ANTI-INFECTION AND ANTI-TUMOR MUCOSAL IMMUNE PREPARATION

Abstract

An immune preparation includes an immune substance formed by polyinosinic-polycytidylic acid, a non-antibiotic amino compound, polyethyleneimine, and at least one metal ion. The immune preparation does not include any antibiotics to prevent potential side effects. The amino compound may be chitosan oligosaccharide. The immune preparation is suitable for mucosal administration. The mucosal immune preparation facilitates mucosal immunity of human body allowing for the activation and proliferation of various immune cells.

Claims

1. An immune preparation, comprising an immune substance formed by polyinosinic-polycytidylic acid, a non-antibiotic amino compound, polyethyleneimine (PEI), and at least one metal ion, wherein the immune preparation is suitable for mucosal administration, and wherein the immune preparation does not contain an antibiotic.

2. The immune preparation of claim 1, wherein the non-antibiotic amino compound is chitosan oligosaccharide, and the chitosan oligosaccharide has a molecular weight of less than 3200 Da.

3. The immune preparation of claim 2, wherein the immune preparation is a spray or an aerosol.

4. The immune preparation of claim 2, wherein the immune preparation comprises a propellant.

5. The immune preparation of claim 2, wherein the metal ion is Ca′.

6. The immune preparation of claim 2, wherein the immune substance has a particle size of less than 1000 nm.

7. The immune preparation of claim 2, wherein the immune preparation further comprises at least one of antigens, chemical drugs and biological immune drugs.

8. A method for anti-infection of a subject having at least one of a viral infection, bacterial infection and parasitic infection, the method comprising: administering, via a mucosal administration route, the immune preparation of claim 2 to the subject in need thereof.

9. The method of claim 8, wherein the mucosal administration route is transnasal.

10. A method of treating a subject having a metastatic carcinoma, the method comprising: administering, via a mucosal administration route, the immune preparation of claim 2 to the subject in need thereof.

11. The method of claim 10, wherein the mucosal administration route is transnasal.

12. An immune preparation, comprising an immune substance formed by polyinosinic-polycytidylic acid, polyethyleneimine (PEI)-grafted chitosan oligosaccharide, and at least one metal ion, wherein the immune preparation is suitable for mucosal administration, and wherein the immune preparation does not contain an antibiotic, and chitosan oligosaccharide in the PEI-grafted chitosan oligosaccharide has a molecular weight of less than 3200 Da.

13. The immune preparation of claim 12, wherein the immune preparation is a spray or an aerosol.

14. The immune preparation of claim 12, wherein the immune preparation comprises a propellant.

15. The immune preparation of claim 12, wherein the metal ion is Ca′.

16. The immune preparation of claim 12, wherein the immune preparation further comprises at least one of antigens, chemical drugs and biological immune drugs.

17. A method for anti-infection of a subject having at least one of a viral infection, bacterial infection and parasitic infection, the method comprising: administering, via a mucosal administration route, the immune preparation of claim 12 to the subject in need thereof.

18. The method of claim 17, wherein the mucosal administration route is transnasal.

19. A method of treating a subject having a metastatic carcinoma, the method comprising: administering, via a mucosal administration route, the immune preparation of claim 12 to the subject in need thereof.

20. The method of claim 19, wherein the mucosal administration route is transnasal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0073] The preferred implementations of the present invention will be described below in detail by embodiments. It should be understood that the following embodiments are merely for illustrative purpose and not intended to limit the scope of the present invention. Without departing from the purpose and spirit of the present invention, those skilled in the art can make various modifications and replacements to the present invention.

[0074] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0075] Unless otherwise specified, the materials, reagents and the like used in the following embodiments are commercially available.

Embodiment 1 Mucosal Immune Preparation and Preparation Method Thereof

[0076] (1) A polyinosinic acid solution and a polycytidylic acid solution were prepared from polyinosinic acid and polycytidylic acid as raw materials by a PBS buffer solution, and the polyinosinic acid solution and the polycytidylic acid solution were stirred at 30° C. to 60° C. to obtain 0.5 mg/mL to 10 mg/mL of a double-stranded polyinosinic-polycytidylic acid solution (PIC solution).

[0077] (2) A chitosan oligosaccharide solution (COS solution) having a concentration of 1.6 mg/mL to 12.8 mg/mL was prepared from chitosan oligosaccharide by the PBS buffer solution.

