A respiratory mucosal immune enhancement pharmaceutical composition

Abstract

The present invention relates to a pharmaceutical composition for enhancing respiratory mucosal immunity and provides a method for preparing the pharmaceutical composition. The composition is based on two novel hexapeptide small molecules, peptide I and peptide II, and is further prepared into an aerosol inhalation preparation. Inhalation administration can effectively increase lysozyme activity and S-IgA content in the saliva of model animals, effectively improving the mucosal immunity of model animals. It has a preventive effect on respiratory tract infections and chronic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a respiratory mucosal immunity enhancing pharmaceutical composition and a preparation method thereof. Background Art

[0002] Mucosal tissues, such as those in the respiratory, intestinal, and urogenital tracts, are constantly exposed to environmental stimulation from non-self substances and various microorganisms. To combat the invasion of harmful substances, mucosal tissues protect the body through physical barriers and immune functions. Mucosal immunity must be able to distinguish between beneficial and harmful foreign substances and also differentiate between symbiotic bacteria and invading pathogens.

[0003] The respiratory system consists of the upper and lower respiratory tracts. As the initial pathway for ambient air to enter the lungs, the upper respiratory tract is constantly exposed to a variety of substances, including commensal bacteria, pathogens, environmental chemicals, and allergen particles. To resist the invasion of harmful foreign substances, respiratory epithelial cells rely on occludin, a protein that tightly connects to the airways through interactions with E-cadherin, providing physical protection. The respiratory epithelium is rich in cilia and mucin (MUC). Within the mucin family, respiratory-associated mucins include membrane-bound MUC1, 4, and 16, and secreted MUC2, 5AC, 5B, and 19.

[0004] In the respiratory tract, large foreign substances are captured by mucins and transported to the mouth through ciliary motility, where they are expelled from the body through coughing. Non-mucin-secreting epithelial cells can produce antibiotics and anti-inflammatory proteins, thereby exerting a protective effect. Basal cells of the respiratory epithelium have properties similar to those of stem cells or precursor cells, which can maintain epithelial cell renewal and epithelial tissue repair.

[0005] As early as 1979, John Bienenstock discovered that adoptive transfer of mesenteric lymph node-derived IgA + B cells can specifically migrate to all mucosa-associated lymphoid tissues, including the respiratory, intestinal, and urogenital mucosa, but not to non-mucosa-associated lymphoid tissues. He thus proposed the concept of a "common mucosal immune system," speculating that the mucosal immune system in the body might be a widely distributed "organ," with the immune systems of various mucosal tissues interconnected and influencing each other. However, the significance of this concept has only recently been recognized.

[0006] Research on mucosal immunity began in the early 20th century. It specifically refers to the local immunity of mucosal surfaces, such as the intestinal tract, respiratory tract, and urogenital tract, which directly connect the body to the outside world. The foundation of mucosal immunity is the mucosal immune system, which is widely distributed throughout the body. This immune response network is composed of lymphoid tissue and immunocompetent cells within the mucosal tissue, primarily including mucosa-associated lymphoid tissue (MALT) and diffuse lymphoid tissue. The mucosal immune system is independent of the systemic immune system, yet is inextricably linked to it.

[0007] The mucosal immune system (MIS) is a local immune system that is limited but very important. It provides protection in the eyes, mouth, respiratory tract, gastrointestinal tract, urogenital system, and mammary glands, and promotes systemic immunity.

[0008] There are differences among macrophages, dendritic cells (DCs), T and B lymphocytes, innate lymphoid cells (ILCs), and other immune cell lineages in the mucosal immune system, which are manifested by differences in quantity and phenotype as well as functional heterogeneity.

[0009] Environmental antigens such as the complex microbial communities residing in the mucosa, antigens in food or inhaled air, and exogenous substances cause mucosal immune cells to be specifically distributed throughout the mucosa of the body, participating in the uptake, processing, and presentation of antigens, and producing cytokines of innate immunity and specific immunity, inducing and regulating acquired immune responses.

[0010] The mucosal system is a crucial physiological barrier in the body's innate immune system, constantly exposed to a vast array of antigens. The vast majority are harmless substances like commensal bacteria or food, but a small number are harmful pathogens. Over 95% of human infections occur in the mucosa or invade the body through the mucosa. Mucosal damage and disruption of mucosal immune function are significant contributors to opportunistic infections and autoimmune diseases. The mucosa is the body's largest immune organ, primarily exerting its effects through the secretion of secretory immunoglobulin A (S-IgA), which prevents microbial proliferation and invasion in the mucosal epithelium. Its lymphocyte population outnumbers the rest of the body combined, with 60% of its cellular workplaces located in the mucosa.

[0011] Mucosal immunity has a shared mechanism. Through the common mucosal immune system, some lymphocytes activated at one mucosal site can migrate to distant sites through the lymphatic and blood circulation, spreading the immune response to other mucosal sites. Therefore, vaccination or medication at one mucosal site can prevent infections in other mucosa.

