CN114903973A - Respiratory tract mucosa immunity enhancing pharmaceutical composition - Google Patents

Abstract

The invention relates to a respiratory tract mucosa immunity-enhancing medicinal composition and provides a preparation method of the medicinal composition. The composition is further prepared into an aerosol inhalation preparation on the basis of two novel hexapeptide small molecules of peptide I and peptide II. The lysozyme activity and the S-IgA content in saliva of the model animal can be effectively improved through inhalation administration, and the mucosal immunity of the model animal is effectively improved. Can be used for preventing respiratory tract related infection and chronic diseases.

Description

Respiratory tract mucosa immunity enhancing pharmaceutical composition

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a respiratory tract mucosa immunity enhancing pharmaceutical composition and a preparation method thereof.

Background

Mucous membrane tissues such as respiratory tract, intestinal tract and genitourinary tract are stimulated by non-self substances and various microorganisms in the environment at any time. In order to resist the invasion of harmful substances, mucosal tissues pass through physical barriers and immune functions to protect the body's safety. Among these, mucosal immunity needs to be able to distinguish not only beneficial and harmful foreign substances but also symbiotic bacteria and invading pathogenic bacteria.

The respiratory system consists of two parts, the upper and lower respiratory tract. The upper respiratory tract, which is the initial path for ambient air to enter the lungs, is constantly exposed to various substances such as commensal bacteria, pathogenic bacteria, environmental chemicals, and allergen particles. In order to resist the invasion of external harmful substances, respiratory epithelial cells are tightly connected by means of the interaction between occludin, blocking protein and E-cadherin, so as to physically protect respiratory tracts. Airway epithelial tissue is rich in cilia and Mucin (mucn, MUC). Among the mucin families, respiratory-related mucins have membrane-bound and secreted MUC1, 4, 16, 2, 5AC, 5B, 19.

In the respiratory tract, a large volume of foreign substances is captured by mucin and then transported to the oral cavity by ciliary movement, and then discharged to the outside of the body by coughing. The epithelial cells secreted by the non-mucin can produce antibiotics and anti-inflammatory proteins, thereby playing a protective role; the airway epithelial basal cells have similar characteristics to stem cells or precursor cells, and can maintain the renewal of epithelial cells and the repair of epithelial tissues.

As early as 1979, John Bienenstock discovered adoptive transfer of mesenteric lymph node-derived IgA ⁺ B cells are able to specifically migrate to all mucosal-associated lymphoid tissues, including respiratory, intestinal and genitourinary mucosa, but not to mucosal-non-associated lymphoid tissues. He hereby presented the concept of a Common mucosal immune system, which presumably exists in the body as a widely distributed "organ", with the immune systems of the individual mucosal tissues being interrelated and interacting. However, the importance of this concept has not really come to be of interest until recently.

The research on mucosal immunity begins at the beginning of the 20 th century, and particularly refers to local immunity of mucosal surfaces of an organism, such as an intestinal tract, a respiratory tract, a genitourinary tract and the like which are directly communicated with the outside. The basis of mucosal immunity is the mucosal immune system widely distributed in the body, which is an immune response network composed of lymphoid tissues and immunocompetent cells in mucosal tissues, and mainly includes Mucosa-associated lymphoid tissues (MALT) and diffuse lymphoid tissues. The mucosal immune system is independent of, but inseparable from, the systemic immune system.

The Mucosal Immune System (MIS) is a local immune system, which is very important though limited, and provides protection in the eyes, oral cavity, respiratory tract, gastrointestinal tract, urogenital system, mammary gland, etc., and has a promoting effect on systemic immunity.

There are differences between macrophages, Dendritic Cells (DCs), T and B lymphocytes, Intrinsic Lymphoid Cells (ILCs), and other immune cell lines in the mucosal immune system, manifested as differences in number and phenotype, as well as heterogeneity in function.

The complex microbial flora colonizing the mucous membrane, the food or the environmental antigens such as antigens, exogenous substances and the like absorbed in the air enable mucous membrane immune cells to be specifically distributed on the whole body mucous membrane, participate in the uptake, processing and presentation of the antigens, generate cytokines of innate immunity and specific immunity, and induce and regulate acquired immune response.

The mucosal system is an important physiological barrier in the innate immune system of the body and is constantly exposed to a large number of antigens, most of which are harmless substances such as symbiotic bacteria or food, but few of which are harmful pathogens. More than 95% of infections of human bodies occur in mucous membranes or invade the body from the mucous membranes, and the mucous membrane injury and the disturbance of the immune function of the mucous membranes are important causes for opportunistic infections or autoimmune diseases. The mucous membrane is the largest immune organ of a human body and mainly plays a role by secreting Secretory immunoglobulin A (S-IgA), and the S-IgA can prevent the reproduction and invasion of microorganisms on the mucous epithelial layer; the number of lymphocytes is greater than the sum of other parts, and 60% of the cellular workplaces are located in the mucosa.

Mucosal immunity has a shared mechanism, and by a common mucosal immune system (common mucosal immune system), part of lymphocytes activated at one mucosal site can migrate to a distant place through the blood circulation of lymph fluid, and an immune response is spread to other mucosal sites, so that vaccination or administration at one mucosal site can prevent other mucosal infections.

