Application of quaternized hyperbranched polylysine in preparation of medicine for preventing and treating superficial fungal infection

Abstract

The invention belongs to the technical field of medical materials, and relates to an application of quaternized hyperbranched polylysine in preparation of medicines for preventing and treating superficial fungal infection, which is characterized in that the quaternized hyperbranched polylysine has a structural general formula as shown in a formula I or a formula II; the quaternized hyperbranched polylysine disclosed by the invention has better inhibiting and killing effects on main dermatophyte strains causing superficial fungal infection, plays an antibacterial role mainly by means of physical membrane rupture, is not easy to induce drug resistance after being used for a long time, has good biocompatibility, does not have skin irritation after being continuously smeared for multiple times, and is safe and reliable. Safety and reliability. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials and relates to application of quaternized hyperbranched polylysine in preparing medicine for preventing and treating superficial fungal infection. Background Art

[0002] The superficial fungal diseases of the skin caused by dermatophytes such as Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans and Trichophyton xulanensis, Microsporum canis of Microsporum and Epidermophyton floccosum of Epidermophyton and Candida albicans, Candida parapsilosis, Candida glabrata of Candida are quite popular and are also the main pathogenic bacteria of tinea pedis, tinea corporis and tinea cruris. These parasitic pathogenic fungi invade skin, hair and finger (toe) nails, parasitize or saprophyte in the keratin tissue of epidermal keratin, hair and nail plate. On the other hand, superficial fungal diseases have certain infectivity, can be transmitted between different body parts of the same person, and can also cross-infect each other between people. The cardinal symptom of superficial fungal infection of the skin is that the skin appears erythema with clear edges, with desquamation and strong pruritus, which has a strong impact on people's quality of life. According to statistics, about 1 / 4 of the world's population suffers from dermatophyte disease, and in the past few decades, the number of cases of superficial fungal infection has been increasing. Superficial fungal infection of the skin has also developed into one of the major problems affecting human health.

[0003] However, most of the current clinical treatment drugs for superficial fungal diseases are topical ointments containing terbinafine, itraconazole, miconazole or clotrimazole, or oral preparations for systemic treatment. Most of these drugs inhibit the synthesis of ergosterol in the fungal cell membrane, destroy the cell membrane structure and function, and then cause fungal death, but there are side effects such as irritation or allergies, and long-term use is also prone to drug resistance. Slow onset or ineffectiveness and toxicity of long-term treatment, as well as the abuse of antibiotics, the development of antifungal resistance and recurrent infections have made the treatment of superficial fungal infections a huge challenge. There is an urgent need to develop new alternative treatment options with clear effects and high safety. Summary of the invention

[0004] The object of the present invention is to provide a quaternary hyperbranched polylysine for use in the preparation of a drug for preventing and treating superficial fungal infections. The quaternary hyperbranched polylysine has a good inhibitory effect on the main dermatophytes of the genus Trichophyton, Microsporum, Epidermophyton and Candida that cause superficial fungal infections. Its bactericidal mechanism is mainly through electrostatic action, physical membrane rupture, destruction of the cell membrane structure, causing cell contents to leak out and leading to cell death. It has a good antifungal effect and is not easy to induce drug resistance after long-term use, and has good biocompatibility. The quaternary hyperbranched polylysine can be used as an active pharmaceutical ingredient for the prevention and treatment of superficial fungal infections.

[0005] The technical solution of the present invention is as follows:

[0006] The quaternized hyperbranched polylysine described in the present invention is divided into copolymerized quaternized hyperbranched polylysine and derived quaternized hyperbranched polylysine, which can be prepared by copolymerizing quaternized lysine monomer with lysine (one-step copolymerization), or can be synthesized by quaternization derivatization of hyperbranched polylysine.

[0007] The preparation of quaternized hyperbranched polylysine by quaternization of hyperbranched polylysine is mainly divided into two steps: 1) firstly synthesizing hyperbranched polylysine by self-polymerization of lysine hydrochloride; 2) then using small molecule quaternary ammonium salt to appropriately replace and modify hyperbranched polylysine to obtain quaternized hyperbranched polylysine. Similarly, the structure and quaternization degree of quaternized hyperbranched polylysine can be regulated by changing the type of small molecule quaternary ammonium salt substituent, the length of alkyl carbon chain, and the feeding ratio of the hyperbranched polylysine. In addition, it can also be regulated by selecting hyperbranched polylysine with different molecular weights and molecular weight distributions. The hydrophilic-hydrophobic balance and surface potential of quaternized hyperbranched polylysine can be regulated by adjusting the structure and quaternization degree of the quaternary ammonium group, thereby realizing the regulation of its antibacterial properties and biosafety performance.

