WO2020114166A1 - 一种氯喹凝胶及其制备方法和应用 - Google Patents

一种氯喹凝胶及其制备方法和应用 Download PDF

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Publication number
WO2020114166A1
WO2020114166A1 PCT/CN2019/115216 CN2019115216W WO2020114166A1 WO 2020114166 A1 WO2020114166 A1 WO 2020114166A1 CN 2019115216 W CN2019115216 W CN 2019115216W WO 2020114166 A1 WO2020114166 A1 WO 2020114166A1
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Prior art keywords
chloroquine
water
chitosan
soluble
group
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PCT/CN2019/115216
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English (en)
French (fr)
Inventor
谢龙旭
李相玲
袁曼莉
王婷
王建瑜
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Guangzhou Hybribio Biotech Ltd
Guangzhou Hybribio Medicine Technology Ltd
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Guangzhou Hybribio Biotech Ltd
Guangzhou Hybribio Medicine Technology Ltd
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Priority to US16/978,713 priority Critical patent/US11147773B2/en
Priority to JP2020549032A priority patent/JP6893288B2/ja
Priority to AU2019391079A priority patent/AU2019391079B2/en
Priority to PL19892996.0T priority patent/PL3760192T3/pl
Priority to EP19892996.0A priority patent/EP3760192B1/en
Priority to RU2020130473A priority patent/RU2760457C1/ru
Application filed by Guangzhou Hybribio Biotech Ltd, Guangzhou Hybribio Medicine Technology Ltd filed Critical Guangzhou Hybribio Biotech Ltd
Priority to KR1020207025970A priority patent/KR102260231B1/ko
Priority to ES19892996T priority patent/ES2938738T3/es
Priority to BR112020019266-0A priority patent/BR112020019266B1/pt
Publication of WO2020114166A1 publication Critical patent/WO2020114166A1/zh
Anticipated expiration legal-status Critical
Priority to US17/472,541 priority patent/US11564891B2/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • 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
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
    • 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
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA 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
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedicine, and more specifically, relates to a chloroquine gel suitable for treating external genital infections and other skin warts, and a preparation method and application thereof.
  • Reproductive infections also called sexually transmitted diseases
  • sexually transmitted diseases are a group of diseases with sexual contact as the main method of transmission.
  • Internationally more than 20 infectious diseases caused by sexual behavior or similar sexual behavior are included in the category of reproductive infections.
  • Reproductive tract infections and sexually transmitted infections in our country have been increasing. They have spread from the coast to the inland and from the city to the countryside. According to statistics, there are about 400,000 AIDS patients in my country today. HIV infection is about one-thousandth, and hundreds of people are infected every day, and the proportion of sexual transmission is large and the characteristics of transmission from high-risk groups to the general population.
  • Chloroquine has always been used as a drug to prevent and treat Plasmodium infections, and is also often used in gene transfection experiments to improve transfection efficiency.
  • In vitro studies have found that high-concentration chloroquine has the effect of inhibiting and killing Cryptococcus in vitro, and the effect of chloroquine on the two variants of Cryptococcus has no difference; at the same time, chloroquine can enhance the ability of amphotericin B to resist cryptococcus, and amphotericin B has a synergistic antibacterial effect.
  • chloroquine has focused on the anti-HIV activity of chloroquine. Studies have shown that chloroquine has a broad-spectrum anti-HIV activity.
  • Chloroquine not only targets the laboratory strain B of HIV-1 evolution, but also has an effect on the primary isolates of HIV-1 evolution A, B, C, D, and E.
  • the main mechanism of chloroquine's anti-HIV effect seems to be to inhibit the glycosylation of the gp120 viral envelope protein, resulting in newly reduced viral particles with severely reduced infectivity.
  • chloroquine is an aminoquinine-type membrane penetrating agent that can be embedded into double-stranded DNA and change the double helix form of DNA by unwinding.
  • chloroquine can be used as a class of intercalating agents such as acridine orange and ethidium bromide to selectively inhibit the covalent bonds of small plasmid DNA.
  • acridine orange and ethidium bromide By changing the density of the DNA supercoil, and then changing the supercoil closure, it can achieve the inhibition of bacteria And the proliferation of virus-infected cells.
  • the DNA of human papillomavirus (HPV), which causes cervical cancer, is such a plasmid. It is a complex DNA of supercoiled assembly, which performs DNA replication and RNA transcription.
  • chloroquine has been shown to inhibit plasmid DNA and interfere with DNA polymerase activity by changing the supercoiled density of plasmid DNA.
  • chloroquine Due to its weak basicity, chloroquine has been shown to cause mitochondrial dysfunction, such as accumulation of endosomes/lysosomes, changes in pH, and possible apoptosis.
  • chloroquine has been shown to reactivate inactivated p53 (HPV degradation/inactivation of P53), connect to melanin (the production of melanin is the same layer as HPV replication in the skin: the basal layer of the epidermis) and prevent the release of iron ions (Energy dependence of iron catalyst/HPV during replication).
  • Iron is necessary for DNA replication, and iron also functions as an electron catalyst in the transportation and storage of oxygen. However, iron is also essential for pathogen survival and virus replication. The suppression of iron particles is also an important mechanism by which chloroquine kills viruses and virus-infected cells.
  • chloroquine has no external medicines, especially no external preparations with little irritation and wider curative effect.
  • chloroquine is mainly used to treat acute malaria attacks and control malaria symptoms. It can also be used to treat liver amoebiasis, clonorchiasis, pulmonary fluke, connective tissue disease and so on. It can also be used to treat photosensitivity disorders such as erythema insolation. Tablets and injections are used to treat malaria. They have a large dosage and a long course of treatment. They may have serious side effects on the gastrointestinal tract and skin.
  • chloroquine has a certain irritation to the skin, which can cause skin damage and various types of rashes. Therefore, how to reduce the irritation of chloroquine, reduce side effects, and increase the application value of chloroquine by changing the dosage form has become the main problem at present.
  • Drug microspheres refer to tiny spherical entities formed by drugs dispersed or adsorbed in a polymer matrix.
  • the microsphere preparation has a long-acting sustained-release or targeting effect, which can greatly improve the convenience and compliance of the patient's medication, and has shown clinical advantages. It is a highly potential dosage form.
  • the added value of microsphere preparation products is large, and the market prospect is broad.
  • the purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art, and to provide a chloroquine nano-microsphere.
  • a natural polymer compound as a microsphere carrier
  • the preparation process is simple, the product side effects are small, and the synergy between chloroquine and microsphere carrier is fully utilized
  • Synergistic effect has significant anti-inflammatory, antibacterial and antiviral effects, reduces the stimulation of chloroquine on the skin, promotes wound healing, and can control the rate of drug release, overcoming the weak antibacterial effect of the existing microsphere carrier and irritating chloroquine Big flaws.
  • Another object of the present invention is to provide a method for preparing the above chloroquine nanospheres suitable for external genitalia and other skin warts.
  • Another object of the present invention is to provide new uses of the above-mentioned chloroquine nanospheres.
  • Another object of the present invention is to provide a product for preventing and treating external genital infections and/or other skin warts
  • a chloroquine nanosphere contains a water-soluble nanosphere carrier and chloroquine or a chloroquine derivative.
  • the inventor unexpectedly discovered that the preparation of nano-spheres by combining water-soluble nano-microsphere carriers with chloroquine or chloroquine derivatives has a long action time, has mucous membrane adhesion, local retention and self-degradability, and reduces the chloroquine Irritating, non-irritating to external genitalia, at the same time overcoming the defects of the existing external genital infection treatment methods that have large side effects and inconvenient use, can effectively solve the side effects and drug resistance of chloroquine; the nanometer microspheres of the present invention can effectively prevent or Treatment of external genital infections and other skin wart diseases, such as vaginitis, flat warts, etc., even produced a significant synergistic effect; not only broaden the use of chloroquine, expand its scope of application, but also can reduce the abuse of existing antibacterial drugs and Lead to the phenomenon of drug resistance.
  • the invention also relates to the preparation method of the above-mentioned chloroquine nanomicrospheres, including the following steps:
  • the mass ratio of the chloroquine or chloroquine derivative to the water-soluble nano-microsphere carrier is 1:3 to 1:5.
  • the mass ratio of the chloroquine or chloroquine derivative to the water-soluble nano-microsphere carrier is 1:3.
  • the chloroquine derivative is selected from one or more of hydroxychloroquine, chloroquine phosphate or chloroquine sulfate.
  • the loading ratio of the chloroquine or chloroquine derivative is 3.0% to 21.6%.
  • the loading rate of the chloroquine or chloroquine derivative is 12.5%.
  • the water-soluble nanosphere carrier is selected from one or more of water-soluble chitosan, water-soluble carrageenan or water-soluble starch.
  • the water-soluble nanosphere carrier is water-soluble chitosan.
  • the water-soluble chitosan has a degree of deacetylation of 80% to 95%, and a viscosity average molecular weight of 3000 to 5000.
  • the volume ratio of the water phase and the oil phase in step S2 is 1:3.
  • the condition of the high-speed centrifugation in step S3 is 1500-2000r/min centrifugation for 5-10 minutes.
  • the condition of the high-speed centrifugation in step S3 is 1500r/min for 10min.
