WO2011103794A1 - 用于治疗人乳头瘤病毒感染引起的疾病的喷剂及其制备方法 - Google Patents

用于治疗人乳头瘤病毒感染引起的疾病的喷剂及其制备方法 Download PDF

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WO2011103794A1
WO2011103794A1 PCT/CN2011/071122 CN2011071122W WO2011103794A1 WO 2011103794 A1 WO2011103794 A1 WO 2011103794A1 CN 2011071122 W CN2011071122 W CN 2011071122W WO 2011103794 A1 WO2011103794 A1 WO 2011103794A1
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acetic acid
spray
acid solution
solution
human
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PCT/CN2011/071122
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English (en)
French (fr)
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孙介光
孙雪然
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Sun Jieguang
Sun Xueran
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the field of medical technology, and more particularly to a spray for the treatment of diseases caused by human papillomavirus infection and preparation thereof. Background technique
  • the human rabies purified vaccine (KI) is found to stimulate the body as a biological antigen to stimulate the body to produce a strong immune response (antigen-specific immunity; non-antigen-specific immunity) while the host uses a biological antigen to stimulate the body.
  • the immune response plays an important role in controlling related diseases.
  • a large number of literature research data prove that: the low immunity of the human body is closely related to the occurrence of cancer. Once the immunity of the body is reduced, various diseases can enter the gap.
  • Clinical data also prove that only the human papilloma virus (HPV) has the most clear relationship with human tumors in the study of the relationship between tumor virus and human diseases. In 1995, WHO and IARC identified HPV as the cause of cervical cancer.
  • HPV mainly invades the epithelial tissue of human skin and mucous membranes, causing benign and malignant hyperplasia of skin and mucous epithelium.
  • HPV is divided into: 13 high-risk HPVs (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) 68), low-risk HPV mainly includes: HPV (6, 11, 32, 40, 42, 43, 54, MM8).
  • HPV high-risk HPV virus
  • HPV high-risk human papillomavirus
  • HPV human papillomavirus
  • HPV human papillomavirus
  • HPV high-risk HPV
  • Subtypes are associated with malignant tumors of the kidney (in which the HPV (16, 18) gene positive rate of renal papillary carcinoma is significantly higher than that of renal clear cell carcinoma and renal granulosa), confirming that high-risk HPV infection is renal cancer.
  • Important carcinogenic causes there is data to prove the relationship between esophageal squamous cell carcinoma and E6 oncogene protein.
  • Low-risk HPV is associated with certain sexually transmitted diseases, including genital herpes, condyloma acuminata, cervical erosion, chronic cervicitis, etc.
  • HPV6 and 11 are the most common low-risk HPV, and 90% of genital warts are associated with genital warts.
  • the current dosage forms and preparation methods for inhibiting human papillomavirus and treating tumors caused by human papillomavirus infection and using human rabies purified vaccine for treating sexually transmitted diseases have not been reported yet. Summary of the invention
  • the object of the present invention is to provide a spray for treating human papillomavirus and treating tumor and sexually transmitted diseases caused by human papillomavirus infection, and a preparation method thereof, and the invention has the obvious therapeutic effect and no toxic side effect on the tumor.
  • the spray of the present invention can preserve the biological activity of the human rabies purified vaccine and does not cause any negative damage to the human body, so that the topical medication is possible.
  • the topical medication according to the present invention refers to the direct administration to the infected patient.
  • the composition of the present invention is 1 ml of human rabies purified vaccine, and is prepared by adding 0.01 g/ml of chitosan-acetic acid solution to 10 ml, wherein the 0.005 to 0.015 g/ml chitosan-acetic acid is: 10 g of shell polycondensation Sugar and 1000 ml acetic acid solution.
  • the virus strain for preparing human rabies purified vaccine is, but not limited to, rabies virus PM strain, PV strain, CTN strain. Or AG strain.
  • a method for preparing a spray for treating a disease caused by human papillomavirus infection according to the following steps: First, preparing a chitosan-acetic acid solution:
  • the pH is adjusted to a pH of 3.8 to 5.0, preferably a pH of 4.9 to 5.0, in a sodium hydroxide solution having a molar concentration of 0.1 mol/L or an acetic acid solution of 0.1 mol/L.
  • the human rabies purified vaccine may be of a solution type or a lyophilized type.
  • glacial acetic acid is analytically pure or chemically pure.
  • the pure water is distilled water.
  • the spray of the invention is used for treating cervical cancer caused by human papillomavirus infection, genital herpes caused by human papillomavirus infection or genital warts, cervix erosion caused by human papillomavirus infection, chronic cervix caused by human papillomavirus infection inflammation.
  • the spray of the present invention is in the form of an external preparation.
  • the human rabies purified vaccine is completely dissolved, and the spray of the present invention has a good aerosol property after being sprayed. Therefore, the drug evenly covers the surface of the mucosa of the lesion after being sprayed, which is beneficial to the absorption of the drug by the body.
  • the external spray and preparation method of the invention focus on the treatment of cervical cancer patients, genital herpes and condyloma acuminata caused by HPV infection, cervical erosion, chronic cervicitis, HPV infection (Tc subgroup detection, HPV detection and Good clinical symptoms have achieved good clinical results.
  • the main component of the present invention is a human rabies purified vaccine, and the direct killing effect of the main component of the present invention on rabies purified vaccine on tumor cells is demonstrated by an in vitro MTT assay on human cervical cancer Hela human tumor cells.
  • the invention enhances the phagocytic ability of mouse monocytes and peritoneal phagocytic cells in the immune regulation of the body, indicating that the phagocytic function of the reticuloendothelial system has obvious activation and enhancement effects. Therefore, the present inventors have found that human rabies vaccine can induce a strong immune response in the body. After systemic treatment of HPV infected patients (without other treatment routes), HPV detects yang conversion.
  • the toxicological test of the present invention proves that the intensity of the sensitization reaction in the repeated contact of the external preparation with the skin is weakly sensitizing. No obvious toxicity was observed in the acute toxicity test of mice and rabbits.
  • the results of mouse micronucleus test showed that the micronucleus rate of bone marrow polychromatic erythrocytes was not induced by mouse muscle injection at ⁇ 375U+25mg/kg.
  • vaginal irritation test of the present invention abnormal changes such as vascular congestion and edema were not observed after continuous vaginal administration of rabbits or rats.
  • the multiple dose stimulation test also proves that the external preparation of the present invention is vaginal adhesion to experimental animals. The membrane did not significantly stimulate the reaction.
  • Spray composition of the invention :
  • the human rabies purified vaccine was prepared in a volume ratio of 1 ml, and diluted to 10 ml by mass concentration of 0.01 g/ml of chitosan-acetic acid solution.
  • the specific equipment is as follows:
  • a sodium hydroxide solution having a molar concentration of 0.1 mol/L or an acetic acid solution having a molar concentration of 0.1 mol/L is used to adjust the pH to 3.2, and the solution has a clarity.
  • the pH value is maintained between 3.8 and 5.0. If the pH exceeds 5.0 or less than 3.8, the chitosan is separated from the acetic acid. The solution becomes opaque and the composition changes. When used, the sprayed solution will not be presented. Mist.
  • Embodiment 1 The difference from Embodiment 1 is that:
  • chitosan-acetic acid solution with a mass concentration of 0.02g/ml: 20g of chitosan is added to the above acetic acid solution and completely dissolved to 1000ml. The solution is not allowed to insoluble particles or insoluble matter, and the mass concentration is 0.02 g/ml of chitosan-acetic acid solution.
  • lyophilized human rabies purified vaccine with 15 IU of activity unit (two in this example, each 0.5 ml, 7.5 IU) was added to a 10 ml volumetric flask, and the virus strain prepared by the human rabies purified vaccine was a rabies virus PV strain.
  • the chitosan-acetic acid solution was diluted to 10 ml.
  • a vaccine solution having an active concentration of 15 IU/10 ml was obtained.
  • the solution has a clarity degree by using a sodium hydroxide solution having a molar concentration of 0.1 mol/L or an acetic acid solution having a molar concentration of 0.1 mol/L to adjust the pH to 4.9.
  • Embodiment 1 The difference from Embodiment 1 is that:
  • chitosan-acetic acid solution with a mass concentration of 0.03g/ml: 30g of chitosan is added to the above acetic acid solution and completely dissolved to 1000ml. The solution is not allowed to have insoluble particles or insoluble matter, and the mass concentration is 0.03 g/ml of chitosan-acetic acid solution.