Respiratory administration and oral administration are currently the major mucosal immune pathways, and many studies have also demonstrated that both uptake pathways induce a protective immune response. However, studies show that respiratory tract immunity is more effective than oral administration, because respiratory tract immunity has no loss of antigens, effective immune response can be induced only by fewer antigens, systemic IgG and respiratory tract S-IgA secretion can be induced, a far-end membrane region can be induced to generate S-IgA, and respiratory tract mucosa and related lymphoid tissues have important functions of limiting infection diffusion, and the immune response is an ideal part for vaccination and drug absorption.

The advantages of mucosal immunity of the respiratory tract are mainly:

the tissue of the respiratory mucosa near the nasopharynx part is distributed with Nasal Associated Lymphoid Tissue (NALT), which is collectively called body mucosa associated lymphoid tissue together with small intestine associated lymphoid tissue, bronchial associated lymphoid tissue and the like. Ciliated cells on NALT, mucosal goblet cells and some non-ciliated epithelial cells, which function similarly to micro-rugate cells (M cells) in the intestinal tract, are capable of taking up vaccines and microparticles (or drugs). Nasal Associated Lymphoid Tissue (NALT) is an important protective barrier for mucosal surfaces, and when drugs (mainly soluble antigens) are administered nasally, they readily cross the nasal mucosal epithelium and contact the inter-epithelial and submucosal lymphocytes to reach superficial lymph nodes, and the elicited mucosal and systemic immunity is critical to defending the body against pathogen invasion. After exposure to the vaccine, the local lymphoid tissue can produce various antibodies which are secreted into the secretory fluids, including the ability to produce secretory IgA with protective effects.

The nasal mucosa has abundant vascularity, and the surface of the epithelial cells of the nasal cavity is covered with a large amount of microvilli, so that the absorption area is increased, and the absorption of the medicament is facilitated.

The nasal cavity immune approach is closer to the natural infection process, can activate mucosal immunity and systemic immunity, and can simultaneously generate humoral and cell-mediated immune responses (IgA and IgG).

Compared with the injection way, the injection can ensure that the organism obtains long-term immunological memory and can resist the re-invasion of pathogens for a long time.

The nasal mucosa immunity also has a long-distance effect, namely after the nasal administration, the nasal mucosa immune mask not only can prevent upper respiratory tract infection, but also can obtain immune response at the far-end intestinal tract and reproductive mucosa, and can also prevent the infection of sexual diseases.

And sixthly, the nasal immunization does not use an injector or a needle, so that the pain and inconvenience caused by intramuscular injection inoculation are avoided, and the nasal immunization is easy to accept by people.

There is no evidence that the function of the lymphatic tissue of the nasal mucosa decreases with age, and the secretion capacity of the human is not obviously weakened with age, so that the immunity of the nasal mucosa is relatively less affected by age.

Mucosal immunity is impaired, causing a series of diseases, as is the case with respiratory mucosal disorders.

There are more than 2 million patients with Chronic Obstructive Pulmonary Disease (COPD) worldwide. COPD has become a public health problem of wide concern all over the world due to the characteristics of a large number of patients, long disease period, high mortality rate, heavy social and economic burden and the like. COPD patients are prone to respiratory tract infections which further exacerbate the disease. A number of recent studies have shown that a decrease in the defense functions of the mucosal epithelium and innate immune cells of the respiratory tract is a major cause of the predisposition to respiratory infections in COPD patients.

In smoking-induced COPD, activation of NF- κ B signaling pathways in airway epithelial cells is inhibited, thereby affecting the function of the innate immune response; the number of alveolar macrophages is obviously reduced, and the phagocytic function is weakened; dendritic Cells (DCs) exhibit an immature phenotype, with a low capacity to recognize and capture antigens; neutrophils mediate an immune response against the lung tissue itself, exacerbating airway obstruction; natural killer cells (NK) express reduced levels of IFN-. gamma.TNF-. alpha.and perforin, and the cell killing activity is reduced. Therefore, in a COPD treatment regimen, boosting the regulation and recovery of the innate immune system is crucial, able to reduce respiratory tract infections and ultimately slow the progression of the disease.

Asthma (Asthma) is another common respiratory disease, manifested as airway hyperresponsiveness. Asthma is divided into a number of types, the more common being allergen-induced asthma, which was thought in previous studies to be mainly caused by helper T cell 2 (Th 2) activation. However, the recent discovery of type II intrinsic lymphocytes (ILC 2s) has changed the awareness of asthma disease. Rag1 deficient in T and B cells ^－/－ In mice, the stimulation of egg protein (Ovalbumin, OVA) can also lead to the development of asthma disease, and the mechanism research finds that the activation of ILC2s is involved in the disease process. In the development of asthma, IL-33 and IL-25 secreted by the damaged airway epithelial cells can stimulate ILC2s to secrete Th2 type cytokines IL-13 and IL-5, thereby causing airway hyperresponsiveness. In humans, ILC2s appears as a lin ^- CD127 ⁺ CRTH2 ⁺ Phenotype, widely distributed throughout the body, long life cycle, but few. Bartemes et al found that blood-derived ILC2s in allergic asthma patients was able to produce large amounts of IL-13 and IL-5 under stimulation by IL-33/IL-2 or IL-25/IL-2, whereas blood-derived ILC2s in allergic rhinitis patients did not respond. This study suggests that ILC2s has a potential differentiation in the progression of human asthma.