[0078] (3) The COS solution was added dropwise in the double-stranded polyinosinic-polycytidylic acid solution obtained in the step (1) while stirring, a PEI solution was then added dropwise while stirring, and a CaCl.sub.2) solution was added dropwise while stirring, to obtain a mucosal immune substance sample solution.

[0079] (4) The prepared mucosal immune substance sample solution was sterilized, filtered, and packaged in a proper spray bottle or aerosol bottle to obtain the product.

Embodiment 2 Mucosal Immune Preparation and Preparation Method Thereof

[0080] (1) A polyinosinic acid solution and a polycytidylic acid solution were prepared from polyinosinic acid and polycytidylic acid as raw materials by a PBS buffer solution, and the polyinosinic acid solution and the polycytidylic acid solution were stirred at 30° C. to 60° C. to obtain 0.5 mg/mL to 10 mg/mL of a double-stranded polyinosinic-polycytidylic acid solution (PIC solution).

[0081] (2) A chitosan oligosaccharide solution (COS solution) having a concentration of 1.6 mg/mL to 12.8 mg/mL was prepared from chitosan oligosaccharide by the PBS buffer solution.

[0082] Or, a PEG-g-COS solution having a concentration of 1.6 mg/mL to 51.2 mg/mL was prepared from PEG-g-COS by the PBS buffer solution.

[0083] (3) The PEG-g-COS solution or the chitosan oligosaccharide solution was added dropwise in the double-stranded polyinosinic-polycytidylic acid solution obtained in the step (1) while stirring, and a CaCl.sub.2) solution was then added dropwise while stirring to obtain a PIC-PEG-g-COS-CaCl.sub.2) or PIC-COS-CaCl.sub.2) compound.

[0084] (4) The PIC-PEG-g-COS-CaCl.sub.2 or PIC-COS-CaCl.sub.2 compound was stirred at a constant speed in a thermostatic magnetic stirrer and then added dropwise with 0.4 mg/mL to 6.0 mg/mL of an aqueous solution of sodium tripolyphosphate until obvious opalescence was observed, the reaction was maintained for 10 min to 60 min to obtain a mucosal immune substance through ionic crosslinking and self-assembly, and the mucosal immune substance was centrifuged at a high speed to obtain a mucosal immune substance having a particle size of less than 1000 nm.

[0085] (5) The prepared mucosal immune substance was re-suspended by distilled water, sterilized, filtered, and packaged in a proper spray bottle or aerosol bottle to obtain the product.

Experimental Example 1 Separate Application of Naristillae Mucosal Immune Preparation in Anti-Influenza Mice Protection Experiments

[0086] Influenza virus: FM1 strain, purchased from the Institute of Virus Disease Control of Chinese Academy of Preventive Medicine.

[0087] Virazole: positive control drug, purchased from Shenyang Yanfeng Pharmacy Factory.

[0088] Mice: Kunming species, where mice 8 g to 10 g were used for passage of FM1 virus, and mice 14 g to 20 g were used for the following normal experiments.

[0089] The influenza virus FM1 strain suspension could lead to fetal pneumonia of mice at a ratio of 5 LD.sub.50/mouse by nasal drip. The mice were infected first and administrated with drugs during the experiment, and then grouped in accordance with Table 1.

TABLE-US-00001 TABLE 1 Application of the mucosal immune preparation of the present invention in anti-influenza mice protection experiments by nasal drip Administration Death Group dosage rate % X.sup.2 P value Influenza virus FM1 strain Normal saline 89 Positive control virazole 0.07 g/kg/d 63 1.64 >0.05 Separate naristillae of the 0.1 ml/mouse 43 3.88 <0.05 present invention

[0090] The experimental results had shown that, in the mice protection experiments, the non-specific anti-influenza effect of the separate naristillae mucosal immune preparation of the present invention was superior to that of the known antiviral drug virazole, and had remarkable anti-influenza virus effects by statistical analysis.

Experimental Example 2

[0091] This experimental example was used for describing the comparison of the influences of the mucosal immune preparation (naristillae) of the present invention in combination with the nasal mucosal immunity and subcutaneous injection immunity of an influenza vaccine and the complete Freund's adjuvant on the titter of the humoral antibody IgA and the influenza virus proliferation.

[0092] The experimental scheme was as follows.

[0093] Influenza virus: FM1, purchased from the Institute of Virus Disease Control of Chinese Academy of Preventive Medicine.