  • a solution-type human rabies purified vaccine with a unit of activity of 10 IU (two tubes in each example, 0.5 ml each, 5 IU) was added to a 10 ml volumetric flask, and the human rabies purified vaccine was prepared as a rabies virus CTN. Strain.
  • the chitosan-acetic acid solution was diluted to 10 ml.
  • a vaccine solution having a concentration of 10 IU/lOml was obtained.
  • the solution has a clarity value by adjusting the pH to 5.0 by using a sodium hydroxide solution having a molar concentration of 0.1 mol/L or an acetic acid solution having a molar concentration of 0.1 mol/L.
  • Embodiment 1 The difference from Embodiment 1 is that:
  • the virus strain prepared by human rabies purified vaccine is rabies virus. AG strain.
  • the chitosan-acetic acid solution was diluted to 10 ml.
  • a vaccine solution having a concentration of 20 IU/10 ml was obtained.
  • the pH of the solution is adjusted to 4.2 by using a sodium hydroxide solution having a molar concentration of 0.1 mol/L or an acetic acid solution having a molar concentration of 0.1 mol/L, and the solution has a clarity.
  • Embodiment 1 The difference from Embodiment 1 is that:
  • the active concentration of the preparation example 4 is 20IU/10ml.
  • the topical application of the spray of the invention is 4 times a day, once sprayed (140 ⁇ 1), administered for 45 days, and HPV test is performed after 20 days of withdrawal, the result shows Turned to yin.
  • Case treatment case 2 Zhou Moumou
  • Specific clinical dosage Take the spray of the invention with the active concentration of 20IU/10ml in the preparation example 4, 6 times a day, once a spray (140 ⁇ 1), 20 days for a course of treatment, a total of four courses, after stopping the drug The body part of the body was shedding, and the HPV test was performed 20 days after the drug was stopped. The result showed that the body was turned negative.
  • Specific clinical dosage Take the spray of the invention with the active concentration of 5IU/10ml in the preparation example 1 for external use, 6 times a day, one spray (140 ⁇ 1), 20 days for one course of treatment, a total of four courses, after stopping the drug Cervical mucosa returned to normal, HPV test was performed 20 days after stopping the drug, and the result showed positive to negative.
  • the external spray of the present invention uses different active concentrations according to actual needs.
  • Positive control drug Fluorouracil injection, batch number 050806, produced by Shanghai Xudong Haipu Pharmaceutical Co., Ltd.
  • Hela human cervical cancer cells
  • the well-grown tumor cells were collected, inoculated in a 96-well culture plate, and cultured for 24 hours.
  • the test sample was cultured for 4 days.
  • the absorbance (OD) value of each well was measured with a 550 type microplate reader at a detection wavelength of 540 nm and a reference wavelength of 405 nm.
  • the experimental data was processed to obtain a half inhibitory concentration ac 50 ).
  • the main ingredient used in the present invention purified human rabies vaccine H e laIC 5 () ⁇ 0.1IU / ml. It is indicated that the main component of the present invention is a human rabies purified vaccine which has an inhibitory effect on proliferation of human cancer cells in vitro.
  • the vaccine solution of the present invention uses different active concentrations according to actual needs.
  • the Hela cells in the experimental examples were used.
  • human rabies vaccine has an effect on the cell cycle distribution of human cervical cancer Hela.
  • Human rabies purified vaccine can arrest human cervical cancer cells in G2/M phase, suggesting that human rabies purified vaccine inhibits tumor growth. Inducing tumor cell cycle arrest is related, see Figure 1.
  • Drugs can inhibit tumor cell proliferation by inducing G2/M phase arrest (the second peak of the map is the cycle arrest inhibitor).
  • Test drug The vaccine solution of Formulation Example 1 has an activity unit of 5 IU/ml.
  • Human cervical cancer cell line Hela autologous cultured cells were inoculated into nude mice for tumor formation. The inventor saved it for generations.
  • Reference drug Doxorubicin batch number: 060303B, Provided by: Zhejiang Haizheng Pharmaceutical Co., Ltd., diluted with physiological syrup 0.1mg/ml for use.
  • the tumor-bearing animals with good tumor growth were selected and the cervical vertebrae were sacrificed by whitening.
  • the tumor block was taken under aseptic conditions, and a tumor piece of 2-3 mm in diameter was cut with a scalpel, and the trocar was inoculated subcutaneously into the ankle of the nude mouse. Tumor growth was seen after 7 days. The length and width of the tumor were measured with a vernier caliper and grouped according to the size of the tumor. 7 animals per group.
  • the negative control group (not administered), the vaccine solution of the present invention 0.2 ml of the local administration of the tumor, the vaccine solution of the present invention 0.1 ml of the local administration of the tumor plus 0.1 ml intramuscular injection of 1 group, the vaccine solution of the present invention 0.1 ml, 0.2 ml and 0.4 ml were intramuscularly administered to each group.
  • Doxorubicin 5 mg/kg was administered intraperitoneally as a positive control. Dosing started on the day of grouping, once a day, for 20 days.
  • the length, width and body weight of the tumors of the experimental animals were measured 2 to 3 times a week, and the tumor growth and general conditions of the animals were observed. After 20 days, the animals were sacrificed and the tumor was removed, and the tumor weight was calculated. The tumor growth inhibition rate was calculated. Calculate tumor volume (TV) and relative tumor volume (RTV), compare the tumor weight of each group of animals Statistical differences in indicators such as volume, tumor volume, and relative tumor volume (RTV) (see Tables 1 and 2).
  • the tissue is visible to the naked eye: mild edema around the tumor, small volume of the tumor tissue, and ulceration or crusting on the surface. When the tumor was removed, the tumor wall was thin, and the contents were yellow-green bean dregs. Muscle administration changed to the tumor of the local injection group, the tumor capsule was thin, the tumor tissue was yellow, and the tumor tissue was soft and tension-free.
  • the main component of the human rabies purified vaccine used in the present invention has a significant inhibitory effect on the growth of human cervical cancer Hela tumor in nude mice xenograft tumors by local administration and local intramuscular injection.
  • Table 1 The inhibitory effect of the vaccine solution of the present invention on human cervical cancer Hela nude mice xenograft tumor
  • Negative control 7 7 21.4 ⁇ 1.05 27.3 ⁇ 2.37 2.94 ⁇ 1.469
  • the invention studies the regulation effect, the synergistic detoxification effect of the drug on the immune index of the tumor-bearing mice, and the regulation and prevention of the damage repair of the radiotherapy and chemotherapy.
  • mice mononuclear phagocytic function the function of mouse mononuclear phagocytic cells was significantly improved after administration, and the phagocytic index and phagocytic activity were 0.3480 and 8.2870, respectively.
  • the results of the study were significantly different by data processing, and the comparison between groups was P ⁇ 0.1.
  • the phagocytosis rate of mouse peritoneal macrophages reached 35.02%, and the phagocytic index also increased significantly, reaching 1.02, which also increased the phagocytic digestion function; increased the half hemolysis value of serum, and had stronger antibody formation after chemotherapy. Regulatory effect (regulatory effect of immune function).
  • the tumor-inhibiting rate of tumor-bearing mice alone was 39.03%, and if used in combination with chemotherapy, it could reach 59.53% (enhanced effect).
  • SOD superoxide dismutase
  • the repair of radiation damage is also obvious.
  • the animal begins to die late, and the skin and mucous membrane damage of the head is lighter.
  • the damage of the control group is heavier, the skin and mucous membrane of the head and face are severely ulcerated, and most of the hair is removed. With symptoms such as diarrhea.
  • the micronucleus rate of bone marrow in the experimental group was not significantly increased.
  • the 30-day survival rate was 82.07%, and the average monthly survival time was 24.62 days, which was higher than that of the control group (regulatory effect of chemoradiotherapy injury repair).
  • the drug can prolong the swimming time of the mice by 75%, which significantly enhances the endurance (preventive effect).
  • Test drug The active unit was prepared according to the preparation method of the present invention: 10 IU/ml external spray.
  • Test animals guinea pigs, female and male, weighing 250-300 g, experimental animal use license number: SCXK (Liao) 2003-0011, provided by Qianmin District Qianmin Farm in Shenyang City.
  • Positive control drug 2, 4-dinitrochlorobenzene (batch number: 20070622) Provided by Tianjin Jinke Fine Chemical Research Institute; diluted with physiological saline to a volume concentration of 1% and 0.1%, ready for use.