Bacterial or viral infections are relatively common respiratory diseases. Recent studies found that there was a large accumulation of ILCs in the lungs of mice in a mouse model of influenza virus infection. In the absence of ILCs, airway epithelial homeostasis, lung tissue function, and airway repair following viral infection are affected; the transfusion of ILCs may restore these functions. This study indicated that ILCs are involved in the maintenance of respiratory homeostasis, but the subset of functional ILCs remains to be explored further. In addition to simple bacterial or viral infections, co-infection of bacteria and viruses is also common in respiratory diseases. Clinically, adoptive bacterial infection of influenza patients is common, and researches show that immunosuppressive effects induced by influenza virus infection are main causes of bacterial secondary infection. However, prior infection with bacteria protects the body against subsequent influenza infection.

Studies have shown that hypoimmunity of the respiratory mucosa is associated with recurrent episodes of Acute Upper Respiratory Infection (AURI). When AURI occurs, under the stimulation of pathogenic microorganisms, local mucosal immune response is activated, synthesis and secretion of S-IgA are promoted to be increased, S-IgA can neutralize virus and inhibit contact of pathogens and mucosal epithelial cells, and can act together with lysozyme and complement to cause bacterial lysis, so that the protective effect of upper respiratory mucosa is exerted. The reduction of the local S-IgA content of the respiratory tract and the activity of lysozyme are important conditions for the occurrence of AURI, and can cause the upper respiratory tract infection to be delayed and not cured, and further develop into bronchitis, pneumonia and even more serious systemic infection.

For the treatment of respiratory diseases, antibacterial, antiviral or hormonal drugs are commonly used. Wherein the antibacterial drugs are as follows: amoxicillin has strong capability of penetrating cell walls, is one of oral penicillins widely applied at present, and has main adverse reactions including anaphylactic reaction and digestive system symptoms. Antiviral drugs such as: ribavirin, an antiretroviral drug, is inhaled and can cause pulmonary function deterioration, bacterial pneumonia, pneumothorax, and cardiovascular reactions (blood pressure drop and cardiac arrest), among others. Meanwhile, respiratory diseases are treated by glucocorticoid (such as prednisone, prednisolone, methylprednisolone and the like) commonly, so that good effects of anti-inflammation, antitoxic and antishock can be achieved, but the side effects cannot be ignored: after long-term use, the medicine can cause metabolic disorders of water, salt, sugar, protein and fat, weaken the body resistance, hinder tissue repair, delay tissue healing and even inhibit the growth and development of children. In summary, the existing chemical drug therapy only aims at killing germs and viruses, does not consider lung qi of organisms, has obvious effect, but has great toxic and side effects, is easy to generate drug resistance, and can generate adverse effect on the health of patients after long-term application. The traditional Chinese medicine considers that the etiology and pathogenesis of respiratory diseases are caused by the pathogenic factors of exogenous pestilence caused by the deficiency of healthy qi of human body.

The "etiology and pathogenesis of diseases and pestilence disease" has a statement: "all diseases are caused by disharmony in the age, cold and warm, and grumpy, so qi changes and looks of the disease are easy to infect, even kill the gate, and extend to other people, so it is necessary to take medicine and prevent it by law. The number and toxicity of the grumpy can directly determine whether a person suffers from a disease or not, and meanwhile, the pestilence treatise clouds the original qi full, so that the pathogenic factors are not easy to enter, and the original qi is suitable for being multiplied by deficiency, respiration and external pathogens. "indicating that the deficiency of healthy qi is the basis of the disease caused by pathogenic factors, the healthy qi is called" healthy qi exists in the interior, and pathogenic factors cannot be dried ".

In summary, there is a need to develop a medicament for treating respiratory diseases, especially a medicament for enhancing the immunity of the respiratory mucosa, which takes into account the lung qi of the body and has a rapid onset of action. Has the functions of filling the essential qi quickly, storing the vital qi internally and keeping the pathogenic qi undried, namely enhancing the immunity of the respiratory mucosa.

Disclosure of Invention

The applicant has studied respiratory mucosal immune diseases and the related potential therapeutic drugs.

First, the present invention provides a pharmaceutical composition for treating respiratory mucosal diseases.

The therapeutic agent comprises both hexapeptide compounds as follows.

The hexapeptide sequence is as follows:

sequence I (peptide I): Ala-Cys-Gln-His-Cys-Ser

Sequence II (peptide II): Phe-Arg-Glu-His-Ala-Asp

The chemical structure of the sequence I (peptide I) is as follows:

the chemical structure of the sequence II (peptide II) is as follows:

the peptides I and II may also be basic salts thereof, preferably potassium, sodium, ammonium, preferably sodium.

The treatment medicine also comprises other pharmaceutically acceptable auxiliary materials, and is used for further preparing a pharmaceutical composition.

The pharmaceutically acceptable auxiliary materials include but are not limited to solvents, solubilizers, cosolvents, preservatives, flavoring agents, aromatics, mucilage agents, coloring agents, antioxidants, fillers, lubricants, glidants and wetting agents.

The solvent is one or more selected from purified water, ethanol, polyethylene glycol, glycerol, propylene glycol, dimethyl sulfoxide and N-methylpyrrolidone.