[0094] Influenza vaccine: split influenza vaccine produced by Hualan Biological Product Co., Ltd.

[0095] Complete Freund's adjuvant: produced by Shanghai Wegene Biotechnology Co., Ltd.

[0096] Mucosal immune adjuvant of the present invention: produced by Xinfu (Beijing) Pharmaceutical Technology Co., Ltd.

[0097] Mice: Kunming species, where mice 8 g to 10 g were used for passage of FM1 virus, and mice 14 g to 20 were used for the following normal experiments.

[0098] Complete Freund's adjuvant influenza vaccine: in a centrifuge tube, the vaccine and the complete Freund's adjuvant at the same volume were uniformly mixed by vortex to obtain water-in-oil emulsion.

[0099] Naristillae of the combined influenza vaccine of the present invention: the influenza vaccine and the mucosal immune adjuvant of the present invention were mixed at the same volume to obtain an aqueous solvent.

[0100] Influenza vaccine: the influenza vaccine and PBS were mixed at the same volume to obtain an aqueous solvent.

[0101] Immunization Method:

[0102] Subcutaneous injection immunization: immune mice were injected subcutaneously for 0 day and injected for 28 days at a ratio of 0.1 mL/mouse; on the 42.sup.rd day, blood was drawn from some mice, the serum was separated, and the titer of the antibody was measured; other mice were infected with the influenza virus FM1 strain suspension at a ratio of 5 LD.sub.50/nasal drop, and the titer of the lung tissue virus was measured on the fifth day after the inflection.

[0103] Nasal immunization: immune mice were injected subcutaneously for 0 day and injected for 28 days at a ratio of 0.1 mL/mouse; on the 42.sup.rd day, blood was drawn from some mice, the serum was separated, and the titer of the antibody was measured; other mice were infected by the influenza virus FM1 strain suspension at a ratio of 5 LD.sub.50/nasal drop, and the titer of the lung tissue virus was measured on the fifth day after the inflection.

[0104] The experimental results of the groups were shown in Table 2:

TABLE-US-00002 TABLE 2 Anti-influenza experiments of the mucosal immune preparation of the present invention in combination with the influenza vaccine by nasal drip Titer of Antibody influenza virus Group index after treatment Subcutaneous injection immunization: Influenza vaccine 10.sup.3.1 10.sup.4   Complete Freund's adjuvant 10.sup.5.5 10.sup.3.5 influenza vaccine Naristillae of the combined 10.sup.4.5 10.sup.2.0 influenza vaccine of the present invention Nasal mucosal immunization: Influenza vaccine 10.sup.2.5 10.sup.4.5 Naristillae of the combined 10.sup.4   <10 .sup.   influenza vaccine of the present invention FM1 virus control 10.sup.5.0

[0105] The complete Freund's adjuvant was a golden standard for detecting the cellular immunity of the body. The experimental results had indicated that, in the subcutaneous immunization, the titer of the antibody generated by the combined influenza vaccine of the mucosal immune preparation of the present invention was lower 10 times than that of the complete Freund's adjuvant influenza vaccine, but the titer of influenza virus was reduced by 31.6 times in comparison to the complete Freund's adjuvant influenza vaccine. Particularly, the nasal mucosal immunization of mice had indicated that the tiger of the antibody generated by the naristillae obtained by combining the mucosal immune preparation of the present invention with the antigen was higher 31.6 times than that of the pure influenza vaccine, and the titer of influenza virus was reduced by above 3100 times, so that the mucosal immune preparation had obvious effects.

Experimental Example 3

[0106] It was detected by Beijing Jingmeng High-tech Stem Cell Technology Co., Ltd. that the present invention had significant effects in the preliminary detection of CIK cell effector target tests.

[0107] Sample No.: JSCIK2016042614; detection date: 2016 Apr. 26; and, report date: 2016 Apr. 28.

[0108] Operation procedure: culturing and analyzing in accordance with the CIK cell effector target test SOP.

[0109] Target cell: A549; effector cell: cultured JSCIK2016042614.

[0110] Conclusion: by a fluorescence microscope and an ELIASA detection method and the experimental error, the killing capability of the cells JSCIK2016042614 was comprehensively analyzed, which was moderate with a ratio of 1:10.

Experimental Example 4

[0111] The results of salvage therapy of four volunteers with end-stage or advanced tumors by the mucosal immune preparation of the present invention will be described below.