  • Excipient group 0.01 g/ml of chitosan-acetic acid solution.
  • each side of the hair removal area is about 3 ⁇ 3cm 2
  • the left hair removal area of the three groups of animals were respectively coated with 0.2 ml of excipient, the vaccine solution of the invention and the volume concentration of 1% 2,4 dinitrochlorobenzene, and fixed for 6 hours after administration.
  • the above administration process was repeated on the 7th and 14th day after the administration, and the above administration process was repeated 14 days after the last administration, the administration site was the right hair removal zone, and the positive control drug volume concentration was changed to 0.1%.
  • the drug was washed off for 6 hours, immediately observed, and then the skin allergic reaction was observed again at 24, 48, and 72 hours.
  • the administrating group and the vehicle group had no erythema or edema on the skin of the guinea pig at the site of administration, and the reaction intensity of 2,4-dinitrochlorobenzene was extremely sensitizing.
  • the topical spray of the present invention was repeatedly contacted with the skin, and no significant allergic reaction to the skin of the test animal was found.
  • the sensitization reaction intensity is weakly sensitizing.
  • the main component of the present invention uses purified rabies vaccine to detect the genetic toxicity of chromosomal aberrations in animals.
  • Test drugs a mixed liquid of purified rabies vaccine and polymyosin injection for lyophilized human, purified rabies vaccine for human lyophilized human (lot number 20050802-4), provided by Liaoning Chengda Biotechnology Co., Ltd.; Injection (production batch number 060418), 2mg/2ml, provided by Zhejiang Wanma Pharmaceutical Co., Ltd. Polymyosin injection 2.0ml Dissolved lyophilized human purified rabies vaccine 30IU/12ml, the maximum dissolved concentration is 15IU + lmg / ml.
  • Test animals mice, male and female, weighing 22-24g, experimental animal use license number: SYXK (Beijing) 2006-0004, provided by the Experimental Animal Center of China National Institute for the Control of Pharmaceutical and Biological Products.
  • Positive control drug cyclophosphamide (batch 06071721), produced by Jiangsu Hengrui Pharmaceutical Co., Ltd., purchased from Beijing Hospital Pharmacy. A solution having a concentration of 2 mg/ml was prepared using water for injection.
  • the test drug was given a high dose at the maximum dissolved concentration ((15IU+lmg)/ml) and the maximum dose (0.5ml/20g). There were three dose groups (375IU+25m)/kg, (125IU+8mg). /kg and (42IU+3mg) /kg), 10 mice per group, the mice were intramuscularly injected into the neck and back, and the second dose was administered 24 hours after the first administration. After 6 hours of administration, The mice were sacrificed from the cervical vertebrae. The femur bone marrow was routinely taken, smeared, and the ratio of 100 polychromatic red blood cells (P) to positive red blood cells (N) was counted. )
  • the micronucleus rate of each dose group was ⁇ 1.9%.
  • the micronucleus rate of the positive control group was >29.0%. .
  • the results showed that the micronucleus rate of bone marrow polychromatic erythrocytes was not induced by intramuscular injection of ⁇ 375 IU+25 mg/kg.
  • Tip The main component of the present invention is a purified rabies vaccine in the test dose range, and the mouse micronucleus test is a negative result.
  • mice 3. Acute toxicity test in mice:
  • Test drug Freeze-dried human rabies purified vaccine (batch number 20050802-4), provided by Liaoning Chengda Biotechnology Co., Ltd. It was dissolved in water for injection to an active concentration of 15 IU/ml before the experiment.
  • mice Male and female, weight 19-21g, experimental animal use license number: SYXK (Beijing) 2006-0004, provided by the Experimental Animal Center of China National Institute for the Control of Pharmaceutical and Biological Products.
  • mice The acute toxicity test in mice shows that the external spray of the present invention has less toxic side effects.
  • Test drug An external spray prepared by the present invention having an activity unit of 10 IU/ml.
  • the animals in the drug-administered group were given 10 IU/ml of the test drug at a dose of 1 ml / kg, and the dose was 10 IU. /kg (1978 times for clinical use, 50 kg for female adults), control animals were given vaginal equal volume excipients.
  • the animals were routinely observed and the general condition of the animals was observed, and the death was recorded for 14 days and weighed once on the 4th, 7th, 11th, and 14th days after administration. At the end, the animals were sacrificed, and the system was autopsied to observe whether the main organs had changed. )
  • the stimulating effect and intensity of the external spray of the present invention on the vaginal mucosa of experimental animals were examined, and the safety evaluation basis was provided for clinical application.
  • the local stimulatory response to vaginal mucosa after continuous vaginal administration of rabbits was determined.
  • Test drug An external spray prepared by the present invention having an activity unit of 1 IU/ml.
  • Control drug 0.01 g/ml of chitosan-acetic acid solution.
  • Test animals Female rabbits, weighing 2.0kg ⁇ 2.5kg, Certificate No.: SCXK [Liao] 2003-011, provided by the Experimental Animal Center of China National Institute for the Control of Pharmaceutical and Biological Products.
  • the chitosan-acetic acid solution control group and the external spray administration group of the present invention have smooth surface of the vaginal tissue, and no obvious vascular congestion and edema are observed. Abnormal changes.
  • the surface of the vaginal tissue of the control group of chitosan-acetic acid solution was smooth, and only one of them showed little hyperemia.
  • the surface of the vaginal tissue of the external spray group of the present invention was smooth, and two of them showed little congestion; Conclusion: The external use of the present invention The spray had no obvious stimulating reaction on the vaginal mucosa of the experimental animals.
  • vaginal topical administration of the external spray of the present invention for 12 weeks due to the toxicity and severity of the accumulation, providing the target organ of the toxic reaction and the reversibility of the damage, determining the non-toxic reaction dose, and providing a reference for the clinical human dose .
  • Test drug An external spray prepared by the present invention having an activity unit of 1 IU/ml.
  • Test animals Female rabbits, weighing 2.0kg ⁇ 2.5kg, Certificate No.: SCXK [Liao] 2003-011, provided by the Experimental Animal Center of China National Institute for the Control of Pharmaceutical and Biological Products.
  • Control group 0.01 g/ml chitosan-acetic acid solution.
  • Female rabbits were divided into two groups: high and low, and one control group.
  • the dose group was 1, 0.3 IU/kg, which was equivalent to 179 and 54 times of the clinical human dose (calculated according to the female adult weight of 50 kg). ), slowly injecting chitosan-acetic acid solution or the spray of the present invention into the vagina of the rabbit directly by a syringe, once a day, once a dose, according to the weight of the rabbit, the dose per kilogram of body weight is 1 ml, continuously given The drug was taken for 12 weeks for histopathological examination.
  • the topical spray of the present invention has no obvious toxic effect, no accumulation or delayed toxicity, and long-term continuous drug safety is better.
  • the main component of the external spray used in the present invention is a rabies purified vaccine which can induce a strong immune response in the body.
  • the T-cell subpopulation is significantly improved before and after the test.
  • HPV-positive virus carriers can be used for cervical cancer, genital herpes, genital warts, cervical erosion, and chronic cervicitis caused by human papillomavirus infection after a course of treatment. treatment.