  • step S3 a precipitant is added to the nanomicrosphere emulsion at a rate of 1 to 5 drops per second.
  • the volume ratio of the emulsifier and precipitant is 1 to 1.5: 30 to 50.
  • the volume ratio of the emulsifier and precipitant is 1:50.
  • the oil phase matrix is a vegetable oil; the precipitant is a basic organic phase.
  • the alkaline organic phase is a sodium hydroxide-n-propanol mixed solution.
  • the pH value of the basic organic phase is 8.5 to 10.0.
  • the pH value of the basic organic phase is 9.0.
  • the vegetable oil is selected from one or more of vegetable oils such as corn oil, olive oil, peanut oil, soybean oil, rapeseed oil and the like.
  • the emulsifier is selected from one or more of Tween-20, Tween-80 and Span80.
  • chloroquine nanospheres in the preparation of antibacterial and/or antiviral products, and the application thereof in the preparation and prevention of products of external genital infections and/or other skin warts are also within the protection scope of the present invention.
  • Products containing the above-mentioned chloroquine nanospheres can significantly reduce the skin irritation of chloroquine, promote wound healing, play a slow-release effect, and at the same time improve its biological activity, suitable for the treatment of external genital infections and other skin warts.
  • the external genital infection includes viral vaginitis, condyloma acuminatum, bacterial vaginosis, fungal infectious vaginitis or trichomonas vaginitis.
  • the other skin warts include flat warts and warts caused by HPV infection in non-genital areas.
  • the chloroquine nanospheres are effective against external genital infections such as viral vaginitis, condyloma acuminatum, bacterial vaginosis, fungal infectious vaginitis, or trichomonas vaginitis, as well as flat warts and non-genital parts of warts caused by HPV infection Other skin warts have good effects.
  • external genital infections such as viral vaginitis, condyloma acuminatum, bacterial vaginosis, fungal infectious vaginitis, or trichomonas vaginitis
  • the present invention also provides a product for preventing and treating external genital infections and/or other skin warts, which contains chloroquine or chloroquine derivatives, and also contains one of water-soluble chitosan, water-soluble carrageenan or water-soluble starch. kind or several.
  • the mass ratio of chloroquine or chloroquine derivative to water-soluble chitosan, water-soluble carrageenan or water-soluble starch does not exceed 1/3.
  • the mass ratio of the chloroquine or chloroquine derivative to water-soluble chitosan, water-soluble carrageenan or water-soluble starch is 1:3 to 1:5.
  • the mass ratio of the chloroquine or chloroquine derivative to water-soluble chitosan, water-soluble carrageenan or water-soluble starch is 1:3.
  • the product for preventing and treating external genital infection and/or other skin warts contains the above-mentioned chloroquine nanospheres.
  • the chloroquine nanospheres account for 1% to 6% of the total weight of the product.
  • the product is a gel, ointment, cream, effervescent tablet, vaginal tablet, capsule, film, or suppository.
  • the product of the present invention can be processed into semi-solid preparations such as gels, creams, etc., or solid preparations such as tablets and effervescent tablets; the products of the present invention can significantly improve the bitter taste and skin irritation of chloroquine itself, Promote wound healing, play a slow-release effect, while improving its biological activity.
  • the product is a gel.
  • the present invention uses a water-soluble nano-microsphere carrier combined with chloroquine to prepare an external preparation that has both bactericidal and anti-viral properties, exerts a significant synergistic effect, and has certain cleaning and killing effects on a variety of bacteria and viruses It has a cleansing effect on male and female external genitalia, reduces the inflammation of external genitalia, and can well improve the skin irritation caused by chloroquine. It has no irritation to the vaginal mucosa and penis. It has mucous membrane adhesion and locality. Retention and self-degradability can not only prevent bacterial and viral infections, but also promote the healing of micro-wounds. It can be used for the effective treatment of external genital infections and warts, especially the health care of external genitals before and after sex. Has a cool and comfortable effect.
  • the present invention mainly uses a water-soluble nano-microsphere carrier combined with a synergist chloroquine as the main component, the prescription is stable, and the drug exists in the form of microspheres, which can prolong the residence time of the drug at the treatment site and make the action time longer.
  • the preparation method is simple, easy to synthesize, and has no toxic and side effects, good safety, can be adapted to the industrial application of new drugs, and provides a new method for the development and application of antibacterial and antiviral active drugs direction.
  • Figure 1 shows chloroquine chitosan nanospheres.
  • Figure 2 shows the standard curve for chloroquine detection by high performance liquid chromatography.
  • Figure 3 is a high-performance liquid detection of chloroquine chitosan microspheres chloroquine content.
  • Figure 4 shows the release of chloroquine chitosan nanospheres and chloroquine microspheres.
  • Figure 5 is a smooth glass plane observation of different ratios of gel products (from 1 to 4 gel products, the mass ratio of chloroquine phosphate: chitosan is 1:5, 1:3, 1:1, 1:0.5, of which No. 5 is a pure chitosan gel product).
  • Figure 6 shows the flow of gel products of different proportions within 1 minute on a smooth glass surface (from gel products No. 1 to No. 4, the mass ratio of chloroquine phosphate to chitosan is 1:5, 1:3, 1:1, and 1: 0.5, of which No. 5 is a pure chitosan gel product).
  • Figure 7 is the HE staining results of the damaged skin irritation test; among them, group A: control group, blank gel matrix, picture A1 (test article side), picture A2 (negative control side); group B: chloroquine shell Glycan nanomicrosphere gel group 1, the mass ratio of chloroquine phosphate to chitosan is 1:3, panel B1 is (test article side), panel B2 is (negative control side); Group C: chloroquine chitosan Nano-microsphere gel group 2, the mass ratio of chloroquine phosphate to chitosan is 1:5, Figure C1 (test article side), Figure C2 is (negative control side); Group D: chloroquine chitosan nanospheres Gel group 3, the mass ratio of chloroquine phosphate to chitosan is 1:0.5, panel D1 (test article side), panel D2 (negative control side); Group E: chloroquine phosphate gel group, panel E1 is (received Sample side), Figure E2 is (negative control side).
  • Figure 8 is the HE staining results of herpes virus vaginitis animal test; among them, Figure A is a blank control group; Figure B is a model control group; Figure C is: chloroquine chitosan nanosphere gel group 1, chloroquine phosphate and shell The mass ratio of glycan is 1:3; Figure D: chloroquine chitosan nanosphere gel group 2, the mass ratio of chloroquine phosphate and chitosan is 1:0.5; Figure E: chloroquine phosphate gel group.
  • Figure 9 is the HE staining results of HPV condyloma acuminata animal experiment; among them, Figure A is the model control group; Figure B is: chloroquine chitosan nanosphere gel group 1, the mass ratio of chloroquine phosphate to chitosan is 1: 3; Figure C: chloroquine chitosan nanosphere gel group 2, the mass ratio of chloroquine phosphate and chitosan is 1:0.5; Figure D: chloroquine phosphate gel group.
  • the prepared chloroquine chitosan nanospheres are shown in FIG. 1 and the particle size is 100-800 nm.
  • the preparation method includes the following steps:
  • chloroquine chitosan nanosphere gel can release chloroquine slowly and smoothly.
  • the chloroquine chitosan nano-microsphere gel prepared by the invention has no sudden release phenomenon in the early stage, and the release speed is almost not affected by the medium, and exhibits a good sustained release effect, which is beneficial to the continuous release of the drug after external administration.
  • Chloroquine Chitosan Nanospheres 5% Carboxymethyl cellulose 0.5% Ethyl paraben 0.5% glycerin 30% peppermint 0.1% purified water Supplement to 100%
  • the preparation method includes the following steps:
  • Figure 5 is a smooth glass plane observation of different shapes of gel product shapes (from 1 to 4 gel products, chloroquine phosphate: chitosan ratios are 1:5, 1:3, 1:1, and 1: 0.5, of which No. 5 is pure chitosan product); the flow of gel products with different ratios in smooth glass plane within 1 min is shown in Fig. 6.
  • test substance groups are:
  • Group A control group, blank gel matrix,
  • Group B chloroquine chitosan nanosphere gel group 1, the mass ratio of chloroquine phosphate to chitosan is 1:3,
  • Group C chloroquine chitosan nanosphere gel group 2, the mass ratio of chloroquine phosphate to chitosan is 1:5,
  • Group D chloroquine chitosan nanosphere gel group 3
  • the mass ratio of chloroquine phosphate to chitosan is 1:0.5
  • Group E Chloroquine phosphate gel group.
  • the skin of the administration site of the animals in each test group did not show any obvious irritant reaction at each observation time point, so it can be judged that the negative control is white petrolatum and the result is negative.
  • Group B 6/6 cases of animal skin on the side of the test substance (left side), slight erythema or edema appeared at the time of visual observation between day 1 and day 12, from day 12 to before necropsy No obvious abnormalities were observed in the administration site of each animal, and the results of histopathological examination at the last 72 hours and 14 days showed that 6/6 animals had skin at the administration site on the side of the test substance, of which 2 cases had mild There were no obvious abnormal changes in the edema of the others. Therefore, the above-mentioned irritation tends to be judged as the skin is damaged, and the drug slightly aggravates the mechanical irritation.