[0012] Respiratory and oral administration are currently the primary mucosal immunization routes, and numerous studies have confirmed that both routes of administration can induce protective immune responses. However, studies have shown that respiratory immunization is more effective than oral administration because it avoids antigen loss and requires less antigen to induce an effective immune response. In addition to inducing systemic IgG and respiratory S-IgA secretion, it can also induce S-IgA production in distal membrane regions. Furthermore, the respiratory mucosa and its associated lymphoid tissues have the important function of limiting the spread of infection, making them ideal sites for vaccination and drug absorption.

[0013] The main advantages of respiratory mucosal immunity are:

[0014] The nasopharyngeal tissues of the respiratory mucosa are lined with nasal-associated lymphoid tissue (NALT). These tissues, along with the small intestine-associated lymphoid tissue and bronchus-associated lymphoid tissue, are collectively referred to as mucosa-associated lymphoid tissue. The NALT is composed of ciliated cells, mucosal goblet cells, and some non-ciliated epithelial cells. These cells function similarly to microfold cells (M cells) in the intestine, capable of taking up vaccines and microparticles (or drugs). The NALT is a crucial protective barrier on the mucosal surface. When drugs (primarily soluble antigens) are administered nasally, they readily cross the nasal epithelium and come into contact with lymphocytes within the epithelium and submucosal compartments, reaching the superficial lymph nodes. The resulting mucosal and systemic immunity is crucial for defending against pathogen invasion. Following vaccine exposure, local lymphoid tissue produces various antibodies, which are secreted into the fluid, including secretory IgA, which has protective effects.

[0015] ② The nasal mucosa is rich in blood vessels, and the surface of nasal epithelial cells is covered with a large number of microvilli, which increases the absorption area and is conducive to drug absorption. Compared with gastrointestinal mucosal immunization, nasal administration of vaccines can reduce degradation by acids, enzymes, etc., is convenient to administer, and can produce effective immune responses.

[0016] ③The nasal immune pathway is closer to the natural infection process, which can activate both mucosal immunity and systemic immunity, and can produce humoral and cell-mediated immune responses (IgA and IgG) at the same time.

[0017] ④ Compared with the injection route, it can enable the body to acquire long-term immune memory and resist the re-invasion of pathogens for a long time.

[0018] ⑤Nasal mucosal immunity also has a "long-range" effect, that is, after administration through the nasal cavity, it can not only protect against upper respiratory tract infections, but also obtain immune responses in the distal intestinal tract and reproductive mucosa, and can also prevent the transmission of sexually transmitted diseases.

[0019] ⑥ Nasal immunization does not use syringes and needles, avoids the pain and inconvenience caused by intramuscular injection, and is easily accepted by people.

[0020] ⑦ There is no evidence that the function of nasal mucosal lymphoid tissue decreases with age, and the human secretion ability does not weaken significantly with age, so nasal mucosal immunity is relatively less affected by age.

[0021] Mucosal immune disorders can cause a series of diseases, and the same is true for respiratory mucosal disorders.

[0022] Chronic obstructive pulmonary disease (COPD) affects over 200 million people worldwide. Due to its high prevalence, prolonged course, high mortality rate, and significant socioeconomic burden, COPD has become a public health concern of widespread concern worldwide. COPD patients are prone to respiratory infections, which further exacerbate their disease. Recent studies have shown that decreased defense function of respiratory epithelial cells and innate immune cells is the primary cause of respiratory infection in COPD patients.

[0023] In smoking-induced COPD, activation of the NF-κB signaling pathway in airway epithelial cells is suppressed, thereby impacting the function of the innate immune response. The number of alveolar macrophages is significantly reduced, impairing their phagocytic function. Dendritic cells (DCs) exhibit an immature phenotype, with reduced ability to recognize and capture antigens. Neutrophils mediate an immune response against the lung's own tissue, exacerbating airway obstruction. Natural killer (NK) cells express decreased levels of IFN-γ, TNF-α, and perforin, weakening their cytotoxic activity. Therefore, strengthening the regulation and restoration of the innate immune system is crucial in COPD treatment, reducing respiratory infections and ultimately slowing the progression of the disease.

[0024] Asthma is another common respiratory disease, characterized by airway hyperresponsiveness. Asthma is divided into many types, the most common of which is allergen-induced asthma. Previous studies believed that it was mainly caused by the activation of T helper 2 cells (Th2). However, the discovery of type II innate lymphoid cells (ILC2s) in recent years has changed people's understanding of asthma. In T and B cell-deficient Rag1 － / － In mice, stimulation by ovalbumin (OVA) can also lead to the development of asthma. Mechanistic studies have found that the activation of ILC2s is involved in this disease process. During the development of asthma, IL-33 and IL-25 secreted by damaged respiratory epithelial cells can stimulate ILC2s to secrete Th2 cytokines IL-13 and IL-5, thereby leading to the development of airway hyperresponsiveness. In humans, ILC2s are lin - CD127 + CRTH2 + phenotype, are widely distributed throughout the body, have a long lifespan, but are small in number. Bartemes et al. found that ILC2s derived from the blood of patients with allergic asthma produced large amounts of IL-13 and IL-5 when stimulated by IL-33 / IL-2 or IL-25 / IL-2, whereas ILC2s derived from the blood of patients with allergic rhinitis did not respond. This study suggests that ILC2s undergo potential differentiation during the course of human asthma.