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Description

用于治疗人乳头瘤病毒感染引起的疾病的喷剂及其制备方法 技术领域
本发明涉及医药技术领域，具体地说是一种用于治疗人乳头瘤病毒感染引起的疾 病的喷剂及制备。 背景技术
人用狂犬病纯化疫苗 (KI)在临床使用过程中， 发现其作为一种生物抗原刺激机体 后， 可诱发机体产生强有力的免疫反应， （抗原特异性免疫； 非抗原特异性免疫） 而 宿主的免疫反应， 对控制相关病变具有十分重要的作用。 大量的文献研究资料证明： 人体自身免疫力低下与癌症的发生有着相当密切的联系， 一旦机体免疫力降低，各种 疾病可以乘隙而入。 临床的资料又证明， 在肿瘤病毒与人类疾病关系的研究中，只有 人乳头瘤病毒 (human papilloma virus, HPV)与人类肿瘤的关系最为明确。 1995年 WHO 和 IARC 已将 HPV确定为是宫颈癌的病因。 HPV主要侵犯人皮肤粘膜的上皮组织， 引起皮肤粘膜上皮的良恶性增生， 墨西哥医学专家指出，人乳头状瘤病毒 (HPV)潜伏 期很长，女性感染这种病毒后如果发现早、 诊治及时，可防止病毒诱发宫颈癌。 目前 HPV已分离出 100多型，根据诱发病变性质的不同，将 HPV分为: 13种高危型 HPV(16、 18、 31、 33、 35、 39、 45、 51、 52、 56、 58、 59、 68)， 低危型 HPV主要包括： HPV(6、 11、 32、 40、 42、 43、 54、 MM8)。 高危型 HPV病毒几乎是所有宫颈癌的元凶， 在细 胞学和分子生物学方面也获得了人乳头状瘤病毒致癌的有力证据。 大量研究表明，高 危型人乳头瘤病毒 (HPV)感染是宫颈癌的主要病因，临床病理检查中，宫颈癌患者几乎 100%的有高危型人乳头瘤病毒 (HPV)的感染，高危型 HPV(16、 18)亚型与肾恶性肿瘤 有关 （其中肾乳头状癌的 HPV(16、 18)基因阳性率明显高于肾透明细胞癌和肾颗粒 癌） ， 证实了高危型 HPV感染是肾癌的重要致癌原因， 有资料证明了食管鳞癌与 E6 癌基因蛋白的关系。 低危型 HPV与某些性病有关， 包括生殖器疱疹、 尖锐湿疣以及 宫颈糜烂、 慢性宫颈炎等， 其中 HPV6和 11是最常见的低危型 HPV， 90%的生殖道 尖锐湿疣与之有关。目前抑制人乳头瘤病毒及治疗人乳头瘤病毒感染引起的肿瘤和采 用人用狂犬病纯化疫苗治疗性病的外用剂型及制备方法还尚未见报道。 发明内容
本发明的目的在于提供一种用于治疗人乳头瘤病毒及治疗人乳头瘤病毒感染引 起的肿瘤和性病的喷剂及其制备方法，采用本发明除对肿瘤具有明显的治疗作用、无 毒副作用外，本发明喷剂可保存人用狂犬病纯化疫苗的生物活性并对人体没有造成任 何负面损伤，使局部用药成为可能，本发明所说的局部用药均指的是直接往感染病患 处用药。
本发明技术方案如下：
本发明喷剂的制备方法：
本发明的成份为 1ml的人用狂犬病纯化疫苗，加为 0.01g/ml的壳聚糖 -醋酸溶液至 10ml制成，其中所述 0.005〜0.015g/ml壳聚糖-醋酸为： 10g壳聚糖及 1000ml醋酸溶液。
制备人用狂犬病纯化疫苗的病毒株为但不限于狂犬病毒 PM株、 PV株、 CTN株 或 AG株。
用于治疗人乳头瘤病毒感染引起的疾病的喷剂的制备方法， 按如下步骤操作： 首先，配制壳聚糖 -醋酸溶液：
1 )制备 0.3〜1.0%醋酸溶液：将冰醋酸 3〜10体积份加入至 1000体积份容量瓶中， 加纯水至刻度， 得到体积百分浓度为 0.3〜1%的醋酸溶液；
2)制备壳聚糖 -醋酸溶液： 称取壳聚糖 10〜30g加入至 1000ml容量瓶中， 加上述 的醋酸溶液， 完全溶解至刻度，制成 0.01〜0.03g/ml的壳聚糖 -醋酸溶液；
3 )根据壳聚糖 -醋酸溶液的 pH值选用体积摩尔浓度为 O.lmol/L的氢氧化钠溶液 或 0.1mol/L的醋酸溶液调节 pH至 3.8〜5.0；
其次， 配制疫苗溶液
取活性单位为 5IU〜20IU的人用狂犬病纯化疫苗 lml， 加壳聚糖-醋酸溶液稀释至 10ml,获得活性浓度为 5IU〜20IU/10ml的疫苗溶液，根据壳聚糖 -醋酸溶液的 pH值选 用体积摩尔浓度为 O.lmol/L 的氢氧化钠溶液或 O.lmol/L 的醋酸溶液调节 pH 至 3.8-5.0, 优选 pH值为 4.9〜5.0。
其中所述人用狂犬病纯化疫苗可以为溶液型或冻干型。
其中所述冰醋酸采用分析纯或化学纯。
其中所述纯水采用重蒸馏水。
本发明的喷剂用于治疗人乳头瘤病毒感染引起的宫颈癌、人乳头瘤病毒感染引起 的生殖器疱疹或生殖器尖锐湿疣、人乳头瘤病毒感染引起宫颈糜烂、人乳头瘤病毒感 染引起的慢性宫颈炎。
本发明的喷剂为外用剂型。
本发明具有如下有益效果：
1. 使用带有正离子并能在酸性条件下可完全溶解的壳聚糖作为粘附剂， 使人用 狂犬病纯化疫苗完全溶解， 并使本发明的喷剂喷出之后具有好的气雾性， 从而使药物 喷出后均匀覆盖于病灶粘膜表层， 有利于机体对药物的吸收。
2. 本发明外用喷剂及制备方法着重对由 HPV感染引起的宫颈癌患者、生殖器疱 疹和尖锐湿疣患者、 宫颈糜烂、 慢性宫颈炎， HPV感染人群进行跟踪治疗 （Tc亚群 检测、 HPV检测及临床症状的改善） ， 均取得良好的临床效果。
3. 采用本发明主要成分为人用狂犬病纯化疫苗， 通过对人宫颈癌 Hela人源性肿瘤 细胞的体外 MTT法检测实验证明本发明主要成份人用狂犬病纯化疫苗对肿瘤细胞的直 接杀伤作用。 通过裸鼠移植性肿瘤抑瘤实验得知： 主要成份人用狂犬病纯化疫苗局部注 射对裸鼠异种移植瘤人宫颈癌 Hela肿瘤生长显示明显抑制作用。 本发明在对机体免疫 调节作用中使小鼠单核细胞、 腹腔吞噬细胞的吞噬能力增强， 说明对网状内皮系统吞噬 功能具有明显的激活、 增强作用。 所以本发明发现人用狂犬病疫苗可诱发机体产生强有 力的免疫反应， HPV感染者经系统治疗后 （未经其它治疗途径)， HPV检测阳转阴。
4. 本发明的毒理实验证明:该外用剂和皮肤重复接触中致敏反应强度为弱致敏 性。 小鼠以及家兔急性毒性实验未见明显毒性反应。 小鼠微核实验结果表明： 小鼠肌 肉注射给药 <375U+25mg/kg剂量下未诱发骨髓嗜多染红细胞微核率的增高。
5. 在本发明阴道刺激试验中， 通过测定家兔或大鼠连续阴道给药后未见明显血 管充血和水肿等异常改变。多次给药刺激试验也证明本发明外用剂对实验动物阴道黏 膜无明显刺激反应。
6. 在对家兔连续给药 12周长期毒性试验中， 确定出无毒性反应的用药剂量， 为 临床人用剂量提供了参考。 附图说明
图 1本发明疫苗溶液对人宫颈癌 Hela细胞周期分布的影响。 具体实施方式
下面通过实施例对本发明作进一步说明， 本发明并不受这些实施例的限制。 制剂实施例 1
本发明喷剂成分：
按体积比计， 成份为 1ml的人用狂犬病纯化疫苗，加质量体积浓度为 0.01g/ml壳聚 糖-醋酸溶液稀释至 10ml。
具体配备方法如下：
1 . 配制壳聚糖-醋酸溶液 （pH=3.2)
( 1 ) 制备 0.3%的醋酸溶液： 将冰醋酸 3ml (分析纯） 加入至 1000ml容量瓶中， 加纯水至刻度， 这里的纯水采用重蒸馏水， 得到体积百分浓度为 0.3 %的醋酸溶液；
(2)制备质量体积浓度为 0.01g/ml的壳聚糖 -醋酸溶液： 称取壳聚糖 10g加入至 上述的醋酸溶液完全溶解至 1000ml, 溶液不允许出现不溶性颗粒或不溶物， 制成质 量体积浓度为 0.01g/ml的壳聚糖 -醋酸溶液；
( 3 )根据壳聚糖 -醋酸溶液的 pH值选用体积摩尔浓度为 O. lmol/L的氢氧化钠溶 液或者体积摩尔浓度为 O. lmol/L的醋酸溶液调节 pH值至 3.2。