The solubilizer is selected from polysorbates or other surfactants.

The cosolvent is selected from salts of organic acids, such as sodium benzoate, sodium salicylate, sodium p-aminobenzoate, etc.

The preservative is selected from one or more of methylparaben, ethylparaben, propylparaben, benzoic acid, sorbic acid, benzalkonium bromide, benzalkonium chloride and chlorhexidine acetate.

The flavoring agent is one or more selected from sucrose, stevioside, saccharin sodium, aspartame, glycerol, sorbitol, and mannitol.

The aromatic is selected from aromatic volatile oils, such as lemon, cherry, fennel, and peppermint. Or essences such as apple essence, banana essence, etc.

The mucilage is selected from one or more of agar, gelatin, sodium alginate, acacia, sodium carboxymethyl cellulose and methyl cellulose.

The colorant is selected from one or more of hematoxylin, alkannin root, madder root, beet red, cochineal carmine, turmeric, gardenia, carotene, folium pini koraiensis, oriental blueberry leaf, caramel, iron oxide red and iron oxide yellow.

The antioxidant is one or more selected from vitamin C, sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin E, tert-butyl p-hydroxyanisole and tert-butyl p-hydroxytoluene.

The filler includes but is not limited to one or more of starch, starch derivatives, cellulose derivatives, mannitol and sorbitol.

The lubricant includes but is not limited to one or more of stearic acid, alkaline earth metal salts of stearic acid (e.g., magnesium stearate, calcium stearate), and sodium stearyl fumarate.

The wetting agent comprises one or more of sodium dodecyl sulfate, polysorbate 80 and poloxamer.

The pharmaceutical composition is a liquid formulation.

The pharmaceutical composition can also be a solid preparation

The pharmaceutical composition is administered by inhalation, preferably by pulmonary nebulization.

The pharmaceutical composition acts by enhancing respiratory mucosal immunity.

The respiratory mucosal disease includes, but is not limited to, respiratory mucosal infections, including but not limited to bacterial infections, fungal infections, viral infections; asthma; chronic obstructive disease of respiratory tract.

The present application further discloses methods for the preparation of peptide I and peptide II, as follows:

s1: swelling the 2-chlorotrityl chloride resin;

s2: connecting the 1 st amino acid of the polypeptide sequence with the 2-chlorotrityl chloride resin, blocking, and then deprotecting;

s3: linking the amino acid at position 2 of the polypeptide sequence through a condensation reaction under the action of an activating agent;

s4: repeating the steps of S2-S3, and sequentially connecting the amino acids in the polypeptide sequence until the sequence is finished;

s5: and (3) cutting the hexapeptide compound from 2-chlorotrityl chloride resin to obtain the hexapeptide compound.

The application further discloses a preparation method of the pharmaceutical composition.

Dissolving the peptide I, the peptide II and other pharmaceutically acceptable auxiliary materials in a solvent, filtering, filling and sterilizing to obtain the liquid preparation.

Mixing peptide I and peptide II with filler and disintegrant, granulating with purified water, drying, grading, mixing the obtained material with glidant, lubricant and wetting agent, tabletting, and coating to obtain oral solid preparation.

The beneficial effects of the present application are further illustrated by the following tests:

test one: in vitro antibacterial action of peptides I and II (see CN 103421084A)

The Minimum Inhibitory Concentrations (MIC) of peptide I and peptide II against bacillus subtilis, staphylococcus aureus and methicillin-resistant staphylococcus aureus were determined using the macrobroth dilution method.

(1) Materials:

bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus, MH broth.

(2) The method comprises the following steps:

a) preparation of test article stock solution

Preparing aqueous solution with the concentration of both the peptide I and the peptide II of 250 mu g/ml, and storing the aqueous solution in an environment at the temperature of 20 ℃ below zero for later use.

b) Preparation of culture Medium

21g of MH broth culture medium is weighed, dissolved in distilled water and fixed to 1L, and sterilized at 121 ℃ for 30 min.

c) Preparation of inoculum

3-5 colonies to be detected with similar shapes are picked by using an inoculating loop, inoculated in 4-5 ml of MH broth and incubated for 2-6 h at 35 ℃. Correcting the concentration of the enriched logarithmic phase bacterial liquid to 0.5 McLeod's turbidimetric standard with MH broth, wherein the concentration is about 1-2 × 10 ⁸ CFU/ml. The bacterial suspension was diluted 1: 100 with MH broth for use.

d) MIC assay

Taking 13 sterile test tubes (13 × 100mm), arranging in a row, adding 1ml MH broth into each tube except 1.6ml MH broth into the 1 st tube, adding the test substance into the 1 st tube, and storing0.4ml of solution (250 mu g/ml) is mixed evenly, then 1ml is sucked into the 2 nd tube, after mixing evenly, 1ml is sucked from the 2 nd tube to the 3 rd tube, and then diluted to the 11 th tube in a continuous multiple ratio way, and 1ml is sucked from the 11 th tube and discarded, wherein the drug concentration of the 1 st tube to the 11 th tube is 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19, 0.098 and 0.049 mu g/ml in sequence. Then 1ml of each of the prepared inocula was added to 1-11 tubes to give a final bacterial liquid concentration of about 5X 10 per tube ⁵ CFU/ml。

The drug concentrations from tube 1 to tube 11 were 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19, 0.098, 0.049, 0.024 μ g/ml, respectively, the 12 th tube was a negative control containing no antibacterial drug, and the 13 th tube was a blank control containing no antibacterial drug and no inoculum.