[0112] 1. Usage: the anti-tumor drug of the present invention is a non-cytotoxic drug. The drug was administrated nasally every other day at a volume of 2 mg to 6 mg and administrated for 1 to 12 months, without other obvious side effects.

[0113] 2. Clinical effects: referring to Table 3.

TABLE-US-00003 TABLE 3 Application of the mucosal immune preparation for the present invention in volunteers with end-stage or advanced tumors by nasal immunization Name Gender Age Disease Before administration After administration Time of duration LIN Female 80 Carcinoma of Lie in bed, low Appetite increased since From June Shulan years submandibular appetite, accompanied October, 3 meals a day, 2016 to old gland, multiple by nausea and put on weight, good June 2017 metastatic emesis, only one meal mental state, lifetime has sites, recurrence a day, spirit prolong 11 months (from after surgery drooping. Need to July 2016 to June 2017) twice, multiple actively support than doctor's prediction. organ metastasis. treatment with Since local pain and The doctor thought she blood and protein. induration were found in only had 2 months injected part, the drug lifetime in May 2016. was then administrated by nasal administration. The good state has kept to the present, and the patient has already been discharged from the hospital. WEN Male 67 Lung adenocarcinoma; Spirit drooping, The drug is administrated From March Qingqiang years he was diagnosed no surgery, nasally at a volume of 6 2017 to old with lung cancer in chemotherapy. mg/each time and June 2017 Peking Union Hospital administrated every other and Tianjin Quanjian day, without side effects. Hospital on February The side effects were and March 2017; there reduced by the mucosal was a duck-egg-sized immune preparation in tumor in the combination with the mediastinum. chemotherapeutic drug (cisplatin), almost no nausea and emesis occurred, and the number of leucocytes was increased from 1000/mm.sup.3 to 2000/mm.sup.3 before administration to 3000/mm.sup.3 after administration. ZHU Female 62 She was diagnosed Severe side Since May 4, 2017, the From May Shuqin years with right effects after wo drug was administrated 2017 to old breast cancer and chemotherapies, nasally every other day at June 2017 metastasis in weight loss, a volume of 4 mg/each liver on May nausea and time, then injected 8, 2017 in emesis, and intramuscularly at a Jilin Tumor increased volume of 2 mg/each Hospital, and tumor. High time. In such cancer nests content of administration in fiber transaminase. circulation, after more texture were than ten times of found in administration, the patient lymph nodes. said that there were no side effects. In combination with the chemotherapeutic drug, the side effects of the chemotherapy were significantly reduced, the appetite was increased, the physical strength was increased, and the breast tumor was softened. The content of glutamic-pyruvic transaminase was decreased from 130.5 U/L to 38.4 U/L. The maximum low-echo halo of the liver was decreased from 7.6 × 5.1 cm to 5.9 × 3.0 cm on Apr. 8, 2017 to 5.9 × 3.0 cm on Jun. 2, 2017. WANG Female 73 Coloretal cancer No appetite, no Since May 27, 2017, the From May Xiufang years surgery in taste, light drug was administrated 2017 to old 2002, breast sleep, tired and nasally every other day at June 2017 cancer surgery in weak, mild a volume of 4 mg/each 2008, lung cancer hair loss, poor time. After six times of surgery in 2015, and gastric kinetic drug administration, the lung cancer energy, cough patient and her family radiotherapy and and take cough members said that there chemotherapy medicines, and were no side effects. The in April 2017. no treatment. appetite was increased, the tumor was decreased, the cough was relieved greatly, and the cough medicines were not administrated.

[0114] 3. It can be known from the above results that the mucosal immune preparation of the present invention has the following advantages: (1) good safety; (2) the mucosal immune preparation can reduce the side effects of the chemotherapy; (3) the clinical effects are significant: the appetite is increased, the physical strength is increased, and the mental state becomes optimistic from pessimistic and becomes confident, and the total survival time is prolonged at least several months in comparison to the survival time predicted by the chief physician; and (4) the applications in the volunteers with advanced tumors have proved that the mucosal immune preparation can obviously reduce the side effects resulted from the chemotherapy, significantly improve the life quality of the patients and prolong the survival time of the patients, so that the mucosal immune preparation is both safe and effective.

[0115] 4. It can be known from ZHU Shuqin's case that the product not only has an anticancer effect on a local part by nasal spray, but also can have a systemic immunity effect after the nasal mucosal immunization and have astonishing effects on metastatic cancers.