2. 疫苗溶液配制
取活性单位为 5IU的溶液型人用狂犬病纯化疫苗 1ml (本实施例采用两支， 每支 0.5ml, 2.5IU) 溶液加入至 10ml量瓶中， 人用狂犬病纯化疫苗制备病毒株采用狂犬病 毒 PM株， 加壳聚糖-醋酸溶液稀释至 10ml。 获得活性浓度为 5IU/10ml的疫苗溶液。 根据疫苗溶液的 pH值选用体积摩尔浓度为 O. lmol/L的氢氧化钠溶液或体积摩尔浓度 为 O. lmol/L的醋酸溶液调节 pH值至 3.2， 溶液具有澄明度。
说明： pH值保持在 3.8到 5.0之间， 如 pH值超过 5.0或低于 3.8壳聚糖与醋酸 脱离， 溶液变成不透明状，成份发生了改变， 在使用时， 喷出的溶液将不呈现雾状。
制剂实施例 2
与实施例 1不同之处在于：
1 . 配制壳聚糖-醋酸溶液 （pH=4.9):
制备 0.4 % 的醋酸溶液采用的冰醋酸 4ml为 （化学纯）； 用体积摩尔浓度为 0.1 mol/L的氢氧化钠溶液或者体积摩尔浓度为 0.1 mol/L的醋酸溶液调节 pH值至 4.9。
制备质量体积浓度为 0.02g/ml的壳聚糖 -醋酸溶液： 称取壳聚糖 20g加入至上述 的醋酸溶液完全溶解至 1000ml, 溶液不允许出现不溶性颗粒或不溶物， 制成质量体 积浓度为 0.02g/ml的壳聚糖 -醋酸溶液。
2. 疫苗溶液配制
取活性单位为 15IU的冻干型人用狂犬病纯化疫苗 1ml (本实施例采用两支， 每 支 0.5ml， 7.5IU)加入至 10ml量瓶中， 人用狂犬病纯化疫苗的制备病毒株为狂犬病毒 PV株。加壳聚糖-醋酸溶液稀释至 10ml。获得活性浓度为 15IU/10ml的疫苗溶液。用 体积摩尔浓度为 O. lmol/L的氢氧化钠溶液或体积摩尔浓度为 O. lmol/L的醋酸溶液调 节 pH值至 4.9， 溶液具有澄明度。
制剂实施例 3
与实施例 1不同之处在于：
1 . 配制壳聚糖-醋酸溶液 （pH=5.0):
制备 1.0% 醋酸溶液采用的冰醋酸 10ml为化学纯； 用体积摩尔浓度为 0.1 mol/L 的氢氧化钠溶液或体积摩尔浓度为 O. lmol/L的醋酸溶液调节 pH至 5.0。
制备质量体积浓度为 0.03g/ml的壳聚糖 -醋酸溶液： 称取壳聚糖 30g加入至上述 的醋酸溶液完全溶解至 1000ml, 溶液不允许出现不溶性颗粒或不溶物， 制成质量体 积浓度为 0.03g/ml的壳聚糖 -醋酸溶液。
2. 疫苗溶液配制
取活性单位为 10IU的溶液型人用狂犬病纯化疫苗 1ml (本实施例采用两支， 每 支 0.5ml， 5IU) 溶液加入至 10ml量瓶中， 人用狂犬病纯化疫苗的制备病毒株为狂犬 病毒 CTN株。 加壳聚糖-醋酸溶液稀释至 10ml。 获得浓度为 lOIU/lOml的疫苗溶液。 用体积摩尔浓度为 O. lmol/L的氢氧化钠溶液或体积摩尔浓度为 O. lmol/L的醋酸溶液 调节 pH值至 5.0， 溶液具有澄明度。
制剂实施例 4
与实施例 1不同之处在于：
1 . 配制壳聚糖-醋酸溶液 （pH=4.2):
制备 0.4% 醋酸溶液采用的冰醋酸 4ml为化学纯；用体积摩尔浓度为 O. lmol/L的 氢氧化钠溶液或体积摩尔浓度为 O. lmol/L的醋酸溶液调节 pH至 4.2。
2. 疫苗溶液配制
取活性单位为 20IU的溶液型人用狂犬病纯化疫苗 1ml (本实施例采用两支， 每 支 0.5ml， 10IU) 溶液加入至 10ml量瓶中， 其中人用狂犬病纯化疫苗的制备病毒株 为狂犬病毒 AG株。 加壳聚糖-醋酸溶液稀释至 10ml。 获得浓度为 20IU/10ml的疫苗 溶液。 用体积摩尔浓度为 O. lmol/L的氢氧化钠溶液或体积摩尔浓度为 O. lmol/L的醋 酸溶液调节 pH值至 4.2， 溶液具有澄明度。
制剂实施例 5
与实施例 1不同之处在于：
1 . 配制壳聚糖-醋酸溶液 （pH=4.9):
制备 0.4% 醋酸溶液采用的冰醋酸 4ml为化学纯；用体积摩尔浓度为 O. lmol/L的 氢氧化钠溶液或体积摩尔浓度为 O. lmol/L的醋酸溶液调节 pH至 4.9。
2. 疫苗溶液配制
取活性单位 15IU的溶液型人用狂犬病纯化疫苗 1ml (本实施例采用两支， 每支 0.5ml, 7.5IU) 溶液加入至 10ml量瓶中， 人用狂犬病纯化疫苗的制备病毒株为狂犬病 毒 PM株。 加壳聚糖-醋酸溶液稀释至 10ml。 获得浓度为 15IU/10ml的疫苗溶液。 用 体积摩尔浓度为 O. lmol/L的氢氧化钠溶液或体积摩尔浓度为 O. lmol/L的醋酸溶液调 节 pH值至 4.9， 溶液具有澄明度。 病情治疗案例一： 孙某某
病理诊断： 宫颈鳞癌前变， CINII; 经宫颈椎切术后， HPV阳性， 干扰素针剂注 射及干扰素药膏局部用药治疗 2个月， HPV依然阳性。
具体临床用量:取制剂实施例 4中活性浓度为 20IU/10ml经本发明喷剂局部用药， 每日 4次， 一次一喷 （ 140μ1)， 用药 45天， 停药 20天后做 HPV检测， 结果显示为 阳转阴。
病情治疗案例二： 周某某
病理诊断： 宫颈鳞癌， II期 Β; 术后化疗两个周期， HPV检测为阳性。
具体临床用量： 取制剂实施例 5中活性浓度为 15IU/10ml的本发明喷剂外用， 每 日 4次， 一次一喷（140μ1)， 20天为一个疗程， 共两个疗程， 停药 20天后做 HPV检 测， 结果显示为阳转阴。
病情治疗案例三： 安某
临床诊断： 生殖器疱疹， HPV阳性；
具体临床用量： 取制剂实施例 3中活性浓度为 lOIU/lOml的本发明喷剂外用， 每 日 4次， 一次一喷（140μ1)， 20天为一个疗程， 共三个疗程， 停药后疱疹消失， 停药 20天后做 HPV检测， 结果显示为阳转阴。
病情治疗案例四： 田某某
临床诊断： 生殖器尖锐湿疣， HPV阳性；
具体临床用量： 取制剂实施例 4中活性浓度为 20IU/10ml的本发明喷剂外用， 每 日 6次， 一次一喷（140μ1)， 20天为一个疗程， 共四个疗程， 停药后疣体干瘪部分脱 落， 停药 20天后做 HPV检测， 结果显示为阳转阴。
病情治疗案例四： 许某某
临床诊断： 宫颈糜烂， HPV阳性；
具体临床用量： 取制剂实施例 1中活性浓度为 5IU/10ml的本发明喷剂外用， 每 日 6次， 一次一喷（140μ1)， 20天为一个疗程， 共四个疗程， 停药后检查宫颈粘膜恢 复正常， 停药 20天后做 HPV检测， 结果显示为阳转阴。
病情治疗案例四： 白某
临床诊断： 慢性宫颈炎， HPV阳性；
具体临床用量： 取制剂实施例 2中活性浓度为 15IU/10ml的本发明喷剂外用， 每 日 6次， 一次一喷（140μ1)， 30天为一个疗程， 共四个疗程， 停药后炎症消失， 停药 20天后做 HPV检测， 结果显示为阳转阴。
实验实施例
本发明对肿瘤细胞的起到直接杀伤的作用通过如下实验可以得到进一步了解。 实验实施例 1
对人源性肿瘤细胞的体外 ΜΤΤ法检测实验
实验试剂与药品：
本发明外用喷剂， 根据实际需要选用不同的活性浓度。
阳性对照药： 氟尿嘧啶注射液， 批号 050806， 为上海旭东海普药业有限公司产
Π
ΡΠ。
细胞株： Hela (人宫颈癌细胞） 由中国医科院药物研究所培养传代。
仪器： BIORAD 550型酶标仪
实验方法： 四氮唑盐 [3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyl-tetrazolium bromide, MTT ] 还原法。