Plugging all 13 branches with plugs, and placing the mixture in a common air incubator at 35 ℃ for incubation for 16-20 h.

The negative control tube (tube 12) was checked for good bacterial growth while the inoculum was checked for subculture to confirm that it was not contaminated. And (4) observing by naked eyes, wherein the lowest concentration tube of the medicament has no bacteria growth, namely the MIC of the tested bacteria.

d) As a result:

the results of the in vitro antimicrobial activity assays for peptide I and peptide II showed that all inoculated tubes had bacterial growth. Namely, the peptide I and the peptide II have no inhibition effect on bacillus subtilis ATCC 6633, staphylococcus aureus ATCC 25923 and methicillin-resistant staphylococcus aureus, namely have no inhibition effect on gram-positive bacteria and gram-negative bacteria.

And (2) test II: peptide I and peptide II protected upper respiratory infection model mice by regulating mucosal immunity (refer to the implementation of the literature: Rena et al, research on the protection of upper respiratory infection model mice by regulating mucosal immunity, J.Chinese Experimental formulary, 2013,19(18), 174-177)

Test animals: healthy Kunming mice, male and female half respectively, clean grade, weight 18-22 g.

Test samples:

peptide group I: dissolving the peptide I with purified water to prepare 0.001g/ml peptide I solution;

peptide group II: dissolving the peptide II with purified water to prepare 0.001g/ml peptide II solution;

peptide I and peptide II groups: dissolving the mixed solution of the peptide I and the peptide II by purified water so that the concentrations of the peptide I and the peptide II in the solution are both 0.0005 g/ml;

first, influence on lysozyme Activity and S-IgA content in saliva of model mouse

Experimental mice were randomly divided into 5 groups, i.e., normal group, model group, peptide I group, peptide II group, and peptide I and peptide II groups, 10 mice per group. The normal group and the model group use purified water as a control, and the other three groups are all administered with the dose of aerosol inhalation for 20min once a day for three consecutive days. On day 3, except for the normal group, all the other groups of experimental animals are placed in a cold environment at the temperature of-20 ℃ for stimulation for 15min, the cold stimulation is copied to an upper respiratory tract mucous membrane hypoimmunity model, 1 time of medicine is immediately given (atomized and inhaled for 20min) after the model is made, 0.3 mL/mouse of pilocarpine injection with the concentration of 0.1 percent is injected subcutaneously into the mouse after 60min, and the saliva of the mouse is taken out by a liquid transfer gun to be stored in an Eppendorf tube at the temperature of-40 ℃ after 2 min. The lysozyme activity and the S-IgA content in the saliva of the mice are respectively determined.

The results show that the lysozyme activity and the S-IgA content in saliva of the model group mice are obviously lower than those of the normal group; peptide I only increased lysozyme activity in saliva of model mice; the peptide II can increase the S-IgA content in saliva of a model mouse and has a tendency of increasing lysozyme activity. And the peptide I and the peptide II can improve the lysozyme activity in saliva of a model mouse and can also improve the content of S-IgA. The results are shown in Table 1.

Table 1 effect of oral administration of peptide I and peptide II on lysozyme and S-IgA in saliva of model mice (n ═ 10)

Group of	Dosage (g/kg)	Lysozyme (U/mL)	S-IgA(ug/L)
				Normal group	/	88.04±28.70	35.16±5.46
Model set	/	40.82±25.65	28.41±8.72
				Peptide group I	Aerosol inhalation	70.42±22.16	29.52±9.16
Peptide group II	Aerosol inhalation	60.05±25.31	34.05±6.78
				Peptide I and peptide II groups	Aerosol inhalation	85.33±27.01	33.45±5.13

Second, the survival protection effect on streptococcus pneumoniae nasal drip model mice

The experimental mice were randomly divided into 6 groups, namely a normal group, a model group, an amoxicillin 1.04g/kg group (positive control group, oral administration), a peptide I group (aerosol inhalation administration), a peptide II group (aerosol inhalation administration), a peptide I and a peptide II group (aerosol inhalation administration)). The aerosol inhalation is carried out for 20min each time, once a day for three consecutive days. On day 3, except for the normal group, other animals replicated the mucous membrane hypoimmunity model (-15 min in cold environment at 20 ℃), and after 60min of molding, 0.05 mL/one streptococcus pneumoniae bacterial suspension was dripped into the nose, with the bacterial liquid concentration of 5 × 10 ⁹ CFU/mL (mortality rate about 90%), and 1 more administration (aerosol inhalation or oral administration) 60min after nasal drip. The mortality of animals 7d after infection was observed and compared among the groups.

The experimental result shows that compared with the model group mice, the death rate of the model mice can be reduced by singly administering the peptide I and the peptide II, and the death of the model mice can be more obviously protected by jointly administering the peptide I and the peptide II. The suggestion that the peptide I and the peptide II can obviously reduce the death rate of mice with mucosal immune hypofunction and nasal cavity infection streptococcus pneumoniae models, and are probably related to the improvement of the local mucosal immune level of respiratory tracts.

