～24g，雌雄各半，取金黄色葡萄球菌，肺炎链球菌作为供试菌种，按体重随机分为正常组、模型组、组合物低剂量组(40mg/kg)、组合物中剂量组(80mg/kg)、组合物高剂量组(160mg/kg)、阿莫西林组(120mg/kg)。以上各组均按20ml/kg腹腔给药，每天1次，除正常组外，各组小鼠均以0.5ml/只腹腔注射金黄色葡萄球菌液(5×10 9CFU/ml)。(肺炎链球菌感染方式及分组情况与金黄色葡萄球菌相同)观察各组小鼠注入菌液后4天内死亡情况，比较组间差异，计算存活率。实验结果如下。
表8受试组合物对感染小鼠具有保护作用(
Figure PCTCN2020107411-appb-000012
n＝10)
Figure PCTCN2020107411-appb-000013
注：与模型对照组比较， *p＜0.05；与模型对照组比较， **p＜0.01
结果表明，对于有明确感染的模型动物，组合物应该加入L-精氨酸。
组合物能够显著提高宿主的存活率，为了进一步验证组合物对淋巴细胞的作用，开展了独立的实验，取ICR小鼠，分别注射0.5ml金黄色葡萄球菌菌液(5×10 9CFU/ml),其中取10只模型小鼠同时腹腔注射5-甲基四氢叶酸、精氨酸的组合物溶液20mg/kg，造模24小时后，处死小鼠，收集脾脏。脾脏总细胞计数、脾淋巴细胞染色分析(表面染色,B细胞、CD4+T细胞、CD8+T细胞、NK细胞。
结果见图20，结果表明组合物可以防止败血症中CD4和CD8T细胞的凋亡，败血症小鼠在造模24小时后，败血症诱导所有类型的免疫效应细胞的凋亡，组合物阻止了CD4T，CD8T细胞以及B细胞的凋亡，但没有阻止NK细胞的减少(n＝11)。这是迄今为止尚未报道组合物的作用，应强烈考虑组合物在脓毒症的治疗前景。
实施例18组合物体内对革兰氏阴性菌的抑制作用
5-甲基四氢叶酸钙、精氨酸、植物血凝素，按:2:8:1比例混合，经过总混后，得到配方C的组合物。
取50只BAL B/C雄鼠，体重18～22g，分为6组，每组8只，其中实验组为2组，其余为各种对照组，分别为组合物低剂量组(40mg/kg)、组合物高剂量组(80mg/kg)、正常组、模型组、青霉素组(450mg/kg)、美罗培南组(75mg/kg)。绿脓肝菌的菌液在LB固体培养基上画线培养后，挑取典型菌落接种于普通LB液体培养基中，经过37℃过夜振荡培养约12h后4000r/min离心3min，弃去上清液，生理盐水重悬菌体后待用。将致死剂量的绿脓杆菌菌液按500μL/只的剂量腹腔注射各实验组和三个对照组中BAL B/C雄鼠。实验组的BAL B/C雄鼠感染菌体30min后，组合物中剂量组、组合物高剂量组灌胃给药，青霉素组和美罗培南组同样灌胃给药，正常组灌胃纯净水。给药24h后，对各实验组和对照组的BAL B/C雄鼠进行二次给药，给药类型和剂量与第一次给药相同。BAL B/C雄鼠给药后每24h观察一次，记录存活情况，第15天处死所有动物。
结果表明，本发明的配方C组合物在动物体内能够起到对革兰氏阴性菌的抑制作用，且毒性小。本发明所述组合物可以使受到致死剂量绿脓杆菌感染的小鼠在14天保持100％的存活。美罗培南组小鼠在14天后存活率100％，而青霉素组全部死亡，模型组也全部死亡。
实施例19组合物治疗和预防给药对H1N1(FM1)流感病毒感染小鼠的死亡保护作用
1.1受试样品
组合物颗粒(5-甲基四氢叶酸钙:精氨酸＝1：4)，连云港金康和信药业有限公司
1.2试验动物
ICR小鼠，SPF级，体重13～15g，雌雄各半。由北京维通利华试验动物有限责任公司提供，许可证号：SCXK(京)2016-0006，动物合格证：1100111911082385。