收集生长良好的肿瘤细胞， 于 96孔培养板内接种， 培养 24小时后加药。 受试样 品培养 4天。用 550型酶标仪在检测波长 540nm， 参考波长 405nm下测定个孔的吸光 度 （OD) 值。 实验数据经处理得到半数抑制浓度 ac50)。
实验结果显示： 本发明所采用的主要成分为人用狂犬病纯化疫苗对 HelaIC5()< 0.1IU/ml。 表明了本发明主要成份为人用狂犬病纯化疫苗对人源癌细胞均有体外增殖 抑制作用。
实验实施例 2
利用流式细胞仪对人宫颈癌 Hela细胞周期分布产生影响的测试。
实验试剂与药品： 本发明疫苗溶液， 根据实际需要选用不同的活性浓度。
细胞株：
采用实验实施例中的 Hela细胞。
结果提示： 人用狂犬病疫苗对人宫颈癌 Hela 细胞周期分布产生影响， 人用狂犬 病纯化疫苗可使人宫颈癌细胞周期阻滞于 G2/M期，提示人用狂犬病纯化疫苗对肿瘤 增殖抑制作用与诱导肿瘤细胞周期阻滞有关， 参见图 1。 药物可通过诱导 G2/M期周 期阻滞抑制肿瘤细胞增殖 (图谱的第二个峰为周期阻滞抑制峰)。
实验实施例 3
本发明疫苗溶液对人宫颈癌 Hela裸鼠异种移植瘤生长抑制作用。
受试药物： 制剂实施例 1中疫苗溶液， 活性单位为 5IU/ml。
实验材料：
1 . 裸鼠， 北京维通利华实验动物有限公司提供， 合格证号： SCXK (京） 2007 一 0001。
2. 人宫颈癌细胞株 Hela, 自体外培养细胞接种于裸鼠成瘤。 发明人传代保存。
3. 伺养小鼠颗粒料， 由沈阳实验动物颗粒伺料厂提供。
4. 对照药： 阿霉素批号： 060303B, 提供单位： 浙江海正药业股份有限公司， 用生理药水稀释 0.1mg/ml备用。
实验方法：
选择肿瘤生长良好的荷瘤动物， 颈椎脱白处死。无菌条件下取出瘤块， 用手术刀 切割成直径 2-3mm的瘤块， 套管针接种于裸鼠腋部皮下。 7天后可见肿瘤生长。 用 游标卡尺测量肿瘤长、 宽径， 按瘤体积大小分层分组。 每组 7只动物。 设阴性对照组 (不给药），本发明疫苗溶液 0.2ml肿瘤局部给药 1组，本发明疫苗溶液 0.1ml肿瘤局 部给药加 0.1ml肌肉注射给药 1组， 本发明疫苗溶液 0.1ml、 0.2ml、 0.4ml肌肉注射 给药各 1组。 阿霉素 5mg/kg腹腔注射给药作为阳性对照。 自分组当日开始给药， 每 日 1次， 给药 20天。
给药后， 每周 2〜3次对实验动物肿瘤的长、 宽径及体重进行测量记录， 观察动 物肿瘤生长及一般情况。 20 天之后将动物处死后剥离肿瘤， 称瘤重， 计算药物对肿 瘤生长抑制率。 计算肿瘤体积 （TV)及相对肿瘤体积 （RTV), 比较各组动物肿瘤重 量、 肿瘤体积、 相对肿瘤体积 （RTV) 等指标的统计学差别 （见表 1与表 2)。 解组织肉眼可见： 肿瘤周围轻度水肿， 瘤组织块体积小， 表面有破溃或结痂。 剥 离肿瘤见肿瘤壁薄， 内容物为黄绿色豆渣样。肌肉给药改局部注射组的肿瘤则可见肿 瘤包膜薄， 瘤组织整体泛黄色，瘤组织质软， 无张力。
结论： 在实验选用的剂量范围内， 本发明所采用的主要成份人用狂犬病纯化疫苗 局部给药和局部肌肉注射给药对裸鼠异种移植瘤人宫颈癌 Hela肿瘤生长显示明显抑 制作用。 表 1 本发明疫苗溶液对人宫颈癌 Hela裸鼠异种移植瘤体重抑制作用
动物数 体重 ( g)
抑制率 组别 开 结 开始 结束 瘤重 ( g)
(%) 始 束
阴性对照 7 7 21.4±1.05 27.3±2.37 2.94±1.469
局部给药 0.2ml 7 7 20.4±0.73 20.7±1.28 0.46±0.337 84.3 局部给药 0.1ml+
7 7 21.0±1.31 20.7±1.03 0.57±0.410 80.6 局部肌肉注射 0.1ml
局部肌肉注射
7 7 21.4±0.49 26.1±1.88 2.99±1.206 0 0.1ml
局部肌肉注射
7 7 20.6±1.29 25.9±2.36 2.93±1.540 0.3 0.2ml
局部肌肉注射
0.4ml 7 7 20.4±1.05 22.7±1.39 1.61±1.219 45.2 改局部给药 0.4ml
阿霉素 5mg/kg 7 7 21.0±1.07 18.4±1.76 1.14±0.663 61.2 注： 与阴性对照组相比， P<0.05 表 2 本发明疫苗溶液对人宫颈癌 Hela裸鼠异种移植瘤体积抑制作用 组别 肿瘤体积 (mm3 ) RTV T/C 开始 结束 (%) 阴性对照 149±76.9 3841±2272.8 30.2±17.03
局部给药 0.2ml 109±46.1 428±329.5 3.43±1.57 11.4 局部给药 0.1ml十局
137±106.8 670±454.7 5.50±2.14 18.2 部肌肉注射 0.1ml
局部肌肉注射 0.1ml 125±85.2 3389±1624.2 30.9±10.63 >100 局部肌肉注射 0.2ml 148±112.6 3016±1346.5 27.7±14.14 91.7 局部肌肉注射 0.4ml
134±97.9 1862±1257.9 20.1±14.26 66.6 改局部给药 0.4ml
阿霉素 5mg/kg 135±74.2 1428±619.0 12.8±8.61 42.4 注： 与阴性对照组相比， P<0.05
实验实施例 4
本发明研究了药物对荷瘤小鼠免疫指标的调节作用、增效解毒作用，对放化疗损 伤修复的调节作用及预防作用。
1. 小鼠单核吞噬细胞功能的实验结果； 用药后小鼠单核吞噬细胞功能明显提高， 吞噬指数与吞噬活性分别为 0.3480和 8.2870。 其研究结果经数据处理具有显著性差 异， 组间比较 P <0.1。
2.用药后使小鼠腹腔巨噬细胞吞噬率达 35.02%，吞噬指数亦明显提高，达 1.02， 还可增加吞噬细胞消化功能； 使血清半数溶血值增加，对化疗后抗体形成有较强的调 节作用 （免疫功能的调节作用）。
3. 可迅速回升外周血白细胞总数达正常水平、 用药后可使白细胞在 7-10天回升 至正常水平 （解毒作用）。
4. 荷瘤小鼠单用药物抑瘤率为 39.03 %， 如与化疗并用， 可达 59.53% (增效作 用）。
5 . 用药后可增强小鼠肝脏超氧化物歧化酶 （SOD ) 的活性， SOD 水平为
662.97ug/ml。 化疗时并用为 452.69 ug/ml ， 高于对照组， 与正常对照组相当 （抗氧 化作用）。
6. 对辐射损伤的修复也较明显， 如照射后动物开始出现死亡较晚， 出现头部皮 肤及粘膜损伤较轻， 而对照组损伤较重， 头面部皮肤、 粘膜溃疡严重， 大部脱毛并伴 有腹泻等症状。 实验组小鼠骨髓微核率未见明显升高。 而且 30天存活率为 82.07%， 平均月生存时间为 24.62天， 均高于对照组 （放化疗损伤修复的调节作用）。
7. 药物可延长小鼠游泳时间达 75%， 明显增强耐力 （预防作用）。
结果提示，通过免疫和抗氧化等动物实验，可对免疫功能产生了明显的抑制作用， 使抗体的形成有所提高， 促进吞噬细胞的趋化、 黏附作用， 促进细胞内活性物质的释 放， 进而促进吞噬和消化功能。
实验实施例 5
毒理实验：
以下试验均按照<新药临床前抗肿瘤药指导原则 >和<第三届全国肿瘤药理及化 疗会议制定方案>进行。
一. 皮肤过敏试验：
受试药物： 根据本发明制备方法制成活性单位： 10 IU/ml外用喷剂。
实验材料：
1.受试动物：豚鼠，雌、雄各半，体重 250-300g，实验动物使用许可证号： SCXK (辽） 2003-0011， 由沈阳市于洪区前民养殖场提供。
2. 对照药：