Table 2 peptide I and peptide II protect the survival of pneumococcal rhinorrhea model mice (n ═ 10)

Group of	Dosage (g/kg)	Death number (only)	Mortality (%)
				Normal group	/	0	0
Model set	/	10	100
				Amoxicillin group	1.04	5	50
Peptide group I	Aerosol inhalation	6	60
				Peptide group II	Aerosol inhalation	7	70
Peptide I and peptide II groups	Aerosol inhalation	5	50

Third, the survival protection function to the intraperitoneal injection pneumococcal model mouse

Experimental mice were randomly divided into 6 groups, namely a normal group, a model group, an amoxicillin 1.04g/kg group (positive control group, oral administration), a peptide I group (aerosol inhalation administration), a peptide II group (aerosol inhalation administration), a peptide I and a peptide II group (aerosol inhalation administration), and aerosol inhalation was performed for 20min, once a day for three consecutive days. On day 3, the other animals replicated a cold-induced mucosal hypoimmunity model, except for the normal group.

After the model is established, mice are adapted for 60min at room temperature, and are respectively injected with 0.5 mL/mouse of streptococcus pneumoniae suspension in the abdominal cavity, and the concentration of the bacterial liquid is 5 multiplied by 10 ⁶ CFU/mL (mortality rate about 90%), and 1 additional administration (aerosol inhalation or oral administration) 60min after intraperitoneal injectionAnd observing the death condition of the animals at 7d after infection, and comparing the death rate of the animals in each group.

The results show that neither peptide I nor peptide II alone or in combination did not reduce the mortality of the i.p. model mice, but that amoxicillin protected the model mice, as shown in table 3. Further, it is demonstrated that the effect of peptide I and peptide II on the prevention and treatment of upper respiratory tract infections is achieved by enhancing local immunity of the respiratory tract, and is not related to bactericidal or bacteriostatic action.

Table 3 protective effect of peptides I and II on the survival of mice model with s.pneumoniae intraperitoneal injection (n ═ 10)

Group of	Dosage (g/kg)	Death number (only)	Mortality (%)
				Normal group	/	0	0
Model set	/	10	100
				Amoxicillin group	1.04	6	60
Peptide group I	Aerosol inhalation	10	100
				Peptide group II	Aerosol inhalation	10	100
Peptide I and peptide II groups	Aerosol inhalation	10	100

In conclusion, cold stimulation can reduce the immune function of the respiratory mucosa of the mice. On the basis of the model, the influence of the peptide I, the peptide II and the compound peptide I and the compound peptide II on mucosal immunity is further investigated in the experiment. Research results show that the compound of the peptide I and the peptide II can obviously improve the S-IgA content and lysozyme activity in saliva of a model mouse; can reduce the death rate of mice with mucous membrane hypoimmunity and streptococcus pneumoniae inoculated by nasal cavities.

The survival protection effect of the compound of the peptide I and the peptide II on the mice infected with streptococcus pneumoniae by nasal drip is related to the improvement of the S-IgA content and the lysozyme activity in saliva of the mice. Meanwhile, the in vitro experiments of the peptide I and the peptide II have no antibacterial effect, and the combined abdominal cavity infection also has no antibacterial effect. The results show that the peptide I and the peptide II have the effect of preventing and treating the upper respiratory tract infection by enhancing the local mucosal immune function of the upper respiratory tract and improving the S-IgA content and the lysozyme activity instead of directly inhibiting or killing bacteria.

Abbreviations and meanings:

MUC: mucin, mucin

IgA is Immunoglobulin A

MALT, mucosae-associated lymphoid tissue

MIS: mucosal immune system, Mucosal immune system

DC: dendritic cells, dendritic cells

ILCs: lnnate lymphoid cells, indigenous lymphoid cells

S-IgA: secretogray Immunoglobulin A, Secretory Immunoglobulin

IgG: immunoglobulin G, Immunoglobulin G

NALT: nasopharynx-associated lymphoid tissue, nasal-related lymphoid tissue

NF-. kappa.B: nuclear factor kappa-B, nuclear factor kappa-B

NK cells: natural killer cells

IFN: interferon

TNF: tumor necrosis factor

Th2 cells: t helper 2cell, helper T cell 2

ILC2 s: type II innate lymphocytes

OVA: ovalbumin, Ovalbumin

IL: colony stimulating factor

AURI: acute upper respiratory infection, acute upper respiratory infection

Ala: alanine

Cys: cysteine

Gln: glutamine

His: histidine

Ser: serine

Phe: phenylalanine

Arg: arginine

Asp: aspartic acid

Fmoc-Pro-OH: fmoc protected proline

Detailed Description

The present invention is further illustrated by the following examples, which are illustrative of the present invention and are not to be construed as being limited thereto.

Example 1: synthesis of peptide I (reference: CN 108530519B)

The synthesis sequence is as follows: from the C-terminal to the N-terminal.

(1) Swelling resin: the 2-chlorotrityl chloride resin was placed in a reaction tube, and methylene chloride (15ml/g) was added thereto and shaken for 30 min.

(2) Grafting with the first amino acid: the solvent was filtered off with suction through a sand core, serine and 3-fold molar excess (compared to the molar amount of serine) of Fmoc-Pro-OH amino acid were added, dimethylformamide was added for dissolution, 10-fold molar excess of N, N-diisopropylethylamine (compared to the molar amount of serine) was added, and shaking was carried out for 60 min. Blocking with methanol.