  • Group C 6/6 cases of the animal's test subject side (left side) skin of the administration site, mild erythema or edema appeared at the time of visual observation between the first day and the 12th day, and the necropsy on the 12th day Before the observation of the administration site of each animal, there was no obvious abnormality, and the results of histopathological examination at the last 72 hours and 14 days showed that there were no obvious abnormal changes in the skin of the administration site on the side of the test substance of 6/6 animals . Therefore, the aforementioned mild irritation tends to be judged as mechanical irritation after skin damage.
  • Group D 6/6 animals on the test substance side (left side) of the administration site, 4/6 cases of the animal showed erythema or edema on the administration site on the 12th day, and the scab fell off during the administration. The skin surface failed to heal completely when she fell off, and 72 hours after the last drug removal, the wound healed significantly.
  • Group E 6/6 animals on the test substance side (left side) of the administration site, 4/6 cases of the animal began to crack at the damaged site on the 11th day, there was a tendency to extend outward, the administration site appeared More obvious erythema or edema, scabs continue to regenerate and then fall off, the skin surface did not heal completely when she fell off, there is a significant difference from the negative control side on the right side of the body, combined with 72 hours and 14 days after the last drug removal The results of histopathological examination showed that 6/6 animals had obvious abnormalities in the skin at the administration site on the side of the test substance.
  • the chloroquine phosphate chitosan nanosphere gel is significantly less irritating to the damaged skin than the chloroquine phosphate gel (general topical preparation);
  • the lower the ratio of chloroquine/nanosphere carrier the smaller the irritant response to the skin, and the better the repair of damaged skin after stopping administration.
  • the chloroquine gel can cause the degree of damage to the damaged skin to increase.
  • the negative control side is completely repaired, the skin on the chloroquine gel side cannot be repaired normally. It resumes 14 days after stopping the administration, and the skin on the chloroquine gel side has recovered to a certain extent.
  • chloroquine has a stimulating effect on damaged skin, and chloroquine chitosan microspheres can significantly reduce this stimulating effect, which may be related to the antibacterial and anti-inflammatory effect of chitosan itself to promote wound healing.
  • Group A As shown in A1 (blank stromal side) and A2 (negative control side) in Figure 7, the skin epidermis, dermis, and accessory structures were intact, and there was no infiltration of inflammatory cells and dilation of small blood vessels.
  • Group B As shown in panel B1 (chloroquino chitosan nanosphere gel) and panel B2 (negative control side) in FIG. 7.
  • Figure B2 shows that the skin epidermis, dermis, and accessories are intact, with no infiltration of inflammatory cells and dilation of small blood vessels.
  • Figure B1 shows that the small focal epidermal layer of the skin in this group is slightly thickened, and a small amount of inflammatory cell infiltration can be seen.
  • Group C As shown in Figure C1 (chloroquino chitosan nanosphere gel side) and Figure C2 (negative control side) in FIG. 7.
  • Figure C2 shows that the skin epidermis, dermis, and accessories are intact, with no infiltration of inflammatory cells and dilation of small blood vessels.
  • Figure C1 shows that the local epidermal layer of this group of skin is slightly thickened, the number of cell layers is significantly increased, and extends to the dermal layer; the arrangement of connective tissue under the epidermis is disordered, with some inflammatory cell infiltration.
  • Group D As shown in panel D1 (chloroquino chitosan nanosphere gel side) and panel D2 (negative control side) in FIG. 7.
  • Figure D2 shows that the skin epidermis, dermis, and accessories are intact, with no infiltration of inflammatory cells and dilation of small blood vessels.
  • Figure D1 shows that the local epidermal layer of this group is slightly thickened, the number of cell layers is increased, and a small amount of inflammatory cell infiltration is involved.
  • Group E As shown in Figure E1 (chloroquine gel side) and Figure E2 (negative control side) in FIG. 7.
  • the skin epidermis, dermis, and attachments were intact, and there was no infiltration of inflammatory cells and dilatation of small blood vessels, and the local epidermal layer of the chloroquine gel had scabs on the skin, and the epidermis was thickened significantly; some scabs were closely connected with the subcutaneous tissue , The epidermal layer is depressed, the structure is fuzzy, a large number of inflammatory cells infiltrate with fibrous tissue hyperplasia; the number of dermal hair follicles is significantly reduced; but there is no obvious abnormality in the muscular layer and subcutaneous tissue.
  • the chloroquine chitosan nanospheres obtained in Example 2 were made into a gel to perform an animal test for herpes virus vaginitis.
  • the specific experimental method is: select a number of Hartley guinea pigs that are qualified for adaptive observation, and randomly select 10 animals as the blank control group (A). The remaining animals are modeled by vaginal infection with herpes virus, and the animals are randomly divided into 5 after modeling Group, the overall grouping is:
  • Group A blank control group
  • Group B Model group
  • Group C chloroquine chitosan nanosphere gel group 1, the mass ratio of chloroquine phosphate to chitosan is 1:3,
  • Group D chloroquine chitosan nanosphere gel group 2, the mass ratio of chloroquine phosphate to chitosan is 1:0.5,
  • Group E Chloroquine phosphate gel group
  • vaginal mucosa tissues were taken for fixation and embedding. Paraffin was continuously sliced at 4 ⁇ m. After hematoxylin-eosin (HE) staining, the pathological conditions of vaginal mucosa tissues were observed under a microscope.
  • HE hematoxylin-eosin
  • Group A The results of the blank control group. As shown in Panel A of Figure 8, the vaginal mucosal epithelial cell structure of the blank control group was intact, and the lamina basement was not dilated and inflammatory cell infiltration.
  • Group B The results of the model control group. As shown in Panel B of FIG. 8, a large number of inflammatory cells infiltrate in the lamina limba submucosa of the model control group, the arrangement of loose connective tissue is disordered, and the edema is obvious.
  • Group C chloroquine chitosan nanosphere gel group 1, the mass ratio of chloroquine phosphate to chitosan is 1:3.
  • panel C in Figure 8 the connective tissue of the serous membrane layer in this group increased and arranged Disorder, obvious edema, accompanied by a small amount of inflammatory cell infiltration.
  • Group D Chloroquine Chitosan Nanosphere Gel Group 2, the mass ratio of chloroquine phosphate to chitosan is 1:0.5.
  • Membrane connective tissue increased, disordered arrangement, obvious edema, and infiltration of inflammatory cells.
  • Group E The results of the chloroquine phosphate gel group. As shown in panel E of Figure 8, the local epithelial cells of the vaginal mucosa in the chloroquine gel group were deformed and necrotic, and the loose connective tissues in the submucosal lamina intestinal were slightly disordered, obvious edema, and accompanied by There is infiltration of inflammatory cells.
  • chloroquine chitosan nanosphere gel can significantly improve the vaginitis caused by herpes virus.
  • the combined application of chloroquine and chitosan can significantly improve the vaginal edema, and the results are similar to the degree of inflammation. This shows that the combined application of chloroquine chitosan microspheres produced a significant increase in treatment.
  • nude mice qualified for adaptive observation were selected, half male and one female, and HPV-infected wart tissue was directly inoculated subcutaneously in nude mice.
  • the transplantation process strictly followed the principle of aseptic surgery.
  • nude mice with good inoculation sites were randomly divided into 4 groups according to body weight and gender, namely:
  • Group A Model control group
  • Group B chloroquine chitosan nanosphere gel group 1, the mass ratio of chloroquine phosphate to chitosan is 1:3,
  • Group C chloroquine chitosan nanosphere gel group 2, the mass ratio of chloroquine phosphate to chitosan is 1:0.5,
  • Group D Chloroquine phosphate gel group
  • each group There are 6 animals in each group, half female. Among them, the model control group was given white petrolatum and the other groups were corresponding gels. Each group was applied through the skin, and the corresponding application area applied to the animal's administration site was about (2cm ⁇ 2cm). Once a day, each contact for 6 hours, after 6 hours of contact, the drug was removed, and the drug was continuously administered for 14 days. The positive control group was exposed 3 times a week for 6 hours each time. After 6 hours of exposure, the drug was removed and given for 14 days.
  • the wart tissue 14 days after the administration, the wart tissue was dissected and fixed the next day, embedded, paraffin wax was continuously sliced at 4 ⁇ m, and after hematoxylin-eosin (HE) staining, the pathology of the wart tissue was observed under a microscope.
  • HE hematoxylin-eosin
  • wart tissues were visible in 6 animals.
  • 3 animals 03, 04, 05; 3/6) localized degeneration and necrosis of epithelial cells of wart tissue, and a few hollow cells were seen; 3 animals (01, 02, 06; 3/6) epithelial cells of wart tissue
  • the structure is complete, with hollow cells partially visible.
  • wart tissues were visible in 6 animals.
  • 4 animals 01, 02, 03, 04; 4/6) had local degeneration and necrosis of the epithelial cells of the wart body tissue, and a few hollow cells were seen; 2 animals (05, 06; 2/6) had epithelial cells of the wart body tissue
  • the structure is complete, with hollow cells partially visible.
  • the wart body the epidermal cells of the wart body are hyperkeratotic, squamous epithelium can be seen under the epidermis, degeneration and necrosis of local epithelial cells, and a small number of hollow cells can be seen locally.
  • chloroquine has the effect of clearing infected cell virus from warts caused by HPV, and as the content of chloroquine increases, the hollow cells infected by HPV decrease.