1 ) 阳性对照药： 2， 4一二硝基氯代苯 （批号： 20070622) 天津市津科精细化工 研究所提供； 用生理盐水稀释为体积浓度为 1%与 0.1%， 待用。
2) 赋形剂组： 0.01g/ml的壳聚糖 -醋酸溶液。
实验方法：
取豚鼠 30只， 雌雄各半， 每组 10只， 用 8%硫化钠脱背部毛， 每侧脱毛面积约 为 3x3cm2， 将三组动物左侧脱毛区分别涂敷 0.2 ml赋形剂、 本发明疫苗溶液和体积 浓度为 1% 2， 4一二硝基氯代苯， 给药后固定 6小时， 于给药后第 7、 14天重复上述 给药过程， 于末次给药 14天后重复上述给药过程， 给药部位为右侧脱毛区， 阳性对 照药物体积浓度改为 0.1%。 6小时洗掉药物， 即刻观察， 然后于 24、 48、 72小时再 次观察皮肤过敏反应。）
结果： 给药组和赋形剂组对豚鼠给药处接触处皮肤无红斑或水肿形成， 2， 4一二 硝基氯代苯反应强度为极度致敏性。
结论： 采用本发明外用喷剂和皮肤重复接触， 未发现对受试动物皮肤引起明显过 敏反应。 致敏反应强度为弱致敏性。
二. 小鼠微核实验：
了解本发明主要成份人用纯化狂犬疫苗是否存在引起动物体内染色体畸变的遗 传毒性。
受试药物： 冻干型人用纯化狂犬疫苗和聚肌胞注射液的混合液体， 冻干型人用纯 化狂犬疫苗 （批号 20050802-4)， 由辽宁成大生物技术有限公司提供； 聚肌胞注射液 (生产批号 060418)， 2mg/2ml， 由浙江万马药业有限公司提供。 聚肌胞注射液 2.0ml 溶解冻干人用纯化狂犬疫苗 30IU/12ml， 其最大溶解浓度为 15IU+ lmg/ml。
实验材料：
1 . 受试动物： 小鼠， 雄、 雌各半， 体重 22-24g， 实验动物使用许可证号： SYXK (京) 2006-0004号， 由中国药品生物制品检定所实验动物中心提供。
2. 对照药
阳性对照药： 环磷酰胺 （批号 06071721 )， 江苏恒瑞医药股份有限公司生产， 购 于北京医院药房。 用注射用水配制成浓度为 2mg/ml的溶液。
实验方法：
受试药物按最大溶解浓度 （（15IU+lmg) /ml) 和最大给药容积 （0.5ml/20g) 作 为高剂量，共设三个剂量组（（375IU+25m) /kg、（125IU+8mg) /kg和（42IU+3mg) /kg), 每组 10只小鼠， 小鼠颈背部肌肉注射给药， 第 1次给药后 24小时进行第 2次 给药， 给药 6小时后， 脱颈椎处死小鼠， 常规取股骨骨髓， 涂片， 计数 100个嗜多染 红细胞 （P) 与正染红细胞 （N) 的比例。）
结果： 受试药物各剂量组的微核率均<1.9%。； 阳性对照组的微核率 >29.0%。。 结果表明： 小鼠肌肉注射给药 <375IU+25mg/kg剂量下未诱发骨髓嗜多染红细胞 微核率的增高。提示： 本发明主要成份人用纯化狂犬疫苗在试验剂量范围内， 小鼠微 核实验为阴性结果。
三. 小鼠急性毒性实验:：
受试药物： 冻干型人用狂犬病纯化疫苗 （批号 20050802-4)， 由辽宁成大生物技 术有限公司提供。 实验前用注射用水溶解为活性浓度为 15IU/ml。
实验材料：
小鼠， 雄、 雌各半， 体重 19-21g， 实验动物使用许可证号： SYXK (京) 2006-0004 号， 由中国药品生物制品检定所实验动物中心提供。
实验方法：
1 )静脉注射最大给药量的测定： 小鼠受试药物按活性单位 （15IU/ml)和最大给 药容积（0.5ml/20g)， 1 日内 2次静脉注射给药， 连续给药 14日， 未见明显毒性反应。 其最大给药量为 750IU+50mg/kg， 相当于临床人肌肉注射拟用量的 （体重按 60kg计 算） 20833倍。
2) 皮下注射最大给药量的测定： 小鼠受试药物活性单位 （15IU/ml)、 最大给药 容积 （0.5ml/20g)， 一日内 2次肌肉注射给药， 连续给药 14日， 未见明显毒性反应。 其最大给药量为 750IU+50mg/kg， 相当于临床人肌肉注射拟用量的 （体重按 60kg计 算） 20833倍。）
结论： 小鼠急性毒性实验表明， 本发明外用喷剂的毒副作用小。
四. 家兔急性毒性实验：
受试药物： 采用本发明所制成的活性单位为 10IU/ml的外用喷剂。
赋形剂： 0.02g/ml的壳聚糖 -醋酸溶液。
实验材料： 雌性家兔， 体重 2.1-2.5kg， 合格证号： SCXK [辽] 2003-011号， 由中 国药品生物制品检定所实验动物中心提供。
实验方法：
取家兔 12只分为给药组和对照组， 每组 6只， 给药组动物经阴道给予 10 IU/ml 浓度受试药物，给药容积为 1 ml /kg，给药剂量为 10 IU/kg (为临床人用剂量 1786倍， 按女性成人 50 kg计算）， 对照组动物经阴道给予等容积赋形剂。 给药后正常伺养并 观察动物的一般状况， 并记录死亡情况， 连续观察 14天， 并于给药后第 4、 7、 11、 14天称重一次。 完毕， 处死动物， 系统尸检观察主要脏器有无改变。）
实验结果：
家兔阴道给予本发明喷剂或赋形剂后， 即刻观察未见有异常体征及死亡动物出 现。 连续观察 14天， 家兔体重正常增长， 未见毛发、 呼吸、 四肢、 皮肤和行为等方 面有异常改变， 也未见死亡动物出现。 系统尸检主要脏器未见肉眼可见的病理改变。
结论： 对家兔阴道局部给药的急性毒性实验结果表明， 本发明喷剂对家兔阴道 局部给药的最大给药量为 10 IU/kg (为临床人用剂量 1786倍，按女性成人 50 kg计算）， 本发明外用喷剂的毒副作用小。
五. 阴道刺激试验
考察本发明外用喷剂对实验动物阴道黏膜的刺激作用和强度，为临床应用提供安 全性评价依据。
测定家兔连续阴道给药后对阴道粘膜局部的刺激反应。
受试药物： 采用本发明所制成的活性单位为 lIU/ml的外用喷剂。
对照组药物： 0.01g/ml的壳聚糖 -醋酸溶液。
实验材料：
受试动物： 雌性家兔， 体重 2.0kg〜2.5kg， 合格证号： SCXK [辽] 2003-011号， 由 中国药品生物制品检定所实验动物中心提供。
实验方法：
1 单次给药刺激试验：
取家兔 6只， 分为对照组组和给药组， 每组 3只， 直接用注射器缓慢注入 2ml 壳聚糖-醋酸溶液或本发明外用喷剂于家兔阴道内，给药后 24 小时后各组分别处死动 物， 纵向切开阴道， 肉眼观察有无水肿， 血管充血后， 作病理组织学检查。 2 多次给药刺激试验
取家兔 6只， 分为对照组组和给药组， 每组 3只， 直接用注射器缓慢注入 2ml 壳聚糖-醋酸溶液或本发明外用喷剂于家兔阴道内， 每天 1次， 共 7天， 末次给药后 24 小时后各组分别处死动物， 纵向切开阴道， 肉眼观察有无水肿， 血管充血后， 作 病理组织学检查。
实验结果：
1 ) 单次给药刺激试验：
采用本发明外用喷剂， 单次给药后 24h阴道组织肉眼观察结果： 壳聚糖-醋酸溶 液对照组与本发明外用喷剂给药组动物阴道组织表面光滑，未见明显血管充血和水肿 等异常改变。
2) 多次给药刺激试验：
壳聚糖-醋酸溶液对照组的动物阴道组织表面光滑， 1只可见极少充血； 本发明外用喷剂给药组的动物阴道组织表面光滑， 2只可见极少充血； 结论： 采用本发明外用喷剂对实验动物阴道黏膜无明显刺激反应。
六. 对家兔连续给药 12周长期毒性试验
观察家兔阴道局部给予本发明外用喷剂 12周由于蓄积产生的毒性反应及其严重 程度， 提供毒性反应的靶器官及其损害的可逆性， 确定无毒性反应剂量， 为临床人用 剂量提供参考。
受试药物： 采用本发明所制成的活性单位为 lIU/ml的外用喷剂。
实验材料：