[0116] 5. Selection of tested populations of the present invention: in accordance with Technical Guidelines for Clinical Trials of Anti-cancer Drugs issued by the State Food and Drug Administration, the score for the performance status (PS) ECOG of the selected cancer patients is 0 to 1 or the score for Karnofsky of the patients is higher than 70 scores, referring to Table 1 and Table 5.

TABLE-US-00004 TABLE 4 Scoring standard for ECOG performance status Score Activity level 0 Completely normal, being able to do all normal activities without any restriction 1 Being unable to do vigorous physical activities, but being able to move around and do light physical activities or office work 2 Being able to move around and take care of themselves in daily life, but being unable to do any job, the time in bed in the daytime being not more than 50% 3 Being able to barely take care of themselves in daily life, the time in bed or on a chair for rest in the daytime being more than 50% 4 Completely incapacitated, being unable to take care of themselves in daily life, being in bed or using a wheelchair 5 Dead

TABLE-US-00005 TABLE 5 Scoring standard for Karnofsky performance status Score Activity level 100 Being able to do normal activities, no complaints or disease symptoms 90 Being able to do normal activities, with slight disease symptoms 80 Being able to barely do normal activities, with some symptoms or signs 70 Being able to take care of themselves in daily life, but unable to maintain normal activities or positive work 60 Occasionally needing help from others, but being able to take care of themselves in daily life most of the time 50 Frequently needing help from others, or needing frequent medical care 40 Being unable to take care of themselves in daily life, and needing special help and care 30 Being completely unable to take care of themselves in daily life, should be in the hospital, without death risk 20 Must be in the hospital, in serious condition, positively supporting the treatment 10 Being terminally ill, impending death 0 Dead

[0117] The selection of treatment objects in the present invention is performed after patients have known ineffective cancer treatment or abandon the treatment and have signed the Informed Consent. The performance requirement is far lower than the above standards. Majority of patients have an ECOG in the fifth level or a Karnofsky score of greater than 20 scores. That is, patents who lose the self-care ability, are in a bad condition and need to positively support treatment are possible, without any age limit. The present invention has certain effects on the all patients.

Experimental Example 5

[0118] This experimental example was used for describing the following situations: precipitation will be generated by increasing the dosage of COS in the mucosal immune preparation of the present invention so that the uniformity of drug administration is affected; after the addition of PEI, the precipitation will be prevented; and, by increasing the dosage of COS, the mucosal immunity is further enhanced (referring to Table 6).

TABLE-US-00006 TABLE 6 Preparation PIC COS PEI CaCl.sub.2 Name (mg/ml) (mg/ml) (mg/ml) (mol/L) Result Group 1 1 0.4 0 0.0004 Clear solution 1 0.8 0 0.0004 Clear solution 1 1.6 0 0.0004 Precipitation Group 2 1 0.8 32 0.0004 Clear solution 1 1.6 32 0.0004 Clear solution 1 3.2 32 0.0004 Clear solution 1 6.4 32 0.0004 Clear solution

Comparative Example 1

[0119] Patent Application No. CN105396130 A disclosed “a polyinosinic-polycytidylic acid, ammonia and calcium adjuvant and a vaccine containing the PIC, ammonia and calcium adjuvant”, and disclosed that the non-antibiotic amino compound is optionally chitosan.

[0120] Chitosan oligosaccharide in Embodiment 1 is replaced with chitosan in this comparison example, and the result of comparison of the influences of the both on the uniformity of drug administration refers to Table 7.

[0121] It is found by researches that the addition of the chitosan solution leads to the generation of precipitation during the preparation process and thus influences the uniformity of drug administration; however, this problem can be solved by using chitosan oligosaccharide. (referring to Table 7).

TABLE-US-00007 TABLE 7 Preparation PIC CS COS CaCl.sub.2 Name (mg/ml) (mg/ml) (mg/ml) (mol/L) Result Group 1 1 0.4 0 0.0004 Precipitation 1 0.8 0 0.0004 Precipitation Group 2 1 0 0.4 0.0004 Clear solution 1 0 0.8 0.0004 Clear solution

[0122] Although the present invention has been described above in detail by general description and specific implementations, it is apparent for those skilled in the art to make some modifications or improvements based on the present invention. Therefore, those modifications or improvements made without departing from the spirit of the present invention shall fall into the protection scope of the present invention.