  • the chloroquine gel group (Group D) can clearly remove infected cells, but can cause local tissue degeneration and necrosis.
  • chloroquine chitosan gel protects the skin while reducing infected cells.
  • the chloroquine chitosan nanosphere gel group (Group B: chloroquine chitosan nanosphere gel, and the mass ratio of chloroquine phosphate to chitosan of 3:1) was tested in vitro for antibacterial activity in Example 6.
  • Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and S. aureus were recovered and diluted.
  • each medium was inoculated with a loop and cultured for 5 to 7 days to start observation.
  • results of the in vitro antibacterial activity test are as follows: different concentrations of chitosan chloroquine nanomicrosphere gel can inhibit the growth of Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Gluconococcus aureus. The higher the inhibition rate. Candida albicans and S. aureus are more sensitive.
  • chloroquine and chitosan are used in combination, which has a synergistic inhibition effect on various vaginal pathogenic microorganisms.
  • the medicine of the present invention can kill or inhibit the effects of various vaginal pathogenic microorganisms, such as Staphylococcus aureus (SA), aeruginosa (EC), hemolytic streptococcus type B (ST), Candida albicans (CA), Ureaplasma urealyticum (Uu), Trichomonas vaginalis and Bacteroides fragilis, herpes simplex virus type 2 (HSV-2).
  • the medicine of the present invention can inhibit the adhesion of pathogens to host vaginal epithelial cells, prevent and treat various vaginitis, such as Candida albicans vaginitis, trichomonas vaginitis, bacterial vaginosis, genital herpes virus and other external genital infections .
  • various vaginitis such as Candida albicans vaginitis, trichomonas vaginitis, bacterial vaginosis, genital herpes virus and other external genital infections .
  • the present invention was applied to the treatment of 30 cases of warts caused by HPV infection. After 2 to 3 weeks of treatment, it was found that 30 cases of warts naturally fell off without any adverse reactions.
  • the chloroquine nanomicrospheres of the present invention are effective for infections of external genitalia such as viral vaginitis, condyloma acuminatum, bacterial vaginosis, fungal infectious vaginitis, or trichomonas vaginitis, as well as flat warts and non-genital warts caused by HPV infection
  • external genitalia such as viral vaginitis, condyloma acuminatum, bacterial vaginosis, fungal infectious vaginitis, or trichomonas vaginitis

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Abstract

一种氯喹凝胶及其制备方法和应用。该氯喹纳米微球，含有水溶性纳米微球载体、以及氯喹或氯喹衍生物，氯喹或氯喹衍生物与水溶性纳米微球载体的质量比不超过1：3。

Description

一种氯喹凝胶及其制备方法和应用 技术领域
本发明属于生物医药技术领域，更具体地，涉及一种适用于治疗外生殖器感染及其他皮肤疣的氯喹凝胶及其制备方法和应用。
背景技术
生殖感染也叫性传播疾病，是以性接触为主要传播方式的一组疾病。国际上将20多种通过性行为或类似性行为引起的感染性疾病列入生殖感染范畴。20世纪80年代以来，流动人口日益增多，性观念的改变，不安全性行为增加。我国生殖道感染和性传播感染不断增加，已从沿海蔓延到内陆，从城市向农村发展，形势相当严峻。据统计，我国现今约有40万艾滋病患者，HIV感染约为千分之一，每天约有数百人受到感染，且性传播比重大以及从高危人群向普通人群传播的特点。据统计，近几年我国两性外生殖器感染的发病人数仍逐年增多，不少地区的发病率在传染病的顺序中名列第二、三位，已婚妇女下生殖道感染发病率高达50％、70％的妇女一生中，至少患过一次外阴阴道念珠菌病，其中5％～10％的妇女患有复发性阴道念珠菌病，90％的女性感染过人乳头瘤状病毒。两性外生殖器感染的发病率增长如此迅速，不易控制已成为对世界危害严重的社会及公共卫生问题，因而有效防治两性外生殖器感染已成为当前一项十分严峻而迫切的任务。
现代医学对于外生殖器感染主要针对病原体进行治疗。在预防及其治疗方面，因引起生殖道感染的病原微生物种类很多，加之病原体的变异与耐药问题，给选用抗生素药物来预防生殖道感染带来了极大的难度。目前在临床实践中，外生殖器感染的治疗多数是基于抗生素或者基于消毒剂给药。然而消毒剂因对局部黏膜刺激性破坏了阴道内环境而导致使用受到限制，且通常只适用于局部治疗。对于细菌性生殖器感染，抗生素治疗法往往带来抗药性和副作用。因此，研制具有抗菌、抗病毒双重作用的新型、高效的外用药物对有效防治外生殖器感染具有重要意义。