1. 受试动物： 雌性家兔， 体重 2.0kg〜2.5kg， 合格证号： SCXK [辽] 2003-011号， 由中国药品生物制品检定所实验动物中心提供。
2. 对照组： 0.01g/ml的壳聚糖 -醋酸溶液。
实验方法：
取雌性家兔， 设高、 低二个剂量组和一个对照组， 剂量组分别为 1、 0.3 IU/kg, 分别相当于临床人用剂量的 179和 54倍（按女性成人体重为 50 kg计算）， 直接用注 射器缓慢注入壳聚糖 -醋酸溶液或本发明喷剂于家兔阴道内， 每天给药 1次， 一次给 药按家兔体重计， 每千克体重给药容积为 lml， 连续给药 12周， 作病理组织学检查。
实验结果： 剂量组、 对照组作病理组织学检查， 各项检查指标在给药 6 周、 12 周后均未见异常， 动物整体发育良好。 在实验期间动物被毛光泽， 自发活动正常， 体 重增长正常。血象检查， 肝、 肾功能等生化检查及主要脏器病理组织学检查均未见异 常变化。主要器官组织学检查未见病理性变化，停药 2周后进行恢复性观察，对照组、 剂量组各项指标仍无异常改变。。
结论： 本发明外用喷剂未见明显毒性作用， 无蓄积性或延迟性毒性， 长期连续用 药安全性较好。
临床治疗过程中，发现本发明外用喷剂所采用的主要成份人用狂犬病纯化疫苗可 诱发机体产生强有力的免疫反应，患者经系统治疗后， T-细胞亚群检测前，后对比有明 显好转， HPV阳性病毒携带者经一疗程给药后，未经其它治疗途径， HPV检测转阴， 可用于人乳头瘤病毒感染引起的宫颈癌、 生殖器疱疹、 生殖器尖锐湿疣、 宫颈糜烂和 慢性宫颈炎的治疗。

Claims

权 利 要 求 书
1. 一种用于治疗人乳头瘤病毒感染引起的疾病的喷剂，其特征在于： 成份为 lml 的人用狂犬病纯化疫苗，加 0.01〜0.03g/ml的壳聚糖-醋酸溶液稀释至 10ml而制成。
2. 按权利要求 1所述的喷剂， 其特征在于： 所述人用狂犬病纯化疫苗的制备病 毒株为但不限于狂犬病毒 PM株、 PV株、 CTN株或 AG株。
3. 一种用于治疗人乳头瘤病毒感染引起的疾病的喷剂的制备方法， 其特征在于 按如下步骤操作：
首先， 配制壳聚糖 -醋酸溶液：
1 ) 制备 0.3〜1.0% 醋酸溶液： 将冰醋酸 3〜10体积份加入至 1000体积份容量瓶 中， 加纯水至刻度， 得到体积百分浓度为 0.3〜1.0%的醋酸溶液；
2)制备壳聚糖 -醋酸溶液： 称取壳聚糖 10〜30g加入至 1000ml容量瓶中， 加上述 的醋酸溶液， 完全溶解至刻度，制成质量体积浓度为 0.01〜0.03g/ml 的壳聚糖-醋酸溶 液；
3 )根据壳聚糖 -醋酸溶液的 pH值选用体积摩尔浓度为 O.lmol/L的氢氧化钠溶液 或 0.1mol/L的醋酸溶液调节 pH值至 3.8〜5.0；
其次， 配制疫苗溶液：
取活性单位为 5IU〜20 IU 的溶液型人用狂犬病纯化疫苗或冻干型人用狂犬病纯 化疫苗 lml， 加上述壳聚糖-醋酸溶液稀释至 10ml， 获得活性浓度为 5IU〜20IU/10ml 的疫苗溶液，根据疫苗溶液的 pH值选用体积摩尔浓度为 O.lmol/L的氢氧化钠溶液或 0.1mol/L的醋酸溶液调节 pH值至 3.8〜5.0， 优选 pH值为 4.9〜5.0。
4. 按权利要求 3所述制备方法， 其特征在于： 其中所述冰醋酸采用分析纯或化 学纯。
5. 按权利要求 3所述制备方法， 其特征在于： 其中所述纯水采用重蒸馏水。
6. 按权利要求 1〜2中任一项所述的喷剂， 其特征在于： 所述的喷剂所治疗的疾 病为人乳头瘤病毒感染引起的宫颈癌。
7. 按权利要求 1〜2中任一项所述的喷剂， 其特征在于： 所述的喷剂所治疗的疾 病为人乳头瘤病毒感染引起的生殖器疱疹或生殖器尖锐湿疣。
8. 按权利要求 1〜2中任一项所述的喷剂， 其特征在于： 所述的喷剂所治疗的疾 病为人乳头瘤病毒感染引起的宫颈糜烂。
9. 按权利要求 1〜2中任一项所述的喷剂， 其特征在于： 所述的喷剂所治疗的疾 病为人乳头瘤病毒感染引起的慢性宫颈炎。
10.按权利要求 1〜2中任一项所述的喷剂，其特征在于：所述的喷剂为外用剂型。
PCT/CN2011/071122 2010-02-26 2011-02-21 用于治疗人乳头瘤病毒感染引起的疾病的喷剂及其制备方法 WO2011103794A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021051685A1 (zh) * 2019-09-20 2021-03-25 江西善行生物科技有限公司 一种用于清除hpv的复合制剂

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102416175A (zh) * 2011-01-18 2012-04-18 辽宁成大生物股份有限公司 一种治疗hpv感染的药物以及该药物在制备治疗hpv感染药物中的应用
CN108324924A (zh) * 2018-04-23 2018-07-27 海南海泽惠生物科技有限公司 苦瓜蛋白在制备抗人乳头瘤病毒感染药物中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1537630A (zh) * 2003-10-22 2004-10-20 翁莹冰 含竹醋、壳聚糖、银离子的药剂及其制备方法和应用
CN1919337A (zh) * 2005-08-22 2007-02-28 上海医药工业研究院 一种胰岛素鼻用粉雾剂及制备方法
WO2007031030A1 (en) * 2005-09-16 2007-03-22 Jieguang Sun Drug for treating tumor and use thereof in the manufature of medicaments for treating tumor
CN101095950A (zh) * 2006-06-30 2008-01-02 辽宁成大生物股份有限公司 一种稳定的人用狂犬病疫苗冻干制剂及其制备

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5980912A (en) * 1997-03-25 1999-11-09 Zonagen, Inc. Chitosan induced immunopotentiation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1537630A (zh) * 2003-10-22 2004-10-20 翁莹冰 含竹醋、壳聚糖、银离子的药剂及其制备方法和应用
CN1919337A (zh) * 2005-08-22 2007-02-28 上海医药工业研究院 一种胰岛素鼻用粉雾剂及制备方法
WO2007031030A1 (en) * 2005-09-16 2007-03-22 Jieguang Sun Drug for treating tumor and use thereof in the manufature of medicaments for treating tumor
CN101095950A (zh) * 2006-06-30 2008-01-02 辽宁成大生物股份有限公司 一种稳定的人用狂犬病疫苗冻干制剂及其制备

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021051685A1 (zh) * 2019-09-20 2021-03-25 江西善行生物科技有限公司 一种用于清除hpv的复合制剂

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