(3) Deprotection: the sand core is filtered by suction, the dimethylformamide is removed, 20 percent piperidine dimethylformamide solution (15ml/g) is added for 5min, and the solution is removed, and then 20 percent piperidine dimethylformamide solution (15ml/g) is added for 15 min.

(4) And (3) detection: and (3) pumping out the piperidine solution, taking dozens of resins, washing with ethanol for three times, adding a ninhydrin detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction. Washing resin: dimethylformamide (10ml/g) was used twice, methylene chloride (10ml/g) was used twice, and dimethylformamide (10ml/g) was used twice.

(5) Condensation: the protected amino acid Fmoc-Pro-OH is in triple excess (compared with the molar amount of serine) and HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) is in triple excess (compared with the molar amount of serine), the protected amino acid Fmoc-Pro-OH and HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) are dissolved in a small amount of dimethylformamide, added into a reaction tube, and N, N-diisopropylethylamine is added in ten-fold excess (compared with the molar amount of serine) at once to react for 30 min.

(6) And (3) detection: taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin detection reagent for detection, heating the resins at 105-110 ℃ for 5min, and taking colorless negative reaction. Washing resin: dimethylformamide (10ml/g) twice, dichloromethane (10ml/g) twice, dimethylformamide (10ml/g) twice.

(7) Repeating the operations from (3) to (6), and sequentially connecting the amino acids in the sequence until the sequence is finished.

(8) The liquid was drained and the resin washed, twice with dimethylformamide (10ml/g), twice with methanol (10ml/g), twice with dimethylformamide (10ml/g) and twice with dichloromethane (10ml/g) for 10min each, and drained.

(9) Cleavage of the polypeptide from the resin: preparing cutting fluid (10/g) trifluoroacetic acid 95%; 1% of water; 2% of 1, 2-ethanedithiol; triisopropylsilane 2%, cleavage time: and (4) 120 min. Drying the cutting fluid with nitrogen as much as possible, washing with diethyl ether for six times, and volatilizing at normal temperature. The crude product is purified by high performance liquid chromatography. Collecting the target polypeptide solution, putting the target polypeptide solution into a freeze dryer for concentration, and freeze-drying the target polypeptide solution into white powder.

The resulting polypeptide ¹ The H NMR data are as follows:

¹ H NMR(500MHz,Chloroform-d)δ8.14(d,J＝8.2Hz,1H),8.05(d,J＝8.4Hz,1H),8.00– 7.95(m,2H),7.83(d,J＝9.3Hz,1H),7.50(dd,J＝5.9,1.6Hz,1H),6.98(dd,J＝4.9,1.7Hz,1H), 6.39(s,2H),5.08(d,J＝5.5Hz,2H),4.57(dt,J＝9.2,5.9Hz,1H),4.46(tt,J＝7.7,4.2Hz,2H), 4.19(dt,J＝9.3,4.6Hz,1H),4.08(dt,J＝8.6,5.5Hz,1H),4.02–3.91(m,2H),3.82–3.71(m, 2H),3.00(d,J＝2.2Hz,1H),3.01–2.94(m,1H),2.92(ddt,J＝6.9,4.2,2.6Hz,4H),2.69(dd,J＝ 5.9,4.8Hz,1H),2.33–2.16(m,2H),1.92(tdd,J＝8.3,5.5,2.9Hz,2H),1.66(t,J＝6.7Hz,2H), 1.32(d,J＝5.1Hz,3H)。

m/z:647.22

example 2 reference for the synthesis of peptide II: CN 108530519B)

The synthesis sequence is as follows: from the C-terminal to the N-terminal.

(1) Swelling resin: the 2-chlorotrityl chloride resin was placed in a reaction tube, and methylene chloride (15ml/g) was added thereto and shaken for 30 min.

(2) Grafting with the first amino acid: the solvent was filtered off with suction through a sand core, aspartic acid and a 3-fold molar excess (compared to the molar amount of aspartic acid) of Fmoc-Pro-OH amino acid were added, dimethylformamide was added to dissolve, then a 10-fold molar excess of N, N-diisopropylethylamine (compared to the molar amount of aspartic acid) was added, and shaking was carried out for 60 min. Blocking with methanol.

(3) Deprotection: the dimethylformamide was filtered off by suction through a sand core, 20% piperidine dimethylformamide (15ml/g) was added for 5min, removed and 20% piperidine dimethylformamide (15ml/g) was added for 15 min.

(4) And (3) detection: and (3) pumping out the piperidine solution, taking dozens of resins, washing with ethanol for three times, adding a ninhydrin detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction. Dimethylformamide (10ml/g) was used twice, methylene chloride (10ml/g) was used twice, and dimethylformamide (10ml/g) was used twice.

(5) Condensation: the protected amino acid Fmoc-Pro-OH is in triple excess (compared with the molar amount of aspartic acid) and HBTU is in triple excess (compared with the molar amount of aspartic acid), all dissolved in a small amount of dimethylformamide, added into a reaction tube, immediately added with N, N-diisopropylethylamine in ten-fold excess (compared with the molar amount of aspartic acid), and reacted for 30 min.