[0008] The copolymerized quaternized hyperbranched polylysine of the present invention is synthesized by copolymerizing quaternized lysine monomer and lysine, and has the general structural formula shown in Formula I:

[0009]

[0010] Wherein, x1, y1, z1, m1 and n1 are the degrees of polymerization, selected from integers of 1 to 100, and 10≤x1+y1+z1+m1+n1≤200; R1 and R2 are selected from substituted or unsubstituted alkyl groups having 1 to 10 carbon atoms; a is selected from integers of 2 to 20;

[0011] The chemical reaction formula of formula I is:

[0012]

[0013] The derivative quaternized hyperbranched polylysine of the present invention is formed by coupling and modifying hyperbranched polylysine and a small molecule quaternary ammonium salt, and has a general structural formula shown in Formula II:

[0014]

[0015] Wherein, x2, y2, z2, m2, n2 and o2 are the degrees of polymerization, selected from integers of 1 to 100, and 10≤x2+y2+z2+m2+n2+o2≤200; R3 and R4 are selected from or hydrogen, and are not hydrogen at the same time; R5 is selected from -CH2-, -CH2CH2-, -CH2CO-, -CO-, -CH2CH2CO-, -CH2CH2CH2CO-, -OCO- or -CH2CH(OH)-; R6 and R7 are selected from substituted or unsubstituted alkyl groups having 1 to 10 carbon atoms; b is selected from an integer of 2 to 20.

[0016] Chemical reaction formula of formula II:

[0017]

[0018] Wherein: R is a coupling group that can react with an amino group.

[0019] Preferably, the quaternization degree of the quaternized hyperbranched polylysine is 0.5%-50%; a and b are independently selected from integers of 2-20.

[0020] Preferably, the quaternization degree of the quaternized hyperbranched polylysine is 1.0%-30%, more preferably 2%-20%, and a and b are independently selected from integers of 4-14, more preferably 6, 8, 10 and 12.

[0021] Preferably, it is characterized in that the anion group of the quaternized hyperbranched polylysine is selected from at least one of a sulfonic acid group, a carboxylic acid group, a phosphoric acid group, a chloride ion, a bromide ion and an iodide ion.

[0022] Preferably, the superficial fungus is one or more of Trichophyton species, Microsporum species, Epidermophyton species and Candida species.

[0023] Preferably, the superficial fungus is one or more of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton schoenleinii, Microsporum canis, Epidermophyton floccosum, Candida albicans, Candida parapsilosis, and Candida glabrata.

[0024] Preferably, the effective concentration of the quaternized hyperbranched polylysine is 0.1 wt‰ to 10 wt%.

[0025] Preferably, the quaternized hyperbranched polylysine is compounded with one or more pharmaceutically acceptable adjuvants, wherein the adjuvants include one or more of diluents, excipients, moisturizers, preservatives, antioxidants, skin penetration enhancers, and pH regulators.

[0026] Preferably, the diluent is selected from one or more of water, ethanol, glycerol, dimethyl sulfoxide, and propylene glycol;

[0027] The excipient is selected from one or more of natural or semi-synthetic polymers such as alginate, gelatin, pectin, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, starch and its derivatives; synthetic polymers such as polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone and sodium polyacrylate;

[0028] The moisturizing agent is selected from one or more of glycerin, propylene glycol, hyaluronic acid, urea, mannitol, polyethylene glycol, sodium lactate and panthenol;

[0029] The preservative is selected from one or more of benzyl alcohol, parabens, benzalkonium chloride, sorbic acid and its salts, benzoic acid and its salts, and ethanol;

[0030] The antioxidant is selected from one or more of ascorbic acid and its derivatives, ethylenediaminetetraacetic acid and its salts, sodium thiosulfate, sulfites, citric acid and its salts, and phenol;

[0031] The skin penetration enhancer is selected from one or more of glycerol, propylene glycol, ethanol, dimethyl sulfoxide, phospholipids and urea;

[0032] The pH regulator is selected from one or more of citric acid, sodium citrate, phosphoric acid, phosphate, lactic acid, acetic acid, sodium bicarbonate, sodium hydroxide, potassium hydroxide and triethanolamine.

[0033] Preferably, the quaternized hyperbranched polylysine is used as an active ingredient in fabrics and shoe materials. The quaternized hyperbranched polylysine can also be used for antibacterial finishing of fabrics and shoe materials, including not only physical blending but also chemical bonding.