一直以来氯喹是作为一种预防和治疗疟原虫感染的药物使用，也常常用于基因转染实验中，以提高转染效率。体外研究发现，高浓度氯喹在体外具有抑制和 杀灭隐球菌的作用，而且氯喹对隐球菌两种变种的作用没有差别；同时氯喹可以增强两性霉素B的抗隐球菌能力，与两性霉素B具有协同抗菌作用。目前，许多研究都集中在氯喹抗HIV的活性上。已有研究表明氯喹的广谱抗HIV活性，氯喹不仅针对HIV-1进化的实验室菌株B，而且对HIV-1进化A、B、C、D、E的初级分离株也很有效果。氯喹的抗HIV作用的主要机制似乎是抑制gp120病毒包膜蛋白的糖基化，导致新产生的病毒粒子具有严重降低的感染性。同时，研究认为氯喹是一种氨基奎宁类膜穿透剂，能嵌入到双链DNA并通过退绕改变DNA的双螺旋形式。细菌学研究认为氯喹可作为一类嵌入剂如吖啶橙和溴乙啶等能选择性的抑制小质粒DNA的共价键，通过改变DNA超螺旋密度，进而改变超螺旋的闭合，达到抑制细菌及病毒感染细胞的增殖。而引起宫颈癌的人乳头瘤状病毒(HPV)的DNA就是这样的一个质粒，是超螺旋组装的复合物DNA，进行DNA复制和RNA转录。反过来，氯喹已经被证明能够通过改变质粒DNA的超螺旋密度，抑制质粒DNA和干扰DNA聚合酶的活性。由于其弱碱性，氯喹已被证实能够导致线粒体功能障碍，如积累核内体/溶酶体、改变pH值和可能引发凋亡。此外，氯喹已被证实能重新激活灭活的p53(HPV降解/灭活P53)，连接到黑色素(黑色素的产生同HPV的复制在皮肤的同一层：表皮的基底层)和防止铁离子的释放(铁催化剂/HPV在复制时的能源依赖)。铁是DNA复制所必需的，铁在氧的运输和储存也起电子催化剂的作用。然而，铁也是病原体存活和病毒复制至关重要的物质。对于铁粒子的抑制也是氯喹杀灭病毒和病毒感染细胞的重要机制。
目前氯喹并没有外用药物，尤其是没有刺激性小，疗效更广泛的外用制剂。在临床应用中，氯喹主要是用于治疗疟疾急性发作，控制疟疾症状。还可用于治疗肝阿米巴病、华支睾吸虫病、肺吸虫病、结缔组织病等。另可用于治疗光敏性疾患，如日晒红斑症。片剂和注射剂治疗疟疾，用药量大，疗程长，对胃肠道和皮肤可能会有较重的副反应，另外氯喹对皮肤具有一定的刺激性，可致皮肤损伤及多种类型皮疹。因此如何通过改变剂型来减少氯喹的刺激性，减少副作用，提高氯喹的应用价值成了目前的主要问题。
药物微球是指药物分散或被吸附在高分子聚合物基质中而形成的微小球状实体。微球制剂具有长效缓释或靶向作用，可以大大提升患者用药的方便性、依从性，在临床上已突显优势，是一种极具潜力的剂型。此外，微球制剂产品附加 值较大，市场前景广阔，近年来已成为药物研发的热点。
发明内容
本发明的目的在于克服上述现有技术的不足，提供一种氯喹纳米微球，通过选用天然高分子化合物为微球载体，制备过程简单，产品副作用小，充分发挥了氯喹与微球载体的协同增效作用，具有显著的消炎、抗菌、抗病毒的作用，减少氯喹对皮肤的刺激，促进伤口的愈合，又可以控制药物的释放速度，克服了现有微球载体抗菌作用弱和氯喹刺激性大的缺陷。
本发明的另一目的是提供上述适用于外生殖器和其他皮肤疣的氯喹纳米微球的制备方法。
本发明的再一目的是提供上述氯喹纳米微球的新用途。
本发明的又一目的是提供一种用于预防和治疗外生殖器感染和/或其他皮肤疣的产品
本发明的上述目的通过如下技术方案予以实现：
一种氯喹纳米微球，含有水溶性纳米微球载体、以及氯喹或氯喹衍生物。
本发明人意外发现，将水溶性纳米微球载体与氯喹或氯喹衍生物联合制备成纳米微球，其作用时间较长，具有粘膜粘附性、局部滞留性以及自身降解性，且减少氯喹的刺激性，对外生殖器无刺激性，同时克服了现有外生殖器感染治疗方法中副作用较大，使用不方便的缺陷，可以有效解决氯喹的副作用和抗药性；本发明的纳米微球能有效预防或治疗外生殖器感染和其他皮肤疣性疾病，如阴道炎、扁平疣等，甚至产生了显著的协同增效的效果；不仅拓宽氯喹的用途，拓宽其应用范围，而且可以减轻现有抗菌药物滥用而导致产生耐药的现象。
本发明还涉及上述氯喹纳米微球的制备方法，包括以下步骤：
S1.将所述水溶性纳米微球载体溶解后，加入氯喹或氯喹衍生物，混合搅拌均匀作为水相，其中氯喹或氯喹衍生物与水溶性纳米微球载体的质量比不超过1/3；
S2.向油相基质中加入乳化剂，作为油相；将水相加到油相中，水相和油相的体积比为1：1～6；于10000～20000r/min乳化10～30min，得到纳米微球乳液；
S3.向纳米微球乳液中加入沉淀剂，混合均匀后，静止2～8min后脱水，高速离心清洗，干燥，即可得到所述氯喹纳米微球。
优选地，所述氯喹或氯喹衍生物与水溶性纳米微球载体的质量比为1：3～1：5。
更优选地，所述氯喹或氯喹衍生物与水溶性纳米微球载体的质量比为1：3。
优选地，所述氯喹衍生物选自羟基氯喹、磷酸氯喹或硫酸氯喹中的一种或几种。
优选地，所述氯喹或氯喹衍生物的负载率3.0％～21.6％。
更优选地，所述氯喹或氯喹衍生物的负载率12.5％。
优选地，所述水溶性纳米微球载体选自水溶性壳聚糖、水溶性卡拉胶或水溶性淀粉中的一种或几种。
优选地，所述水溶性纳米微球载体为水溶性壳聚糖。
更优选地，所述水溶性壳聚糖的脱乙酰度为80％～95％，其粘均分子量为3000～5000。
优选地，步骤S2所述水相和油相的体积比为1：3。
优选地，步骤S3所述高速离心的条件为1500～2000r/min离心5～10min。
更优选地，步骤S3所述高速离心的条件为1500r/min离心10min。
优选地，步骤S3中，向纳米微球乳液中以每秒1～5滴的速度加入沉淀剂。
优选地，所述乳化剂和沉淀剂的体积比为1～1.5：30～50。
更优选地，所述乳化剂和沉淀剂的体积比为1：50。
优选地，所述油相基质为植物油；所述沉淀剂为碱性有机相。
更优选地，所述碱性有机相为氢氧化钠-正丙醇混合溶液。
更优选地，所述碱性有机相的pH值为8.5～10.0。
更进一步优选地，所述碱性有机相的pH值为9.0。
优选地，所述植物油选自玉米油、橄榄油、花生油、大豆油、菜籽油等植物油中的一种或多种。
优选地，所述乳化剂选自吐温-20、吐温-80、Span 80中的一种或多种。
上述氯喹纳米微球在制备抗菌和/或抗病毒产品中的应用，及其在制备预防和治疗外生殖器感染和/或其他皮肤疣的产品中的应用，也在本发明的保护范围之内。
含有上述的氯喹纳米微球的产品能明显降低氯喹皮肤刺激性，促进伤口愈合，起到缓释的效果，同时提高其生物活性，适用于治疗外生殖器感染和其他皮 肤疣体。
优选地，所述外生殖器感染包含病毒性阴道炎、尖锐湿疣、细菌性阴道病、真菌感染性阴道炎或滴虫性阴道炎。
优选地，所述其他皮肤疣包含扁平疣、非生殖器部位由HPV感染引起的疣体。
所述氯喹纳米微球对病毒性阴道炎、尖锐湿疣、细菌性阴道病、真菌感染性阴道炎或滴虫性阴道炎等外生殖器感染，以及扁平疣、非生殖器部位由HPV感染引起的疣体等其他皮肤疣均有良好的疗效。
本发明还提供了一种用于预防和治疗外生殖器感染和/或其他皮肤疣的产品，包含氯喹或氯喹衍生物，此外还包含水溶性壳聚糖、水溶性卡拉胶或水溶性淀粉中一种或几种。
优选地，氯喹或氯喹衍生物与水溶性壳聚糖、水溶性卡拉胶或水溶性淀粉的质量比不超过1/3。
更优选地，所述氯喹或氯喹衍生物与水溶性壳聚糖、水溶性卡拉胶或水溶性淀粉的质量比为1：3～1：5。
更进一步优选地，所述氯喹或氯喹衍生物与水溶性壳聚糖、水溶性卡拉胶或水溶性淀粉的质量比为1：3。
进一步地，在本发明较佳的实施例中，所述用于预防和治疗外生殖器感染和/或其他皮肤疣的产品，含有上述氯喹纳米微球。
更优选地，所述氯喹纳米微球占产品总重量的1％～6％。
进一步地，在本发明较佳的实施例中，所述产品是凝胶剂、软膏剂、乳膏剂、泡腾片、阴道片、胶囊剂、膜剂或栓剂。
本发明所述产品可加工制成半固体制剂如凝胶剂、乳膏剂等，或者固体制剂如片剂、泡腾片等；本发明所述产品能明显改善氯喹本身的苦味，皮肤刺激性，促进伤口愈合，起到缓释的效果，同时提高其生物活性。
更进一步地，在本发明较佳的实施例中，所述产品为凝胶剂。
与现有技术相比，本发明的有益效果是：
(1)本发明应用水溶性纳米微球载体结合氯喹，制备出同时具有杀菌抗病毒的外用制剂，发挥了显著的协同增效的效果，对多种细菌、病毒均有一定的清洗、杀灭作用，对男性及女性外生殖器均具有清洁护理的作用，减轻外生殖器的 炎症反应，且能很好地改善氯喹引起的皮肤刺激性，对阴道粘膜、阴茎无刺激，具有粘膜粘附性、局部滞留性以及自身降解性，不仅能预防细菌病毒感染，亦能促进微伤口的愈合，可以用于外生殖器感染和疣体的有效治疗，尤其是性生活前后的外生殖器的卫生护理，效果显著并有清凉舒适的作用。
(2)本发明主要以水溶性纳米微球载体结合增效剂氯喹为主要成分，处方稳定，药物以微球的形式存在，可延长药物在治疗部位的滞留时间，使其作用时间较长，提高药物的生物利用度和治疗效果，其制备方法简便、易于合成，且无毒副作用，安全性好，可适应新药的产业化应用，为抗菌、抗病毒活性药物的开发和应用提供了新的方向。
附图说明
图1为氯喹壳聚糖纳米微球。
图2为高效液相检测氯喹的标准曲线。
图3为高效液相检测氯喹壳聚糖微球的氯喹含量。
图4为氯喹壳聚糖纳米微球与氯喹微球的释药情况。
图5为光滑玻璃平面观察不同比例凝胶产品(从1到4号凝胶产品，磷酸氯喹：壳聚糖的质量比分别为1：5、1：3、1：1、1：0.5，其中5号为纯壳聚糖的凝胶产品)。