(6) And (3) detection: taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin detection reagent for detection, heating the resins at 105-110 ℃ for 5min, and taking colorless negative reaction. Washing resin: dimethylformamide (10ml/g) once, dichloromethane (10ml/g) twice, dimethylformamide (10ml/g) twice;

(7) repeating the operations from (3) to (6), and sequentially connecting the amino acids in the sequence until the sequence is finished.

(8) The solvent was drained and the resin washed twice with dimethylformamide (10ml/g), twice with methanol (10ml/g), twice with dimethylformamide (10ml/g) and twice with dichloromethane (10ml/g) for 10min each time, and drained.

(9) Cleavage of the polypeptide from the resin: preparing cutting fluid (10/g) trifluoroacetic acid 95%; 1% of water; 2% of 1, 2-ethanedithiol; triisopropylsilane 2%, cleavage time: and (4) 120 min. Drying the cutting fluid with nitrogen as much as possible, washing with diethyl ether for six times, and volatilizing at normal temperature. The crude product is purified by high performance liquid chromatography. Collecting the target polypeptide solution, putting the target polypeptide solution into a freeze dryer for concentration, and freeze-drying the target polypeptide solution into white powder.

The resulting polypeptide ¹ The H NMR data are as follows:

¹ H NMR(500MHz,Chloroform-d)δ8.47(d,J＝9.3Hz,1H),7.97(dd,J＝8.8,4.0Hz,2H), 7.88(dd,J＝8.2,6.4Hz,2H),7.61(t,J＝3.7Hz,1H),7.50(dd,J＝5.9,1.6Hz,1H),7.31–7.19 (m,5H),6.98(dd,J＝4.9,1.7Hz,1H),6.78(s,1H),6.26(s,2H),4.61(dt,J＝9.2,7.7Hz,1H), 4.56(dt,J＝9.2,5.8Hz,1H),4.53(s,1H),4.52(s,1H),4.26–4.17(m,2H),4.13(dt,J＝8.6,5.8 Hz,1H),3.88(p,J＝5.9Hz,1H),3.15(tdd,J＝5.0,3.8,0.9Hz,2H),3.09–2.94(m,4H),2.74–2.68(m,1H),2.71–2.66(m,1H),2.63(dd,J＝15.6,7.7Hz,1H),2.22–2.06(m,2H),1.83–1.57(m,5H),1.55–1.43(m,1H),1.42(s,1H).

m/z:773.35

example 3: aerosol inhalation solution comprising peptide I and peptide II

Raw and auxiliary materials	Formulation 1	Formulation 2	Formulation 3	Formulation 4
					Peptide I	/	/	3.85g	3.85g
Peptide II	/	3.85g	/	3.85g
					Benzalkonium chloride	0.1g	0.1g	0.1g	0.1g
Sodium dihydrogen phosphate	1.2g	1.2g	1.2g	1.2g
					0.1M sodium hydroxide	Proper amount of	Proper amount of	Proper amount of	Proper amount of
pH	6.0	6.0	6.0	6.0
					The purified water is fixed to the constant volume	100ml	100ml	100ml	100ml

The preparation method comprises the following steps:

1) taking purified water with the prescription amount of 70%, and sequentially dissolving benzalkonium chloride, sodium dihydrogen phosphate, peptide I and peptide II for later use;

2) adding 0.1M NaOH solution into the solution obtained in the step 1), and adjusting the pH value of the solution to 6.0;

3) adding the rest purified water into the solution obtained in the step 2), and fixing the volume to 100 ml;

4) filtering, filling and sterilizing the solution obtained in the step 3).

The above summary and examples describe the basic principles and main features of the present invention and the technical effects of the present invention, and it should be understood by those skilled in the art that the present invention is not limited by the above examples, and the above examples and descriptions only describe the best technical solution of the present invention, and the present invention can be subject to various changes and modifications without departing from the spirit and scope of the present invention, i.e. the pharmaceutical formulation compounded by peptide I and peptide II and the preparation process thereof fall within the scope of the claimed invention, which is defined by the appended claims and their equivalents.

Claims (10)

1. A respiratory tract mucosa immunity enhancing medicine composition comprises peptide I, peptide II and pharmaceutically acceptable auxiliary materials, and is characterized in that the chemical structures of the peptide I and the peptide II are as follows:

peptide I:

peptide II:

2. the pharmaceutical composition according to claim 1, wherein the peptides I and II are also basic salts thereof, preferably potassium, sodium, ammonium, further preferably sodium salts.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable excipient is one or more selected from the group consisting of solvents, solubilizers, preservatives, flavoring agents, fragrances, mucilages, colorants, antioxidants, fillers, lubricants, glidants, and wetting agents.

4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a liquid formulation.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a solid formulation.

6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered by inhalation, preferably by pulmonary nebulization.

7. Use of the pharmaceutical composition of claim 1 for the preparation of a medicament for enhancing the immunity of the respiratory mucosa.

8. Use of the pharmaceutical composition of claim 1 for the preparation of a medicament for the treatment of a respiratory mucosal disease.

9. The pharmaceutical composition of claim 8, wherein the respiratory mucosal disease comprises a respiratory mucosal infection, asthma, or chronic respiratory obstructive disease.

10. The pharmaceutical composition of claim 9, wherein the respiratory mucosal infection is one or more of a bacterial infection, a viral infection, and a fungal infection.