1.3接种毒株
购买FM1毒株(浓度为100.TICD 50)。由本实验室传代，-80℃冰箱保存。
2试验方法及结果
2.1剂量设计
试验中受试物均采用1000倍FM1毒株稀释液进行小鼠滴鼻感染，组合物分高、中、低三个剂量组。另外，设置预防给药组，采用低剂量一次性给药，设置维生素组与中剂量组进行比较。
2.2菌液准备
取FM1毒株0.2ml，冻融后用生理盐水进行梯度稀释获得试验所需浓度(1000倍量)。
3.3动物感染量确定
将不同浓度的FM1病毒液分别滴鼻感染小鼠，每个浓度组10只，每只45μl，观察感染后12天内动物的死亡情况，选用引起小鼠80±5％死亡的病毒液浓度作为正式试验的感染浓度。结果见表9。
表9流感病毒FM1致小鼠死亡感染毒液浓度的确定
Figure PCTCN2020107411-appb-000014
Figure PCTCN2020107411-appb-000015
根据以上结果：正式试验用浓度为1000倍FM1稀释液，45μl/只，滴鼻感染。
4.1动物感染及分组
取ICR小鼠130只，动物按体重等级随机分为7组，分别为正常对照组、模型对照组、组合物高、中、低剂量组、组合物预防组、组合物后期治疗组。取10只做为正常对照组外，其余各组20只动物按45ul/只滴鼻感染H1N1流感病毒。感染后各给药组灌胃给药，0.1ml/10g。
4.2治疗给药及预防给药剂量设计
正常对照组：给与等量的生理盐水
模型对照组：给与等量的生理盐水
高剂量组：组合物(5-甲基四氢叶酸钙：精氨酸＝1：4)0.346g/kg体重，给药时间分别为感染后12h、24h各给药一次，第二次给药是第一次给药剂量的一半。
中剂量组：组合物(5-甲基四氢叶酸钙：精氨酸＝1：4)0.173g/kg体重，给药时间分别为感染后12h、24h各给药一次，第二次给药是第一次给药剂量的一半。
低剂量组：组合物(5-甲基四氢叶酸钙：精氨酸＝1：4)0.087g/kg体重，给药时间分别为感染后12h、24h各给药一次，第二次给药是第一次给药剂量的一半。
预防组：预防作用，即造模前12h一次性给予低剂量组用量，即组合物(5-甲基四氢叶酸钙：精氨酸＝1：4)0.087g/kg体重。
后期治疗组：组合物(5-甲基四氢叶酸：精氨酸＝1：4)0.173g/kg体重，给药时间分别为感染后12h、24h各给药一次，第二次给药是第一次给药剂量的一半。组合物(5-甲基四氢叶酸：精氨酸：维生素C＝3：12：1)0.173g/kg体重，给药两次时间分别为感染后第3天、第6天。
观察感染后14天内动物的死亡情况，计算死亡率、死亡保护率(对照组死亡率-实验组死亡率)/对照组死亡率。肺指数＝肺湿重(g)/体重(g)。结果采用组间比较X2检验和t检验进行统计学处理。结果见下表。
表10对初次感染FM1流感病毒致小鼠死亡的保护作用
Figure PCTCN2020107411-appb-000016
表11对FM1流感病毒致小鼠肺部炎症的影响
Figure PCTCN2020107411-appb-000017
Figure PCTCN2020107411-appb-000018
5.组合物对治疗后存活小鼠重复感染的死亡保护作用
在上述实验中，于给药后第15天，对于经过上述流感病毒感染或药物干预后存活的小鼠，将继续复感实验，采用同种流感病毒再次感染存活动物1次，观察再次感染7天内死亡情况，比较不同处理组对流感病毒再次感染的小鼠的死亡率和死亡保护率的影响。复感实验各组不进行药物干预。
表12组合物对复感FM1流感病毒的小鼠死亡保护作用
Figure PCTCN2020107411-appb-000019