图6为不同比例凝胶产品1min内在光滑玻璃平面流动情况(从1到4号凝胶产品，磷酸氯喹：壳聚糖的质量比分别为1：5、1：3、1：1、1：0.5，其中5号为纯壳聚糖的凝胶产品)。
图7为破损皮肤刺激性试验的H-E染色结果；其中，A组：对照组，空白凝胶基质，图A1为(受试物侧)、图A2为(阴性对照侧)；B组：氯喹壳聚糖纳米微球凝胶组1，磷酸氯喹和壳聚糖的质量比为1：3，图B1为(受试物侧)、图B2为(阴性对照侧)；C组：氯喹壳聚糖纳米微球凝胶组2，磷酸氯喹和壳聚糖的质量比为1：5，图C1(受试物侧)、图C2为(阴性对照侧)；D组：氯喹壳聚糖纳米微球凝胶组3，磷酸氯喹和壳聚糖的质量比为1：0.5，图D1(受试物侧)、图D2(阴性对照侧)；E组：磷酸氯喹凝胶组，图E1为(受试物侧)、图E2为(阴性对照侧)。
图8为疱疹病毒阴道炎动物试验的H-E染色结果；其中，图A为空白对照组；图B为模型对照组；图C为：氯喹壳聚糖纳米微球凝胶组1，磷酸氯喹和壳 聚糖的质量比为1：3；图D为：氯喹壳聚糖纳米微球凝胶组2，磷酸氯喹和壳聚糖的质量比为1：0.5；图E为：磷酸氯喹凝胶组。
图9为HPV尖锐湿疣动物实验的H-E染色结果；其中，图A为模型对照组；图B为：氯喹壳聚糖纳米微球凝胶组1，磷酸氯喹和壳聚糖的质量比为1：3；图C为：氯喹壳聚糖纳米微球凝胶组2，磷酸氯喹和壳聚糖的质量比为1：0.5；图D为：磷酸氯喹凝胶组。
具体实施方式
下面结合具体实施方式对本发明作进一步的说明，但实施例并不对本发明做任何形式的限定。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的简单修改或替换，均属于本发明的范围；若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 氯喹壳聚糖纳米微球的制备
1、制备方法
S1.称取水溶性壳聚糖3g加入100mL水中溶解后，加入磷酸氯喹1.0g，混合搅拌均匀作为水相；其中水溶性壳聚糖的脱乙酰度为80％，其粘均分子量为5000；
S2.量取300mL玉米油作为油相基质，向油相基质中加入1mL乳化剂吐温-20，作为油相；将水相加到油相中，水相和油相的体积比为1：3，于15000r/min乳化30min，得到纳米微球乳液；
S3.以氢氧化钠-正丙醇混合溶液为沉淀剂，先取10mL正丙醇加入数滴饱和氢氧化钠溶液调节pH＝9，混合均匀后以每秒1～5滴的速度加入纳米微球乳液中，控制乳化剂和沉淀剂的体积比为1：50，静止5min，加入适量正丙醇脱水；1500r/min离心10min，清洗，干燥，即可得到所述氯喹壳聚糖纳米微球。
2、产品表征
(1)所制备得到的氯喹壳聚糖纳米微球如图1所示，其粒径为100～800nm。
(2)如图2和图3所示，高效液相检测，氯喹壳聚糖微球中氯喹的负载率为12.5％。
实施例2 氯喹壳聚糖纳米微球的制备
1、制备方法
S1.将水溶性壳聚糖用水溶解后，加入羟基氯喹，混合搅拌均匀作为水相，控制羟基氯喹与水溶性壳聚糖的质量比为1：5；其中水溶性壳聚糖的脱乙酰度为90％，其粘均分子量为3000；
S2.量取300mL玉米油作为油相基质，向油相基质中加入1mL乳化剂吐温-20，作为油相；将水相加到油相中，水相和油相的体积比为1：1，于10000r/min乳化30min，得到纳米微球乳液；
S3.以氢氧化钠-正丙醇混合溶液为沉淀剂，取10mL正丙醇，加入数滴饱和氢氧化钠溶液调节pH＝8.5，混合均匀后缓慢加入纳米微球乳液中，控制乳化剂和沉淀剂的体积比为1.5：50，静止2min，加入适量正丙醇脱水；2000r/min离心5min，清洗，干燥，即可得到所述氯喹卡拉胶纳米微球，其粒径为200～800nm，氯喹的负载率为10.3％。
实施例3 氯喹壳聚糖纳米微球的制备
1、制备方法
S1.将水溶性壳聚糖用水溶解后，加入硫酸氯喹，混合搅拌均匀作为水相，控制硫酸氯喹与水溶性壳聚糖的质量比为1：7；其中水溶性壳聚糖的脱乙酰度为90％，其粘均分子量为4000；
S2.取橄榄油作为油相基质，向油相基质中加入乳化剂吐温-80，作为油相；将水相加到油相中，水相和油相的体积比为1：6，于20000r/min乳化10min，得到纳米微球乳液；
S3.以氢氧化钠-正丙醇混合溶液为沉淀剂，先取正丙醇加入数滴饱和氢氧化钠溶液调节pH＝10，混合均匀后缓慢加入纳米微球乳液中，控制乳化剂和沉淀剂的体积比为1.5：30，静止8min，加入适量正丙醇脱水；1500r/min离心10min，清洗，干燥，即可得到所述氯喹淀粉纳米微球，其平均粒径为400nm，氯喹的负载率为8.6％。
实施例4 氯喹壳聚糖纳米微球凝胶的制备
1、凝胶处方：
氯喹壳聚糖纳米微球 1％
羧甲基纤维素 2％
羟苯乙酯 0.2％
甘油 30％
薄荷油 0.1％
纯化水 补充至100％
2、制备方法，包括如下步骤：
(1)微球的制备：按实施例1的氯喹壳聚糖纳米微球的制备步骤进行；
(2)取羧甲基纤维素20g，加入处方量甘油300g，羟苯乙酯2g，搅拌均匀，待其充分溶胀后作为凝胶基质；取氯喹壳聚糖纳米微球10g，用100mL水溶解，分次加入到凝胶基质中并搅拌；加入薄荷油1ml，加水至1000mL，制得适用于外生殖器抗菌、抗病毒的氯喹壳聚糖纳米微球凝胶。
3、用同样方法配制单纯的氯喹凝胶，比较氯喹壳聚糖纳米微球凝胶与氯喹凝胶中氯喹释放率。
如图4所示，氯喹壳聚糖纳米微球凝胶能缓慢平稳得的释放氯喹。本发明制备的氯喹壳聚糖纳米微球凝胶早期无突释现象，释放速度也几乎不受介质影响，呈现良好的缓释效应，有利于外用给药后药物的持续释放。
实施例5 氯喹壳聚糖纳米微球凝胶的制备
1、凝胶处方：
氯喹壳聚糖纳米微球 5％
羧甲基纤维素 0.5％
羟苯乙酯 0.5％
甘油 30％
薄荷油 0.1％
纯化水 补充至100％
2、制备方法，包括如下步骤：
(1)微球的制备：按实施例1的氯喹壳聚糖纳米微球的制备步骤进行；
(2)取羧甲基纤维素加入处方量甘油，加入800mL水，搅拌均匀，待其充分溶胀后作为凝胶基质；取氯喹壳聚糖纳米微球，用100mL水溶解，分次加入到凝胶基质中并搅拌，用50mL的80～85℃热水将羟苯乙酯溶解，待温度降低至40℃以下加入凝胶基质中，搅拌均匀；加入薄荷油，加水至1000mL，制得适用于外生殖器抗菌、抗病毒的氯喹壳聚糖纳米微球凝胶。
3、按照上述步骤，其余工艺条件不变，改变磷酸氯喹和壳聚糖的质量比分别制备得到凝胶1、2、3、4；同时，按照上述实施例的制备步骤，其余工艺条件不变，不加入氯喹或其衍生物，以纯壳聚糖为主要活性成分制备得到凝胶5，如下表：
表1 不同氯喹壳聚糖比例制备纳米微球凝胶的结果
Figure PCTCN2019115216-appb-000001
其中，图5为光滑玻璃平面观察不同比例凝胶产品形状图(从1到4号凝胶产品，磷酸氯喹：壳聚糖的比例分别为1：5、1：3、1：1、1：0.5，其中5号为纯壳聚糖产品)；不同比例凝胶产品1min内在光滑玻璃平面流动情况如图6所示。
实验发现，当磷酸氯喹和壳聚糖的比例在为1：1或1：0.5时，制备出的凝胶质地不均匀，含有小颗粒，流动性不够好。当磷酸氯喹和壳聚糖的比例为1：5或1：3时，制备出凝胶质地均匀，pH适合阴道及皮肤给药，粘度适中色泽气味均符合不同人群需求，氯喹含量稳定的产品。但当磷酸氯喹和壳聚糖的质量比为1：5时，氯喹含量较低，药效受到一定程度的限制，综合考虑当磷酸氯喹和壳聚糖的质量比为1：3时，产品性能更好。
实施例6 氯喹壳聚糖纳米微球凝胶对破损皮肤的刺激性试验
1、实验方法
(1)以新西兰兔为实验对象，采用同体左右侧皮肤自身对比法，分别在左右侧的给药部位皮肤上划“井”字(面积约3.0cm×3.0cm)，左侧给予1.0g受试物，给药剂量为9cm 2/只，每次的贴敷面积为3.0cm×3.0cm，右侧给予1.0g的白凡士林作为阴性对照侧。各组动物每天给药1次，连续28天。
受试物组分别为：
A组：对照组，空白凝胶基质，
B组：氯喹壳聚糖纳米微球凝胶组1，磷酸氯喹和壳聚糖的质量比为1：3，
C组：氯喹壳聚糖纳米微球凝胶组2，磷酸氯喹和壳聚糖的质量比为1：5，
D组：氯喹壳聚糖纳米微球凝胶组3，磷酸氯喹和壳聚糖的质量比为1：0.5，
E组：磷酸氯喹凝胶组。
每次涂布前以及每次去除药物后1小时、第28天末次去除药物后1小时、24小时、48小时、72小时和14天，上述各组动物的各个观察时间点内分别观察并记录给药部位红斑及水肿情况并对红斑及水肿进行评分，同时还需观察给药部位是否有色素沉着、出血点、皮肤粗糙或皮肤菲薄情况，并记录其发生及消退时间。
(2)上述五组动物均在末次去除药物后的第14天，分别对各组动物给药部位的左右侧皮肤进行病理组织学检查。
2、结果
(1)各组皮肤观察结果
试验结果：