[0025] Bacterial or viral infections are relatively common respiratory diseases. The latest study found that in the mouse influenza virus infection model, a large number of ILCs accumulated in the mouse lungs. In the absence of ILCs, airway epithelial homeostasis, lung tissue function, and airway repair will be affected after viral infection; the transfer of ILCs can restore these functions. This study shows that ILCs are involved in the maintenance of respiratory homeostasis, but the subpopulation of ILCs that play a role still needs further exploration. In addition to simple bacterial or viral infections, co-infection of bacteria and viruses is also common in respiratory diseases. Clinically, adoptive bacterial infection is relatively common in influenza patients. Studies have shown that the immunosuppressive effect induced by influenza virus infection is the main cause of bacterial secondary infection. However, prior bacterial infection can protect the body against subsequent influenza virus infection.

[0026] Studies have shown that impaired respiratory mucosal immune function is associated with recurrent acute upper respiratory tract infections (AURIs). During AURIs, pathogenic microorganisms activate the local mucosal immune response, leading to increased synthesis and secretion of S-IgA. S-IgA protects the upper respiratory tract mucosa by neutralizing viruses and inhibiting contact between pathogens and mucosal epithelial cells. It also works with lysozyme and complement to lyse bacteria, thereby exerting a protective effect. Reduced local S-IgA levels and lysozyme activity in the respiratory tract are key factors in the development of AURIs, potentially leading to persistent upper respiratory tract infections and progression to bronchitis, pneumonia, and even more severe systemic infections.

[0027] For the treatment of respiratory diseases, antibacterial, antiviral or hormonal drugs are commonly used. Among them, antibacterial drugs such as amoxicillin have a strong ability to penetrate cell walls and are one of the most widely used oral penicillins. Their main adverse reactions include allergic reactions and digestive system symptoms. Antiviral drugs such as ribavirin are anti-non-retroviral drugs. Inhalation of the drug can lead to lung function deterioration, bacterial pneumonia, pneumothorax and cardiovascular reactions (decreased blood pressure and cardiac arrest). At the same time, respiratory diseases are often treated with glucocorticoids (such as prednisone, prednisolone, methylprednisolone, etc.), which can have good anti-inflammatory, anti-toxic and anti-shock effects, but their side effects cannot be ignored: long-term use can cause disorders in water, salt, sugar, protein and fat metabolism, weaken the body's resistance, hinder tissue repair, delay tissue healing, and even inhibit children's growth and development. Overall, existing chemical drug treatments often focus solely on eliminating bacteria and viruses, without considering the body's lung qi. While effective, these treatments can be toxic and have significant side effects, are prone to developing drug resistance, and can negatively impact patient health with long-term use. Traditional Chinese Medicine (TCM) believes that the etiology and pathogenesis of respiratory diseases stem from a deficiency of the body's vital energy, coupled with the influx of external pathogenic pathogens.

[0028] The Treatise on the Causes and Symptoms of Various Diseases and Epidemics states: “It is because of the disharmony of seasons and the irregularity of temperature and coolness that people are affected by the evil qi and become ill. The evil qi can then spread easily to others and even wipe out a whole family and spread to outsiders. Therefore, it is necessary to take medicine in advance and practice magic to prevent it.” It points out that the amount and toxicity of evil qi can directly determine whether a person will become ill and the degree of illness. At the same time, the Treatise on Epidemics says, “When the original qi is full, it is not easy for evil to enter. When the original qi is deficient, external evil will take advantage of it during a breath.” It points out that the deficiency of the body’s positive qi is also the basis for being affected by evil qi and becoming ill. As the saying goes, “When the positive qi is inside, evil qi cannot interfere.”

[0029] In summary, there is an urgent need to develop a drug for treating respiratory diseases that takes into account the body's lung qi and acts quickly, especially one that can enhance the immune system of the respiratory mucosa. This can quickly replenish the body's qi, retaining positive energy and preventing pathogens from invading, thereby enhancing the body's respiratory mucosal immunity. Summary of the Invention

[0030] The applicant has conducted research on respiratory mucosal immune diseases and related potential therapeutic drugs.

[0031] First, the present invention provides a pharmaceutical composition for treating respiratory mucosal diseases.

[0032] The therapeutic drug also includes the following two hexapeptide compounds.

[0033] The hexapeptide sequence is as follows:

[0034] Sequence I (Peptide I): Ala-Cys-Gln-His-Cys-Ser

[0035] Sequence II (Peptide II): Phe-Arg-Glu-His-Ala-Asp

[0036] The chemical structure of the sequence I (peptide I) is as follows:

[0037]

[0038] The chemical structure of the sequence II (peptide II) is as follows:

[0039]

[0040] The peptide I and peptide II may also be in the form of basic salts thereof, preferably potassium salts, sodium salts, ammonium salts, preferably sodium salts.