结果表明组合物高剂量组，在初次感染和重复感染后对动物死亡均有保护作用。预防给药组也有一定的保护作用。提示组合物除了降低流感病毒导致的小鼠死亡率，对动物有较好的治疗和死亡保护作用外，其预防给药也显示出一定的保护作用，能够延长小鼠存活时间。
实施例20配方A组合物对内毒素引起的发热的抑制作用
配方A组合物的配制：5-甲基四氢叶酸钙与维生素C按1：1质量比混合，经三维混合总混后，制得配方A的组合物。
内毒素配制：根据以往报道，本实验经文献报道，预实验后将内毒素致热量确定为250ng/ml/kg，实验前用生理盐水配制。
家兔的选择：新西兰兔35只，体重2.0-3.0Kg，每日测肛温1次，连续2日，使兔适应此测温操作。选择体温范围在37.5-38.5℃，且体温波动在0.5℃以内者，用于实验。
每兔均从耳缘静脉注射内毒素，于注射后1小时，测肛温，根据体温变化均衡分组，分别为模型组、阳性药组、本发明药物组合物A配方大(40mg/kg)、中(20mg/kg)、小(10mg/kg)剂量组。各个给药组灌胃给药一次，2ml/kg，模型组在同等条件下给予蒸馏水。分别测定给药后0.5h、1h、1.5h、2h肛温。实验结果见表13。
表13本发明配方A组合物对内毒素致热家兔体温变化的影响(n＝6)
Figure PCTCN2020107411-appb-000020
结果显示，注射内毒素后，各组家兔体温均明显升高，用药后，各组家兔体温均有所下降，显示组合物具有一定的解热作用。
实施例21C配方体内抗肺炎支原体试验
肺炎支原体国际标准株(ATCCFH15531),购自美国菌种保藏中心。
C配方组合物，实验室自制，5-甲基四氢叶酸钙、精氨酸、植物血凝素，按:2:8:1比例混合，经过总混后，得到配方C的组合物。
BALB/C小鼠50只，雌雄各半，体重16-20g，购自广东省医学实验动物中心。
阳性药组，阿奇霉素分散片，哈药集团制药六厂，批号160303，规格，0.25g/片。
BALB/C小鼠适应性饲养一周后，分层随机分为5组，雌雄各半，分别为正常对照组、模型对照组、阳性药对照组(40mg/kg)、C组合物高剂量组(80mg/kg)、C(40mg/kg)组合物低剂量组。除正常对照组外，其余各组小鼠经乙醚麻醉后，滴鼻感染浓度为10 6CCU/ml的肺炎支原体(MP)菌液50μL，连续感染3天。之后开始灌胃给药，每日一次，连续10天。最后一次给药4h后，小鼠眼球取血处死，取肺脏、脾脏、胸腺称重后进行病理学观察，另取一小块肺组织，研磨后使用PCR定量检测MP的含量。结果如下。
表14对小鼠脾指数和胸腺指数的影响
Figure PCTCN2020107411-appb-000021
注：与模型对照组比较， *p＜0.05
与模型组比较，空白对照组小鼠脾脏指数具有显著性差异，各给药组小鼠脾指数具有显著差异，提示给药后，机体内MP被杀灭。对免疫器官刺激减轻，降低脾指数。
小鼠肺脏组织病理学检查，解剖时可见于正常组相比模型组肺部组织病变明显，肺部外观充血、水肿，并且在肺叶分布有不等的坏死灶，病理学检查可见病变主要在肺内，主要为间质性肺炎和细支气管肺炎，支气管有明显的淋巴细胞浸润，空白组肺组织基本正常；阿奇霉素对照组可见轻度的间质性肺炎；C组合物组炎症明显减轻，细支气管周围可见少许炎性细胞浸润，间质性肺炎的程度随着剂量的增加而逐渐减轻。结果表明，C组合物具有控制小鼠肺炎支原体感染的作用，使肺组织病 变情况减轻。
实施例22一氧化氮组合物作为免疫佐剂对狂犬病毒疫苗效果的影响