[0034] The quaternized hyperbranched polylysine of the present invention has good inhibitory and killing effects on superficial fungi that can cause skin infection. The quaternized hyperbranched polylysine of the present invention has low cytotoxicity and better biological safety than organic small molecule quaternary ammonium salt compounds.

[0035] The quaternary ammonium hyperbranched polylysine of the present invention is a cationic polyamino acid compound, which mainly exerts antibacterial effects through charge interaction and physical membrane breaking, has good antifungal effects and is not easy to induce the generation of microbial resistance, has good biocompatibility, and is non-irritating to the skin. The quaternary ammonium hyperbranched polylysine of the present invention can be used as an active pharmaceutical ingredient for preparing drugs for treating superficial fungal infections, and can be compounded with pharmaceutically acceptable carriers to prepare lotions, sprays, liniments, ointments, creams, powders, granules and other preparation forms for use. It can be used as a pharmaceutical preparation and applied to the skin or mucosal surface in various ways such as spraying, smearing, applying or soaking for the treatment of superficial fungal infections, and can also be used as a disinfectant for the disinfection of clothing, shoes and boots, etc., and can also be used to disinfect and prevent the local environment while treating skin diseases, thereby preventing or limiting the spread of infection. The various dosage forms can be prepared according to conventional process methods in the pharmaceutical field. In addition, the quaternized hyperbranched polylysine of the present invention can also be used as an antibacterial finishing agent for antibacterial finishing of fabrics and shoe materials, such as cotton socks, underwear, bed sheets, insoles, shoe linings, etc.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The quaternized hyperbranched polylysine of the present invention has a good inhibitory and killing effect on the main skin fungi that cause superficial fungal infections, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans and Trichophyton schrenckii of the genus Trichophyton; Microsporum canis of the genus Microsporum and Epidermophyton floccosum of the genus Epidermophyton; and Candida albicans, Candida parapsilosis, Candida glabrata, etc. of the genus Candida.

[0038] (2) The quaternized hyperbranched polylysine of the present invention mainly relies on physical membrane disruption to cause cell death to exert its antibacterial effect, and is not easy to induce fungal resistance; and it has no skin irritation even after multiple consecutive application, and is safe and reliable.

[0039] (3) The quaternized hyperbranched polylysine of the present invention can be used as an antifungal active drug, which can be compounded with one or more pharmaceutically acceptable adjuvants or additives. The adjuvants and additives include conventional diluents, excipients, moisturizers, skin penetration enhancers, pH regulators, etc. in the pharmaceutical field. The drug for treating superficial fungal infections can be made into various forms such as solutions, sprays, ointments, creams, powders, granules, etc. The drug can be applied to the skin or mucous membrane surface by spraying, smearing, applying or soaking to exert a therapeutic effect. It can also be used as an antibacterial agent, a disinfectant, or an antibacterial finishing agent for materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG1 is a H-NMR spectrum a (400 MHz) and a C-NMR spectrum b (400 MHz) of the quaternized hyperbranched polylysine HPL-QAS1 prepared in Example 1.

[0041] Figure 2 It is the gel permeation chromatogram (GPC curve) of the quaternized hyperbranched polylysine HPL-QAS1 prepared in Example 1.

[0042] Figure 3 This is the H-NMR spectrum (400 MHz) of the hyperbranched poly-lysine prepared in Example 2.

[0043] Figure 4 This is the H NMR spectrum (400 MHz) of the quaternized hyperbranched polylysine HPL-QAS17 prepared in Example 3.

[0044] Figure 5 It is the GPC curve of the quaternized hyperbranched polylysine HPL-QAS17 prepared in Example 3.

[0045] Figure 6 This is the H NMR spectrum (400 MHz) of the quaternized hyperbranched polylysine HPL-QAS18 prepared in Example 4.

[0046] Figure 7 This is a photograph of the quaternary ammonium hyperbranched polylysine gel prepared in Example 5 after being cultured on an agar plate containing Trichophyton rubrum for 7 days.

[0047] Figure 8 These are photos of the control group and the antibacterial fabric group cultured with Trichophyton rubrum at different times. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0049] Example 1

[0050] The structural formula of the quaternized lysine monomer of this embodiment is as follows:

[0051]

[0052] 10g of quaternized lysine monomer, 30g of lysine and 0.5g of FeCl3 were added to a 500mL single-necked flask, connected to a water separator, and nitrogen was replaced three times. Finally, the nitrogen atmosphere was maintained, and the temperature was raised to 180°C and heated and stirred for 6h. After cooling to room temperature, the polymer product was dissolved in methanol and precipitated with ether, and filtered and dried to obtain quaternized hyperbranched polylysine HPL-QAS1. The H NMR spectrum of HPL-QAS1 is shown in Figure 1. GPC characterization: Mn = 6900g / mol, PDI = 1.23, GPC curve is as shown Figure 2 shown.