[0041] The therapeutic drug also includes other pharmaceutically acceptable excipients for further preparation into a pharmaceutical composition.

[0042] The pharmaceutically acceptable excipients include, but are not limited to, solvents, solubilizers, cosolvents, preservatives, flavoring agents, fragrances, mucilage agents, colorants, antioxidants, fillers, lubricants, glidants, and wetting agents.

[0043] The solvent is selected from one or more of purified water, ethanol, polyethylene glycol, glycerol, propylene glycol, dimethyl sulfoxide, and N-methylpyrrolidone.

[0044] The solubilizer is selected from polysorbates, polysorbates or other surfactants.

[0045] The cosolvent is selected from salts of organic acids, such as sodium benzoate, sodium salicylate, sodium p-aminobenzoate, etc.

[0046] The preservative is selected from one or more of methylparaben, ethylparaben, propylparaben, benzoic acid, sorbic acid, benzalkonium bromide, benzalkonium chloride and chlorhexidine acetate.

[0047] The flavoring agent is selected from one or more of sucrose, stevioside, saccharin sodium, aspartame, glycerol, sorbitol, and mannitol.

[0048] The fragrance is selected from aromatic volatile oils, such as lemon, cherry, fennel, and mint volatile oils, or essences, such as apple essence, banana essence, and the like.

[0049] The slurry agent is selected from one or more of agar, gelatin, sodium alginate, gum arabic, sodium carboxymethyl cellulose, and methyl cellulose.

[0050] The colorant is selected from one or more of hematoxylin, lithospermum root, madder root, beet red, cochineal red, turmeric, gardenia, carotene, pine needle orchid, blueberry leaf, caramel, red iron oxide, and yellow iron oxide.

[0051] The antioxidant is selected from one or more of vitamin C, sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin E, butylated hydroxyanisole, and butylated hydroxytoluene.

[0052] The filler includes but is not limited to one or more of starch, starch derivatives, cellulose derivatives, mannitol, and sorbitol.

[0053] The lubricant includes but is not limited to one or more of stearic acid, alkaline earth metal salts of stearic acid (such as magnesium stearate, calcium stearate), and sodium stearyl fumarate.

[0054] The wetting agent includes but is not limited to one or more of sodium lauryl sulfate, polysorbate 80, and poloxamer.

[0055] The pharmaceutical composition is a liquid preparation.

[0056] The pharmaceutical composition can also be a solid preparation

[0057] The pharmaceutical composition is administered by inhalation, preferably by pulmonary aerosol inhalation.

[0058] The pharmaceutical composition exerts its effects by enhancing respiratory mucosal immunity.

[0059] The respiratory mucosal diseases include, but are not limited to, respiratory mucosal infections, including but not limited to bacterial infections, fungal infections, viral infections; asthma; and chronic obstructive airway diseases.

[0060] The present application further discloses a method for preparing peptide I and peptide II, which is as follows:

[0061] S1: Swelling 2-chlorotrityl chloride resin;

[0062] S2: connecting the first amino acid of the polypeptide sequence to the 2-chlorotrityl chloride resin and blocking the connection, and then deprotecting the connection;

[0063] S3: connecting the second amino acid of the polypeptide sequence through a condensation reaction under the action of an activating agent;

[0064] S4: Repeat steps S2 to S3 to sequentially connect the amino acids in the polypeptide sequence to the end of the sequence;

[0065] S5: The hexapeptide compound is cleaved from the 2-chlorotrityl chloride resin.

[0066] This application further discloses a method for preparing the pharmaceutical composition.

[0067] Peptide I and peptide II and other pharmaceutically acceptable excipients are dissolved in a solvent, filtered, filled, and sterilized to obtain a liquid preparation.

[0068] Peptide I and peptide II are mixed with fillers and disintegrants, granulated with purified water, dried, and granulated. The resulting material is mixed with a glidant, a lubricant, and a wetting agent, tableted, and coated to obtain an oral solid preparation.

[0069] The following experiments further illustrate the beneficial effects of this application:

[0070] Experiment 1: In vitro antibacterial effects of peptides I and II (performed with reference to CN 103421084A)

[0071] The minimum inhibitory concentration (MIC) of peptide I and peptide II against Bacillus subtilis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus was determined using the constant broth dilution method.

[0072] (1) Materials:

[0073] Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus, MH broth medium.

[0074] (2) Methods:

[0075] a) Preparation of test substance storage solution

[0076] Aqueous solutions of peptide I and peptide II were prepared with a concentration of 250 μg / ml and stored at -20°C until use.

[0077] b) Preparation of culture medium

[0078] Weigh 21 g of MH broth medium, dissolve it in distilled water and make up to 1 L, and sterilize it at 121°C for 30 min.