成年昆明小鼠30只，体重20～28g，雌雄各半够自新疆医科大学实验动物中心，狂犬病rSRV 9减毒口服冻干活疫苗(北京中联康生物科技有限公司)，一氧化氮组合物B配方制备：5-甲基四氢叶酸钙与精氨酸按质量比1：4混合，值得B配方组合物。将30只小鼠雌雄各半分成3组，每组10只。分别为空白对照组，病毒口服免疫组，B配方+病毒口服免疫组。分别于试验第1天，7天，14天口服对应的疫苗，免疫后0，14，21，35，42，70天眼眶采血300μL/只，静置1h后，5000r/min离心5min，吸取血清；同步采集小鼠粪便0.05g左右，放入PBS(pH值约7.4)500μL，粉碎形成浑浊液，离心吸取上清，于-20℃冰箱保存，血清IgG抗体采用ELISA检测试剂盒检测，粪便IgA抗体采用小鼠血清狂犬病特异性IgA抗体ELISA检测试剂盒检测。结果如下表。
表15各组小鼠初免后不同时间检测血清抗狂犬病特异性IgG水平(U/ml)
Figure PCTCN2020107411-appb-000022
结果表明，组合物B能够提高血清中IgG的抗体水平，口服疫苗与组合物C联用后，第14天既具有相当程度的抗体，免疫21天后，不同组之间抗体差异明显。
表16各组小鼠初免后不同时间检测粪便SIgA水平(U/ml)
Figure PCTCN2020107411-appb-000023
以上结果表明，配方B组合物能够，提高疫苗的免疫活性，在配方B组合物作为免疫佐剂的协同作用下，rSRV 9病毒口服减毒疫苗能够明显提高小鼠体内的抗体表达，具有降低免疫接种次数，提高免疫效果的作用。
实施例23配方C组合物应用在非洲猪瘟的治疗
江苏省动物疫病防控中心送检样品经中国动物卫生与流行病学研究中心确诊了一例非洲猪瘟，阳性样品来源于江苏省连云港赣榆区某养殖户，该户存栏生猪300头、发病130头、死亡120多头。对死亡病猪进行解剖，病理学检查发现：肺出血，间质性肺炎等症状，对脾脏解剖发现，脾脏严重肿大，严重的脾脏肿大7倍；胃部解剖发现胃浆膜面弥漫性出血；肾脏肿胀明显，符合非洲猪瘟的症状。
将取该户3头发病猪、及10头健康猪血液，3000r/min离心，取血清加有陶珠的Roche中，加入PBS缓冲液，使用病毒DNA试剂盒提取DNA，检测确定为ASFV非洲猪瘟基因II型，为2017年俄罗斯远东地区和东欧地区播放的病毒属。使用配方C组合物介入非洲猪瘟的治疗。
配方C组合物注射剂的制备：5-甲基四氢叶酸钙与L-精氨酸与植物血凝素按按2:8:1比例混合，经过总混后，得到配方C的组合物。配方C组合物经过灭菌处理后，经生理盐水溶解，过微滤膜过滤，活性炭吸附热源，之后制备成注射剂。
对18头发病初期的猪进行隔离，相关饲料和废水、粪便进行无害化处理。患病猪体温平均在40℃，部分病猪出现皮肤充血、紫绀，并且在耳部、腹下出现多处出血点或红点，全部病猪进食不正常，食欲不振。对病猪进行血液检测发现白细胞水平较正常猪下降。
根据以上情况，对18头病猪进行配方C组合物介入治疗，每头猪每天注射含有配方C的注射液，剂量为50mg/公斤，持续2天，期间检测各猪的体温。
结果如下：
表17 18头病猪7天存活率统计汇总
Figure PCTCN2020107411-appb-000024
结果表明：对非洲猪瘟患病猪具有超乎预期的效果，提示配方C组合物具有非常好的抗病毒效果。结果表明7天内仅有1头猪死亡，之后由于政策要求，所有感染猪进行处死，该养殖户其他染病猪均在发病后3至4天死亡。
非洲猪瘟死亡病例解剖检测
对病死猪进行解剖，发现病死猪脾脏严重肿大，而且脾脏充血，质地变脆，易碎，肺部出现出血的现象，肺部出血较大规模出血，肺组织观察定性为间质性肺炎；胃部也同样出血，胃浆膜面弥漫性出血；肾脏肿胀明显。
取血液，静置1h后，5000r/min离心5min，吸取血清。检测IgG抗体水平。血液加入Krebs-HEPES缓冲液，维持在37℃，静置30分钟，加入L-NAME(100μM)，使用电化学方法检测超氧化物，亚硝酸盐及NO的含量。