[0053] The structure and degree of quaternization of the quaternized hyperbranched polylysine in Table 1 are mainly controlled by changing the types of substituents R1 and R2 of the quaternized lysine monomers, the length of the alkyl carbon chain a, and the feed ratio of the quaternized lysine monomers to lysine.

[0054] The process of determining the degree of quaternization is as follows: HPL-QAS is titrated with a standard hydrochloric acid solution by potentiometric titration to calculate the total amine molar number n1; the polymer solution is then masked with salicylaldehyde for primary amines and titrated with a standard hydrochloric acid solution to determine the secondary amine molar number n2. The calculation formula for the degree of quaternization is as follows:

[0055] Quaternization degree % = n2 / n1×100%

[0056] The preparation methods of copolymerized quaternized hyperbranched polylysines with other structures and quaternization degrees are basically the same as above. The specific structures and characterizations of HPL-QAS1 and other quaternized hyperbranched polylysines are shown in Table 1.

[0057] Table 1 Synthesis of quaternized hyperbranched polylysine

[0058]

[0059]

[0060] Example 2

[0061] The synthesis steps of hyperbranched poly-lysine are as follows:

[0062] 100 g of lysine hydrochloride and 22 g of sodium hydroxide were added to a 500 mL single-necked flask, a water separator was connected, nitrogen was replaced three times, and finally the nitrogen atmosphere was maintained, the temperature was raised to 180 ° C, heated and stirred for 6 h, and after cooling to room temperature, the polymer product was dissolved in methanol and precipitated with ether, and filtered and dried to obtain 62.5 g of hyperbranched polylysine (HPL), a light yellow solid powder, whose nuclear magnetic hydrogen spectrum is shown as follows: Figure 3 As shown, GPC characterization: Mn=5800 g / mol, PDI=2.34.

[0063] Other hyperbranched polylysines with different molecular weights and molecular weight distributions are prepared mainly by adjusting the catalyst, polymerization temperature and reaction time.

[0064] Example 3

[0065] Preparation of quaternized hyperbranched polylysine by quaternization of hyperbranched polylysine

[0066] The structural formula of the small molecule quaternary ammonium salt of the present embodiment is as follows:

[0067]

[0068] 30 g of hyperbranched polylysine, 2.7 g of small molecule quaternary ammonium salt, 2.86 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.62 g of N-hydroxysuccinimide sulfonic acid sodium salt were dissolved in 50 mL of water and placed in a 250 mL round-bottom flask. The mixture was reacted at room temperature for 24 h, dialyzed and freeze-dried to obtain a light yellow solid powder of quaternized hyperbranched polylysine HPL-QAS17, the H NMR spectrum of which is shown in FIG. Figure 4 GPC characterization: Mn = 6300 g / mol, PDI = 2.45, GPC curve as shown Figure 5 The amine value was measured by potentiometric titration and the degree of quaternization was calculated to be 3.1%.

[0069] Example 4

[0070] Preparation of quaternized hyperbranched polylysine by quaternization of hyperbranched polylysine

[0071] The structural formula of the small molecule quaternary ammonium salt of the present embodiment is as follows:

[0072]

[0073] 30 g of hyperbranched polylysine and 3.6 g of small molecule quaternary ammonium salt were dissolved in 50 mL of ethanol, placed in a 250 mL round-bottom flask, and refluxed at 80 ° C for 24 h. After the reaction, the mixture was dialyzed and freeze-dried to obtain a light yellow solid powder of quaternized hyperbranched polylysine HPL-QAS18. Its H NMR spectrum is shown in Figure 6 GPC characterization: Mn = 6500 g / mol, PDI = 2.36. The amine value was measured by potentiometric titration, and the degree of quaternization was calculated to be 2.9%.

[0074] Example 5

[0075] Preparation of quaternized hyperbranched polylysine by quaternization of hyperbranched polylysine

[0076] The structural formula of the small molecule quaternary ammonium salt of the present embodiment is as follows:

[0077]

[0078] 30g of hyperbranched polylysine and 3.0g of small molecule quaternary ammonium salt were dissolved in 50mL of isopropanol, placed in a 250mL round-bottom flask, refluxed at 85°C for 24h, dialyzed and freeze-dried to obtain pale yellow solid powder quaternized hyperbranched polylysine HPL-QAS19, GPC characterization: Mn = 6600g / mol, PDI = 2.22. The amine value was measured by potentiometric titration, and the degree of quaternization was calculated to be 3.3%.