[0079] c) Preparation of inoculum

[0080] Use an inoculating loop to pick 3 to 5 colonies of similar morphology to be tested, inoculate them into 4 to 5 ml of MH broth, and incubate at 35°C for 2 to 6 hours. After the growth, the logarithmic growth phase of the bacterial solution is calibrated with MH broth to a concentration of 0.5 McFarland turbidimetric standard, containing approximately 1 to 2 × 10 8 CFU / ml. Dilute the bacterial suspension 1:100 with MH broth for later use.

[0081] d) MIC determination

[0082] Take 13 sterile test tubes (13×100 mm) and arrange them in a row. Add 1 ml of MH broth to each tube except the first tube, which has 1.6 ml of MH broth. Add 0.4 ml of the test substance stock solution (250 μg / ml) to the first tube, mix well, then pipette 1 ml into the second tube. After mixing well, pipette 1 ml from the second tube into the third tube. Repeat this process of serial dilutions to the 11th tube, and discard 1 ml from the 11th tube. The drug concentrations in tubes 1-11 are 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19, 0.098, and 0.049 μg / ml, respectively. Then add 1 ml of the prepared inoculum to each tube 1-11, so that the final bacterial solution concentration in each tube is approximately 5×10 5 CFU / ml.

[0083] The drug concentrations in tubes 1 to 11 were 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19, 0.098, 0.049, and 0.024 μg / ml, respectively. Tube 12 was a negative control without antibiotics, and tube 13 was a blank control without antibiotics and inoculum.

[0084] All 13 tubes were plugged and placed in a 35°C ordinary air incubator for 16 to 20 h.

[0085] Check the negative control tube (tube 12) for good bacterial growth. Also, check the inoculum subculture to ensure it is not contaminated. Visually inspect the tube with the lowest drug concentration for the absence of bacterial growth, which is the MIC for the test bacteria.

[0086] d) Results:

[0087] The results of in vitro antibacterial activity assays of peptides I and II showed bacterial growth in all inoculated test tubes. In other words, peptides I and II had no inhibitory effect on Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, or methicillin-resistant Staphylococcus aureus, meaning they had no inhibitory effect on either Gram-positive or Gram-negative bacteria.

[0088] Experiment 2: Peptide I and peptide II protect mice with upper respiratory tract infection model by regulating mucosal immunity (refer to the literature: Lei Na et al., Study on the protection of upper respiratory tract infection model mice by regulating mucosal immunity with Jiebiao Fang, Chinese Journal of Experimental Traditional Chinese Medicine, 2013, 19(18), 174-177)

[0089] Experimental animals: healthy Kunming mice, half male and half female, clean grade, weighing 18-22 g.

[0090] Test sample:

[0091] Peptide I group: Dissolve peptide I in purified water to prepare a 0.001 g / ml peptide I solution;

[0092] Peptide II group: Dissolve peptide II in purified water to prepare a 0.001 g / ml peptide II solution;

[0093] Peptide I and Peptide II group: a mixed solution of Peptide I and Peptide II was dissolved in purified water so that the concentrations of Peptide I and Peptide II in the solution were both 0.0005 g / ml;

[0094] 1. Effects on lysozyme activity and S-IgA content in saliva of model mice

[0095] The experimental mice were randomly divided into 5 groups: normal group, model group, peptide I group, peptide II group, and peptide I and peptide II group, with 10 mice in each group. The normal group and the model group were treated with purified water as controls, while the other three groups were given a 20-minute aerosol inhalation once a day for three consecutive days. On the third day, except for the normal group, the experimental animals in the other groups were placed in a -20°C cold environment for 15 minutes to replicate the cold stimulation to create an upper respiratory mucosal immunodeficiency model. Immediately after modeling, the animals were given a single dose of medication (20-minute aerosol inhalation). 60 minutes later, the mice were subcutaneously injected with 0.1% pilocarpine injection (0.3 mL / mouse). 2 minutes later, the mouse saliva was collected with a pipette into an Eppendorf tube and stored at -40°C. Lysozyme activity and S-IgA content in the mouse saliva were measured.

[0096] The results showed that compared with the normal control group, the saliva lysozyme activity and S-IgA content of the model mice were significantly lower. Peptide I only increased the lysozyme activity in the saliva of the model mice, while peptide II increased the S-IgA content in the saliva of the model mice and showed an upward trend in lysozyme activity. Both peptides I and II increased both the lysozyme activity and the S-IgA content in the saliva of the model mice. The results are shown in Table 1.