阴性性对照侧(右侧)，各受试物组的动物给药部位皮肤，在各个观察时间点内均未见明显的刺激性反应，故可判断阴性对照品为白凡士林的结果为阴性。
A组：在各个观察时间点内均未见明显的刺激性反应，故可判断空白凝胶基质对皮肤无刺激性。
B组：6/6例动物的受试物侧(左侧)给药部位皮肤，在第1天到第12天之间肉眼观察时间点出现了轻微红斑或水肿，第12天至剖检前观察各例动物的给药部位均未见明显异常，且末次72小时和14天的组织病理检查结果显示，6/6例动物受试物侧的给药部位皮肤，其中有2例有轻度的水肿，其他均未见明显异 常改变。所以上述刺激倾向于判断为破损皮肤后，药物对机械刺激有轻度加重。
C组：6/6例动物的受试物侧(左侧)给药部位皮肤，在第1天到第12天之间肉眼观察时间点出现了轻度红斑或水肿，第12天至剖检前观察各例动物的给药部位均未见明显异常，且末次72小时和14天的组织病理检查结果显示，6/6例动物受试物侧的给药部位皮肤，均未见明显异常改变。所以上述轻度刺激倾向于判断为破损皮肤后的机械刺激。
D组：6/6例动物受试物侧(左侧)的给药部位有4/6例动物在第12天开始给药部位出现了红斑或水肿较明显，结痂在给药时脱落，脱落时皮肤表面未能完全愈合，结合末次除药后72小时，伤口明显愈合。
E组：6/6例动物受试物侧(左侧)的给药部位有4/6例动物在第11天开始出现破损部位开裂，有向外延伸的趋势，给药部位出现了比前期更明显的红斑或水肿，结痂不断重新生成后又脱落，脱落时皮肤表面未能完全愈合，与同体右侧的阴性对照侧有着显著性的差异，结合末次除药后72小时和14天的组织病理学检查结果显示6/6例动物受试物侧的给药部位皮肤均出现明显异常，其中72小时剖检的3例动物可见一定程度的损伤，表皮层明显增厚，表皮下结缔组织排列紊乱、纤维组织增生，炎细胞浸润，表皮结痂，部分表皮层凹陷，结构模糊等；末次14天剖检的3例动物则可见局部表皮增厚，表皮下结缔组织排列紊乱、纤维组织增生，伴有炎细胞浸润。
由上述实验结果可见，对新西兰兔破损皮肤进行多次给药，磷酸氯喹壳聚糖纳米微球凝胶对破损皮肤的刺激性明显低于磷酸氯喹凝胶(一般外用制剂)；对磷酸氯喹壳聚糖纳米微球凝胶而言，氯喹/纳米微球载体的比例越低，对皮肤产生刺激性反应越小，停止给药破损皮肤修复越好。而氯喹凝胶可引起破损皮肤损伤程度加重，阴性对照侧完全修复的情况下，氯喹凝胶侧皮肤不能正常修复，停止给药后恢复14天，氯喹凝胶侧皮肤才见一定程度的恢复，差异明显。说明氯喹对破损皮肤有刺激作用，而氯喹壳聚糖微球能明显减少这种刺激作用，这可能与壳聚糖本身具有抗菌消炎促进伤口愈合的作用有关。
(2)病理检测结果
A组：如图7中的A1(空白基质侧)、图A2(阴性对照侧)所示显示，皮肤表皮、真皮及附件结构完好，未见炎细胞浸润和小血管扩张。
B组：如图7中的图B1(氯喹壳聚糖纳米微球凝胶)、图B2(阴性对照侧) 所示。图B2显示，皮肤表皮、真皮及附件结构完好，未见炎细胞浸润和小血管扩张。与阴性对照侧相比，图B1显示，该组皮肤小灶性表皮层稍增厚，可见少量炎细胞浸润。
C组：如图7中的图C1(氯喹壳聚糖纳米微球凝胶侧)、图C2(阴性对照侧)所示。图C2显示，皮肤表皮、真皮及附件结构完好，未见炎细胞浸润和小血管扩张。图C1显示，该组皮肤局部表皮层稍增厚，细胞层数明显增多，并向真皮层延伸性生长；表皮下结缔组织排列紊乱，伴一些炎细胞浸润。
D组：如图7中的图D1(氯喹壳聚糖纳米微球凝胶侧)、图D2(阴性对照侧)所示。图D2显示，皮肤表皮、真皮及附件结构完好，未见炎细胞浸润和小血管扩张。与阴性对照侧相比，图D1显示，该组局部表皮层稍增厚，细胞层数增多，伴少量炎细胞浸润。
E组：如图7中的图E1(氯喹凝胶侧)、图E2(阴性对照侧)所示。在阴性对照侧，皮肤表皮、真皮及附件结构完好，未见炎细胞浸润和小血管扩张，而氯喹凝胶侧皮肤局部表皮层有结痂，表皮明显增厚；部分痂皮与皮下组织紧密相连，表皮层凹陷，结构模糊，大量炎细胞浸润伴纤维组织增生；真皮层毛囊数量明显减少；但肌层及皮下组织未见明显异常。
上述病理检测结果显示：氯喹壳聚糖微球组的凝胶与氯喹凝胶相比，其刺激性，以及停药后的愈合都具有明显的优势。在各氯喹壳聚糖微球组中，氯喹纳米微球载体的比例越低，对皮肤产生刺激性反应越小。
综合皮肤观察结果和病理检测结果，说明氯喹壳聚糖纳米微球凝胶对皮肤产生的刺激性反应明显小于氯喹凝胶，说明氯喹壳聚糖纳米微球大大减轻了氯喹对皮肤的刺激性副作用，并在一定程度上促进伤口的愈合。当氯喹与壳聚糖的质量比不超过1：3时，刺激作用较小，含药量最高。
实施例7 疱疹病毒阴道炎动物试验
1、实验方法
将实施例2中所得到的氯喹壳聚糖纳米微球作成凝胶，做疱疹病毒阴道炎动物试验。
具体实验方法为：选用适应性观察合格的Hartley豚鼠若干只，随机选取10只动物为空白对照组(A)，其余动物均通过阴道感染疱疹病毒进行造模，造模后动物随机分为5个组，总体分组情况为：
A组：空白对照组，
B组：模型组，
C组：氯喹壳聚糖纳米微球凝胶组1，磷酸氯喹和壳聚糖的质量比为1：3，
D组：氯喹壳聚糖纳米微球凝胶组2，磷酸氯喹和壳聚糖的质量比为1：0.5，
E组：磷酸氯喹凝胶组；
每组10只，均采用阴道给药。空白对照组、模型对照组给予0.9％氯化钠注射液，C组、D组、及E组给予相应的受试药物，每日给药1次，各组连续给药2周。给药14天后，次日取阴道粘膜组织进行固定，包埋，石蜡连续4μm切片，苏木素-伊红(HE)染色后，镜下观察阴道黏膜组织的病理情况。
2、结果
(1)病理检测
A组：空白对照组的结果，如图8中的A图所示，空白对照组的阴道黏膜上皮细胞结构完整，固有层血管无扩张及炎性细胞浸润。
B组：模型对照组的结果，如图8中的B图所示，模型对照组的阴道黏膜下固有层可见大量炎性细胞浸润，疏松结缔组织排列紊乱，水肿明显。
C组：氯喹壳聚糖纳米微球凝胶组1，磷酸氯喹和壳聚糖的质量比为1：3的结果如图8中的C图所示，该组浆膜层结缔组织增多，排列紊乱，水肿明显，伴有少量炎细胞浸润。
D组：氯喹壳聚糖纳米微球凝胶组2，磷酸氯喹和壳聚糖的质量比为1：0.5的结果如图8中的D图所示，该组黏膜下固有层水肿明显，浆膜层结缔组织增多，排列紊乱，水肿明显，并可见炎细胞浸润。
E组：磷酸氯喹凝胶组的结果，如图8中的E图所示，氯喹凝胶组的阴道黏膜局部上皮细胞变形坏死，黏膜下固有层疏松结缔组织排列稍乱，明显水肿，并伴有炎细胞浸润。
(2)试验动物组织学检查病变程度结果
表3 试验动物组织学检查病变程度统计表
Figure PCTCN2019115216-appb-000002
Figure PCTCN2019115216-appb-000003
注：“-”表示组织结构未见明显异常；“±”表示极轻度损伤；“+”表示轻度损伤；“++”表示中度损伤；“+++”表示严重损伤。
上述研究结果显示，相对于氯喹凝胶，氯喹壳聚糖纳米微球凝胶能明显改善疱疹病毒引起的阴道炎。各组炎症程度比较：模型对照组＞磷酸氯喹E组＞氯喹 壳聚糖纳米微球凝胶D组>氯喹壳聚糖纳米微球凝胶C组>空白对照组。而且，氯喹与壳聚糖联合应用能显著改善阴道的水肿情况，且结果与炎症程度类似。说明氯喹壳聚糖微球联合应用产生了显著的治疗增加的效果。
实施例8 治疗HPV引起的尖锐湿疣
1、实验方法
选用适应性观察合格的裸小鼠若干只，雌雄各半，将HPV感染的疣体组织直接接种于裸小鼠皮下，移植过程严格按照无菌手术原则。疣体接种5～6天后，选择接种部位良好的裸鼠按体重和性别随机均衡分成4组，即：
A组：模型对照组，
B组：氯喹壳聚糖纳米微球凝胶组1，磷酸氯喹和壳聚糖的质量比为1：3，
C组：氯喹壳聚糖纳米微球凝胶组2，磷酸氯喹和壳聚糖的质量比为1：0.5，
D组：磷酸氯喹凝胶组，
每组6只，雌性各半。其中模型对照组给予白凡士林、其他各组均是相应的凝胶，各组均经皮肤涂抹给药，涂抹在动物给药部位的相应给药区域约为(2cm×2cm)。每日1次，每次接触6小时，接触6小时后除药，连续给药14天。阳性对照组每周3次，每次接触6小时，接触6小时后除药，给药14天。给药14天后，次日剖取疣体组织进行固定，包埋，石蜡连续4μm切片，苏木素-伊红(HE)染色后，镜下观察疣体组织的病理情况。
2、结果
病理检测结果发现：
(1)A组疣体结果显示：各例动物可见疣体组织，疣体组织上皮细胞结构完整，局部可见凹空细胞。
如图9中的A图所示，疣体：局部上皮细胞细胞核周围有明显空泡，细胞核核仁明显。(H-E染色×200)
(2)B组疣体结果显示：6例动物可见疣体组织。其中2例动物(03、04；2/6)疣体组织上皮细胞局部变性坏死，并可见少量凹空细胞；4例动物(01、02、05、06；4/6)疣体组织上皮细胞结构完整，局部可见凹空细胞。
如图9中的B图所示，疣体：局部上皮细胞细胞核周围有明显空泡，细胞核核仁明显。(H-E染色×200)