对治愈猪进行处死解剖进行病理学观察，发现治愈猪除了脾脏略微大之外，肺部局部有出血现象。取血液，静置1h后，5000r/min离心5min，吸取血清。检测IgG抗体水平。血液加入Krebs-HEPES缓冲液，维持在37℃，静置30分钟，加入L-NAME(100μM)，使用光化学方法检测亚硝酸盐及NO的含量。
结果如下：
表18病死组及治愈猪的生化指标
Figure PCTCN2020107411-appb-000025
结果显示，治愈猪体内存在大量的抗体，提升组合物C能够提高猪的免疫系统水平，起到抗病毒的效果。
并且还发现了病死猪的NO水平也并不低，但是其中的活性氮水平大大提高，这提示我们由病毒引起的急性症状及死亡与活性氮的水平有关，高强度的免疫系统反而加剧了个体死亡，这个在病毒的治疗过程中是常见的。对于某些病毒，免疫基因敲除小鼠的存活时间反而大大超过正常小鼠。因此我们推测非洲猪瘟的死亡与免疫系统过度反应有关，通过比较发现病死猪血液中RNS/NO的比值是治愈猪血液RNS/NO比值的3倍之多。
提示，本发明组合物能够通过降低RNS，而减少恶性病毒由于免疫过度表达而导致的死亡，并且维持免疫系统正常运作，达到清除病毒的目的。本发明组合物的实施效果大大超乎预期。
实施例24组合物对家猪免疫细胞的影响
取3只普通家猪，猪龄约3个月，体重25kg左右，分别给予含有5-甲基四氢叶酸的组合物，组合物的制备过程如下，5-甲基四氢叶酸钙与L-精氨酸与植物血凝素按1:4:0.1比例混合，得到药物组合物。服用组合物前取耳血，测血常规。之后每日按猪体重每公斤30mg口服组合物，分别在第一周，第二周取耳血，测血常规。血常规检测主要指标LYMP(淋巴细胞)、NEUP(中性粒细胞)。
血常规检测指标如下：
Figure PCTCN2020107411-appb-000026
免疫指数LYMP/NEUP较高的病人感染病毒症状会比较轻。在[Zhang B,Zhou X,Qiu Y,et al.Clinical characteristics of 82 death cases with COVID-19[J].MedRxiv,2020.]文章中对新冠肺炎患者的临床分析，死亡的患者基本都是淋巴细胞/中性粒细胞的比值较低。在图21中，表明了组合物具有提高LYMP/NEUP比值，能够限制感染病毒的症状的严重程度。一定程度说明组合物对病毒具有预防作用。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种用于预防和治疗猪瘟的药物组合物，其包括NO减毒剂和NO增量剂，所述NO减毒剂选自5-甲基四氢叶酸或其盐，去氢抗坏血酸，NMN中的一种或者几种，所述NO增量剂选自精氨酸、瓜氨酸或精氨酸生素中的一种或几种。
  2. 根据权利要求1的组合物，其包括5-甲基四氢叶酸和精氨酸。
  3. 根据权利要求2的组合物，其还包括植物血凝素。
  4. 根据权利要求3的组合物，5-甲基四氢叶酸：精氨酸：植物血凝素的质量比为2:8:1。
  5. 根据权利要求1的组合物，所述猪瘟为非洲猪瘟。
  6. 根据权利要求1的组合物，所述组合物能够提高病毒感染宿主的T细胞水平，特别是CD4以及CD8T细胞，降低炎症因子的表达，抗病毒感染。
  7. 根据权利要求1或权利要求4的组合物，所述组合物单剂量的活性成分为至少30mg/公斤，例如50mg/公斤。
  8. 权利要求1-7任一项的组合物在制备预防或治疗猪瘟的药物组合物中的用途。
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