[0097] Table 1 Effects of oral administration of peptide I and peptide II on lysozyme and S-IgA in saliva of model mice (n=10)

[0098] Group Dosage (g / kg) Lysozyme (U / mL) S-IgA (ug / L) Normal group / 88.04±28.70 35.16±5.46 Model Group / 40.82±25.65 28.41±8.72 Peptide Group I Nebulized inhalation 70.42±22.16 29.52±9.16 Peptide II group Nebulized inhalation 60.05±25.31 34.05±6.78 Peptide I and Peptide II Group Nebulized inhalation 85.33±27.01 33.45±5.13

[0099] 2. Protective Effect on the Survival of Mice Induced by Nasal Drops of Streptococcus Pneumoniae

[0100] The experimental mice were randomly divided into six groups: a normal group, a model group, an amoxicillin 1.04g / kg group (positive control, oral administration), a peptide I group (administered by nebulization), a peptide II group (administered by nebulization), and a peptide I and peptide II group (administered by nebulization). Nebulization was administered for 20 minutes once daily for three consecutive days. On the third day, except for the normal group, the remaining animals were subjected to a mucosal immunodeficiency model (stimulated in a -20°C cold environment for 15 minutes). Sixty minutes after modeling, 0.05 mL of a pneumococcal suspension (5×10 50 μg / mL) was administered intranasally. 9 CFU / mL (mortality rate of about 90%), and 60 minutes after nasal drip, the drug was administered once more (nebulized inhalation or oral administration). The mortality of the animals was observed 7 days after infection, and the mortality rate of the animals in each group was compared.

[0101] The experimental results showed that compared with the model group mice, the administration of peptide I and peptide II alone could reduce the mortality of the model mice, and the combined administration of peptide I and peptide II had a more significant protective effect against the death of the model mice. This suggests that peptide I and peptide II can significantly reduce the mortality of mice with mucosal immunodeficiency and nasal infection with Streptococcus pneumoniae, which may be related to their ability to enhance the local mucosal immunity level of the respiratory tract.

[0102] Table 2 Protective effects of peptide I and peptide II on survival of mice in the pneumococcal nasal drop model (n=10)

[0103] Group Dosage (g / kg) Number of deaths (individuals) mortality rate(%) Normal group / 0 0 Model Group / 10 100 Amoxicillin group 1.04 5 50 Peptide Group I Nebulized inhalation 6 60 Peptide II group Nebulized inhalation 7 70 Peptide I and Peptide II Group Nebulized inhalation 5 50

[0104] 3. Protective effect on survival of mice injected with pneumococcal bacteria

[0105] The mice were randomly divided into six groups: a normal group, a model group, an amoxicillin 1.04 g / kg group (positive control, oral administration), a peptide I group (administered by nebulization), a peptide II group (administered by nebulization), and a peptide I and peptide II group (administered by nebulization). Nebulization was performed for 20 minutes once daily for three consecutive days. On the third day, the remaining animals, except the normal group, were subjected to a cold-induced mucosal immunodeficiency model.

[0106] After the model was established, mice were acclimated to room temperature for 60 min and then intraperitoneally injected with 0.5 mL of Streptococcus pneumoniae suspension per mouse at a concentration of 5 × 10 6 CFU / mL (mortality rate of about 90%), and administer the drug again 60 minutes after intraperitoneal injection (nebulized inhalation or oral administration). Observe the death of animals 7 days after infection and compare the mortality of animals in each group.

[0107] The results showed that neither peptide I nor peptide II, administered alone or in combination, could reduce the mortality of mice injected intraperitoneally with Streptococcus pneumoniae, but amoxicillin had a protective effect on the model mice, as shown in Table 3. This further demonstrates that the effects of peptide I and peptide II in preventing and treating upper respiratory tract infections are achieved by enhancing local immunity in the respiratory tract and are unrelated to bactericidal or antibacterial effects.

[0108] Table 3 The protective effects of peptide I and peptide II on the survival of model mice injected with Streptococcus pneumoniae (n=10)

[0109] Group Dosage (g / kg) Number of deaths (individuals) mortality rate(%) Normal group / 0 0 Model Group / 10 100 Amoxicillin group 1.04 6 60 Peptide Group I Nebulized inhalation 10 100 Peptide II group Nebulized inhalation 10 100 Peptide I and Peptide II Group Nebulized inhalation 10 100

[0110] In summary, cold exposure can reduce respiratory mucosal immune function in mice. Based on this model, this study further investigated the effects of aerosolized inhalation of peptide I, peptide II, and a combination of peptide I and peptide II on mucosal immunity. The results showed that the combination of peptide I and peptide II significantly increased saliva S-IgA levels and lysozyme activity in model mice and reduced mortality in mice with mucosal immunodeficiency and nasal inoculation with Streptococcus pneumoniae.

[0111] The protective effect of the peptide I and peptide II combination on the survival of mice infected with Streptococcus pneumoniae via intranasal drip was associated with increased levels of S-IgA and lysozyme activity in the mice's saliva. Furthermore, peptides I and II showed no antibacterial activity in vitro and also lacked antibacterial activity against concurrent intraperitoneal infection. These results suggest that peptides I and II do not exert their effects through direct bacteriostasis or bactericidal action, but rather by enhancing local mucosal immunity in the upper respiratory tract, increasing S-IgA levels and lysozyme activity, thereby preventing and treating upper respiratory tract infections.