(3)C组疣体结果显示，6例动物可见疣体组织。其中3例动物(03、04、 05；3/6)疣体组织上皮细胞局部变性坏死，并可见少量凹空细胞；3例动物(01、02、06；3/6)疣体组织上皮细胞结构完整，局部可见凹空细胞。
如图9中的C图所示，疣体：局部上皮细胞细胞核周围有明显空泡，细胞核核仁明显。(H-E染色×200)
(4)D组疣体结果显示，6例动物可见疣体组织。其中4例动物(01、02、03、04；4/6)疣体组织上皮细胞局部变性坏死，并可见少量凹空细胞；2例动物(05、06；2/6)疣体组织上皮细胞结构完整，局部可见凹空细胞。
如图9中的D图所示，疣体：疣体表皮细胞角化过度，表皮下可见鳞状上皮，局部上皮细胞变性坏死，局部可见少量凹空细胞。
上述研究结果显示，氯喹对于HPV引起的疣体有清除感染细胞病毒的作用，且随着氯喹含量的增大，被HPV感染的凹空细胞减少。氯喹凝胶组(D组)能明显的清除感染的细胞，但能引起组织局部变性坏死。氯喹壳聚糖凝胶与单纯的氯喹凝胶相比，在减少感染细胞的同时，对皮肤起到保护作用。
实施例9 对多种阴道致病微生物的杀灭或抑制作用
将实施例6中氯喹壳聚糖纳米微球凝胶组(B组：氯喹壳聚糖纳米微球凝胶，且磷酸氯喹和壳聚糖的质量比为3：1)做体外抗菌活性检测。将20g凝胶产品加入100mL pH 7.0缓冲液中(阴性对照不加供试品)，使其充分溶解。取加入供试品的pH 7.0缓冲液1mL、0.5mL、0.25mL、0.125mL加入相应菌种对应的培养基中，倒平皿培养。铜绿假单胞菌，白色念珠菌，大肠杆菌，金黄色葡糖球菌进行复苏稀释，菌浓度为100cfu/mL时，接种环接种各个培养基，培养5～7天开始观察。
体外抗菌活性检测结果为：不同浓度的壳聚糖氯喹纳米微球凝胶对铜绿假单胞菌，白色念珠菌，大肠杆菌，金黄色葡糖球菌都能抑制其生长，浓度越高，菌的抑制率越高。其中白色念珠菌与金黄色葡糖球菌的敏感性更强。
结果说明，氯喹壳聚糖纳米微球凝胶对多种细菌均有很好的抑制作用，但敏感性有所差别。
本发明将氯喹与壳聚糖联合应用，对多种阴道致病微生物具有协同抑制作用。本发明的药物可以杀灭或抑制多种阴道致病微生物的作用，如金黄色葡萄球菌(SA)、铜绿(EC)、乙型溶血性链球菌(ST)、白色念珠菌(CA)、解脲脲原体(Uu)、阴道毛滴虫及脆弱拟杆菌、单纯性疱疹病毒2型(HSV-2)。另 外，本发明的药物可抑制病原体与宿主阴道上皮细胞的黏附、防治多种阴道炎，如白色念珠菌阴道炎、滴虫性阴道炎、细菌性阴道病、生殖器疱疹病毒等多种外生殖器感染。
实施例10 初步临床研究结果
将本发明的氯喹壳聚糖纳米微球用于由念珠菌感染而引起的阴道炎的妇女10例发现，在治疗过程中没有患者报告副作用和不良反应，随访6个月，也没有发现副作用和不良反应，且氯喹壳聚糖纳米微球治疗组的总有效率比单独氯喹治疗组的总有效率提高明显30％。
将本发明用于30例由HPV感染引起的疣体的治疗，经过2～3周的治疗发现30例患者的疣体自然脱落，且无任何不良反应。
本发明的氯喹纳米微球对病毒性阴道炎、尖锐湿疣、细菌性阴道病、真菌感染性阴道炎或滴虫性阴道炎等外生殖器感染，以及扁平疣、非生殖器部位由HPV感染引起的疣体等其他皮肤疣均有良好的疗效。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (17)

  1. 一种氯喹纳米微球，其特征在于，含有水溶性纳米微球载体、以及氯喹或氯喹衍生物。
  2. 根据权利要求1所述的氯喹纳米微球，其特征在于，其制备方法包括以下步骤：
    S1.将所述水溶性纳米微球载体溶解后，加入氯喹或氯喹衍生物，混合搅拌均匀作为水相，其中氯喹或氯喹衍生物与水溶性纳米微球载体的质量比不超过1/3；
    S2.向油相基质中加入乳化剂，作为油相；将水相加到油相中，水相和油相的体积比为1：1～6；于10000～20000r/min乳化10～30min，得到纳米微球乳液；
    S3.向纳米微球乳液中加入沉淀剂，混合均匀后，静止2～8min后脱水，高速离心清洗，干燥，即可得到所述氯喹纳米微球。
  3. 根据权利要求1或2所述的氯喹纳米微球，其特征在于，所述氯喹或氯喹衍生物与水溶性纳米微球载体在制备时的质量比为1：3～1：5。
  4. 根据权利要求1或2所述的氯喹纳米微球，其特征在于，所述氯喹衍生物选自羟基氯喹、磷酸氯喹或硫酸氯喹中的一种或几种。
  5. 根据权利要求1或2所述的氯喹纳米微球，其特征在于，所述水溶性纳米微球载体选自水溶性壳聚糖、水溶性卡拉胶或水溶性淀粉中的一种或几种。
  6. 根据权利要求1或2所述的氯喹纳米微球，其特征在于，所制备的氯喹纳米微球中氯喹或氯喹衍生物的负载率为3.0％～21.6％。
  7. 根据权利要求2所述的氯喹纳米微球，其特征在于，乳化剂和沉淀剂的体积比为1～1.5：30～50。
  8. 根据权利要求2所述的氯喹纳米微球，其特征在于，所述油相基质为植物油；所述沉淀剂为碱性有机相。
  9. 根据权利要求8所述的氯喹纳米微球，其特征在于，所述碱性有机相为氢氧化钠-正丙醇混合溶液；所述碱性有机相的pH值为8.5～10.0。
  10. 权利要求1或2所述氯喹纳米微球在制备抗菌和/或抗病毒产品中的应用。
  11. 权利要求1或2所述氯喹纳米微球在制备预防和治疗外生殖器感染和/ 或其他皮肤疣的产品中的应用。.
  12. 根据权利要求11所述的应用，其特征在于，所述外生殖器感染包含病毒性阴道炎、尖锐湿疣、细菌性阴道病、真菌感染性阴道炎或滴虫性阴道炎。
  13. 根据权利要求11所述的应用，其特征在于，所述其他皮肤疣包含扁平疣、非生殖器部位的疣体。
  14. 一种用于预防和治疗外生殖器感染和/或其他皮肤疣的产品，其特征在于，包含氯喹或氯喹衍生物，此外还包含水溶性壳聚糖、水溶性卡拉胶或水溶性淀粉中一种或几种。
  15. 根据权利要求14所述的产品，其特征在于，含有权利要求1～9任一所述的氯喹纳米微球。
  16. 根据权利要求15所述的产品，其特征在于，所述氯喹纳米微球占产品总重量的1％～6％。
  17. 根据权利要求14～16任一所述的产品，其特征在于，所述产品的剂型是凝胶剂、软膏剂、乳膏剂、泡腾片、阴道片、胶囊剂、膜剂或栓剂。
PCT/CN2019/115216 2018-12-04 2019-11-04 一种氯喹凝胶及其制备方法和应用 Ceased WO2020114166A1 (zh)

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AU2019391079A AU2019391079B2 (en) 2018-12-04 2019-11-04 Chloroquine gel and preparation method and application therefor
PL19892996.0T PL3760192T3 (pl) 2018-12-04 2019-11-04 Żel chlorochinowy oraz sposób jego przygotowania i zastosowanie
EP19892996.0A EP3760192B1 (en) 2018-12-04 2019-11-04 Chloroquine gel and preparation method and application therefor
US16/978,713 US11147773B2 (en) 2018-12-04 2019-11-04 Chloroquine gel and preparation method and application thereof
BR112020019266-0A BR112020019266B1 (pt) 2018-12-04 2019-11-04 Produto para prevenir e tratar infecção externa de genitália e/ou outras verrugas cutâneas, nanosfera de cloroquina e sua aplicação
RU2020130473A RU2760457C1 (ru) 2018-12-04 2019-11-04 Хлорохин-гель, способ его изготовления и применения
ES19892996T ES2938738T3 (es) 2018-12-04 2019-11-04 Gel de cloroquina y procedimiento de preparación y aplicación del mismo
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