[0112] Abbreviations and meanings:

[0113] MUC: mucin

[0114] IgA: Immunoglobulin A, immunoglobulin A

[0115] MALT: Mucosa-associated lymphoid tissue

[0116] MIS: Mucosal immune system

[0117] DC: dendritic cell

[0118] ILCs: innate lymphoid cells

[0119] S-IgA: Secretory Immunoglobulin A, secretory immunoglobulin

[0120] IgG: Immunoglobulin G

[0121] NALT: Nasopharynx-associated lymphoid tissue

[0122] NF-κB: nuclear factor kappa-B

[0123] NK cells: natural killer cells

[0124] IFN: interferon

[0125] TNF: tumor necrosis factor

[0126] Th2 cells: T helper 2 cells, helper T cells 2

[0127] ILC2s: type II innate lymphoid cells

[0128] OVA: Ovalbumin, egg protein

[0129] IL: colony stimulating factor

[0130] AURI: acute upper respiratory infection

[0131] Ala: alanine

[0132] Cys: Cysteine

[0133] Gln: glutamine

[0134] His:histidine

[0135] Ser: serine

[0136] Phe: Phenylalanine

[0137] Arg: Arginine

[0138] Asp: Aspartic acid

[0139] Fmoc-Pro-OH: Fmoc-protected proline DETAILED DESCRIPTION

[0140] The present invention will be further described in detail below by way of examples. The following examples are intended to explain the present invention but the present invention is not limited to the following examples.

[0141] Example 1: Synthesis of Peptide I (Reference: CN 108530519 B)

[0142] Synthesis order: from C-terminus to N-terminus.

[0143] (1) Resin swelling: Place 2-chlorotrityl chloride resin in a reaction tube, add dichloromethane (15 ml / g), and shake for 30 minutes.

[0144] (2) Add the first amino acid: Filter the solvent through a sand core, add serine and a 3-fold molar excess (compared to the molar amount of serine) of Fmoc-Pro-OH amino acid, add dimethylformamide to dissolve, then add a 10-fold molar excess of N,N-diisopropylethylamine (compared to the molar amount of serine), and shake for 60 minutes. Block with methanol.

[0145] (3) Deprotection: Filter the sand core to remove dimethylformamide, add 20% piperidine dimethylformamide solution (15 ml / g) for 5 minutes, remove it, and add 20% piperidine dimethylformamide solution (15 ml / g) for 15 minutes.

[0146] (4) Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add ninhydrin detection reagent, and heat at 105-110°C for 5 minutes. A dark blue color indicates a positive reaction. Wash the resin: twice with dimethylformamide (10 ml / g), twice with dichloromethane (10 ml / g), and twice with dimethylformamide (10 ml / g).

[0147] (5) Condensation: A three-fold excess (compared to the molar amount of serine) of the protected amino acid Fmoc-Pro-OH and a three-fold excess (compared to the molar amount of serine) of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) were dissolved in as little dimethylformamide as possible and added to the reaction tube. Immediately, a ten-fold excess (compared to the molar amount of serine) of N,N-diisopropylethylamine was added and the reaction was allowed to proceed for 30 min.

[0148] (6) Detection: Take a dozen pellets of resin, wash them three times with ethanol, add ninhydrin detection reagent for detection, and heat at 105-110°C for 5 minutes. A colorless reaction indicates a negative reaction. Wash the resin: dimethylformamide (10 ml / g) twice, dichloromethane (10 ml / g) twice, and dimethylformamide (10 ml / g) twice.

[0149] (7) Repeat steps (3) to (6) to connect the amino acids in the sequence in sequence until the sequence ends.

[0150] (8) Drain the liquid and wash the resin with dimethylformamide (10 ml / g) twice, methanol (10 ml / g) twice, dimethylformamide (10 ml / g) twice, and dichloromethane (10 ml / g) twice, each for 10 min, and drain.

[0151] (9) Cleavage of the peptide from the resin: Prepare a cleavage solution (10 / g): 95% trifluoroacetic acid; 1% water; 2% 1,2-ethanedithiol; and 2% triisopropylsilane. Cleavage time: 120 min. Blow the cleavage solution dry with nitrogen, wash it six times with ether, and then evaporate it to dryness at room temperature. Purify the crude product by high-performance liquid chromatography. Collect the target peptide solution, place it in a freeze dryer, concentrate it, and freeze-dry it to a white powder.

[0152] The obtained peptide 1 The H NMR data are as follows:

[0153] 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).

[0154] m / z:647.22

[0155] Example 2: Synthesis of Peptide II (Ref: CN 108530519 B)

[0156] Synthesis order: from C-terminus to N-terminus.

[0157] (1) Resin swelling: Place 2-chlorotrityl chloride resin in a reaction tube, add dichloromethane (15 ml / g), and shake for 30 minutes.

[0158] (2) First amino acid: Remove the solvent by filtration through a sand core, add aspartic acid and a 3-fold molar excess (compared to the molar amount of aspartic acid) of Fmoc-Pro-OH amino acid, dissolve in dimethylformamide, then add a 10-fold molar excess of N,N-diisopropylethylamine (compared to the molar amount of aspartic acid), and shake for 60 minutes. Block with methanol.

[0159] (3) Deprotection: Dimethylformamide was filtered off through a sand core, 20% piperidine dimethylformamide solution (15 ml / g) was added for 5 min, removed, and 20% piperidine dimethylformamide solution (15 ml / g) was added again for 15 min.

[0160] (4) Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add ninhydrin detection reagent for detection, heat at 105℃-110℃ for 5 minutes, and turn dark blue for a positive reaction. Add dimethylformamide (10ml / g) twice, dichloromethane (10ml / g) twice, and dimethylformamide (10ml / g) twice.

[0161] (5) Condensation: A three-fold excess (compared to the molar amount of aspartic acid) of the protected amino acid Fmoc-Pro-OH and a three-fold excess (compared to the molar amount of aspartic acid) of HBTU were dissolved in as little dimethylformamide as possible and added to the reaction tube. Immediately, a ten-fold excess (compared to the molar amount of aspartic acid) of N,N-diisopropylethylamine was added and the reaction was allowed to proceed for 30 min.

[0162] (6) Detection: Take a dozen resin pellets, wash them three times with ethanol, add ninhydrin detection reagent for detection, heat at 105℃-110℃ for 5 minutes, and a colorless reaction indicates a negative reaction. Wash the resin: dimethylformamide (10ml / g) once, dichloromethane (10ml / g) twice, and dimethylformamide (10ml / g) twice;

[0163] (7) Repeat steps (3) to (6) to connect the amino acids in the sequence in sequence until the sequence ends.

[0164] (8) Drain the solvent and wash the resin with dimethylformamide (10 ml / g) twice, methanol (10 ml / g) twice, dimethylformamide (10 ml / g) twice, and dichloromethane (10 ml / g) twice, each for 10 min, and drain.

[0165] (9) Cleavage of the peptide from the resin: Prepare a cleavage solution (10 / g): 95% trifluoroacetic acid; 1% water; 2% 1,2-ethanedithiol; and 2% triisopropylsilane. Cleavage time: 120 min. Blow the cleavage solution dry with nitrogen, wash it six times with ether, and then evaporate it to dryness at room temperature. Purify the crude product by high-performance liquid chromatography. Collect the target peptide solution, place it in a freeze dryer, concentrate it, and freeze-dry it to a white powder.

[0166] The obtained peptide 1 The H NMR data are as follows:

[0167] 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).

[0168] m / z:773.35

[0169] Example 3: Nebulized inhalation solution containing peptide I and peptide II

[0170] Raw materials Recipe 1 Recipe 2 Recipe 3 Recipe 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 0.1 M sodium hydroxide appropriate amount appropriate amount appropriate amount appropriate amount pH 6.0 6.0 6.0 6.0 Dilute to volume with purified water 100ml 100ml 100ml 100ml

[0171] The preparation method is as follows:

[0172] 1) Take 70% of the prescribed amount of purified water and dissolve benzalkonium chloride, sodium dihydrogen phosphate, peptide I, and peptide II in sequence for later use;

[0173] 2) Adding 0.1 M NaOH solution to the solution obtained in step 1) to adjust the pH of the solution to 6.0;

[0174] 3) Take the solution obtained in step 2) and add the remaining purified water to make up to 100 ml;

[0175] 4) Taking the solution obtained in step 3), filtering, filling, and sterilizing to obtain.

[0176] The above invention contents and embodiments describe the basic principles and main features of the present invention patent application and the technical effects of the present invention patent application. Those skilled in the art should understand that the present invention patent application is not limited by the above embodiments. The above embodiments and descriptions only illustrate the optimal technical solutions of the present invention patent application. Without departing from the spirit and scope of the present invention patent application, the present invention patent application will also have various changes and improvements, that is, the drug formulation of the combination of peptide I and Tai II and the preparation process thereof, all fall within the scope of the present invention patent application for protection, and the scope of protection required by the present invention patent application is defined by the attached claims and their equivalents.

Claims

1. A pharmaceutical composition for enhancing respiratory mucosal immunity, comprising peptide I and peptide II and pharmaceutically acceptable excipients, characterized in that: The chemical structures of peptide I and peptide II are as follows: Intestinal I: , Peptide II: .

2. The pharmaceutical composition according to claim 1, wherein The peptide I and peptide II are basic salts thereof.

3. The pharmaceutical composition according to claim 2, wherein The basic salt is one of potassium salt, sodium salt and ammonium salt.

4. The pharmaceutical composition according to claim 2, wherein The basic salt is a sodium salt.

5. The pharmaceutical composition according to claim 1, wherein The pharmaceutically acceptable excipients are selected from one or more of solvents, solubilizers, cosolvents, preservatives, flavoring agents, aromatics, mucilage agents, colorants, antioxidants, fillers, lubricants, glidants, and wetting agents.

6. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is a liquid preparation.

7. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is a solid preparation.

8. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is administered by inhalation.

9. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is administered by pulmonary aerosol inhalation.

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