[0079] The specific structures and characterizations of HPL-QAS17, HPL-QAS18 and HPL-QAS19 together with other quaternized hyperbranched polylysines are shown in Table 2:

[0080] Table 2 Synthesis of quaternized hyperbranched polylysine

[0081]

[0082]

[0083] Example 6

[0084] The preparation steps of the gel are as follows:

[0085] Using conventional technology, firstly, 3 parts of hydroxypropyl methylcellulose are dissolved in partially purified water and stirred evenly until completely dissolved; 2 parts of glycerol and 10 parts of propylene glycol are added to the above solution and continued to stir; then 1 part of quaternized hyperbranched polylysine HPL-QAS6 solution is added to the above mixture and stirred evenly; 0.1 part of sodium benzoate is added and stirred evenly; water is added to the formula amount and the pH value of the mixture is adjusted to 5-6 with citric acid, and stirred evenly to obtain a gel preparation.

[0086] Example 7

[0087] The steps for preparing the spray are as follows:

[0088] Heat the formulated amount of water to about 50°C, gradually add 0.5 parts of polyvinyl alcohol, and stir until completely dissolved. After cooling to room temperature, add 1 part of quaternized hyperbranched polylysine HPL-QAS6 to the above mixture and stir evenly; add 0.1 parts of potassium sorbate, and use acetic acid to adjust the pH value to between 5-7, mix thoroughly, and put into a spray bottle.

[0089] Example 8

[0090] The preparation steps of antibacterial fabric are as follows:

[0091] 100 g of fabric was first soaked in a 2% 3-chloropropyltrimethoxysilane methanol solution for 10 min, and then the fabric was baked at 60°C for activation pretreatment for 1 h; then the pretreated fabric was soaked in a 2% quaternary ammonium hyperbranched polylysine HPL-QAS6 aqueous solution, air-dried at room temperature, and then baked at 160°C for 10 min for curing. After completion, the antibacterial fabric was washed with water and air-dried to obtain.

[0092] Example 9

[0093] The preparation steps of antibacterial EVA masterbatch are as follows:

[0094] Put 1kg EVA (ethylene-vinyl acetate copolymer) masterbatch and 50g quaternized hyperbranched polylysine HPL-QAS6 into a mixer and stir to premix evenly. Then add the premix into a twin-screw extruder, control the temperature to 110-120°C, extrude and granulate to obtain antibacterial EVA masterbatch. It can be further compounded with other materials for the manufacture of insoles, soles, etc.

[0095] Test Example 1

[0096] Drug sensitivity test of quaternary ammonium hyperbranched poly-lysine

[0097] The test strains used in the test examples are: Trichophyton rubrum (ATCC MYA-4438), Trichophyton mentagrophytes (BNCC340405), Trichophyton tonsurans (BNCC366566), Trichophyton schoenleinii (BNCC366571), Microsporum canis Bodin anamorph (BNCC259680), Epidermophyton floccosum (BNCC366550) and Candida albicans (ATCC10231), Candida parapsilosis (AS2.590), Candida glabrata (ATCC 15216). Among them, ATCC stands for American Type Culture Collection, BNCC stands for Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center, and AS stands for Institute of Microbiology, Chinese Academy of Sciences.

[0098] The antibacterial activity of quaternized hyperbranched poly-lysine against superficial fungi (Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton schrenckii, Microsporum canis, Epidermophyton floccosum and Candida albicans, Candida parapsilosis, Candida glabrata) was detected, and the clinically used antifungal drugs fluconazole and terbinafine were used as controls. Fluconazole and terbinafine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0099] The antibacterial test method was carried out in accordance with the broth microdilution method for antifungal drug sensitivity test protocol CLSI-M38-A2 recommended by the National Committee for Clinical Laboratory Standards of the United States. Unless otherwise specified, the strains were cultured according to conventional culture methods. All test strains were eluted with sterile saline on the day of the test, counted with a hemocytometer, and diluted to obtain a bacterial suspension concentration of 1×104 CFU / mL is used as inoculum. The minimum inhibitory concentration (MIC) is determined as the lowest drug concentration at which the turbidity of the experimental group is significantly reduced or there is no obvious hyphae growth compared with the control group under naked eye observation after culturing for 48h (Candida albicans, Candida parapsilosis, Candida glabrata) or 5 days (Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton xulanensis, Microsporum canis and Epidermophyton floccosum) at an appropriate culture temperature. The antibacterial experimental results of quaternized hyperbranched polylysine are shown in Table 3.

[0100] Table 3 Antibacterial activity of quaternized hyperbranched polylysine

[0101]

[0102]

[0103] The results in Table 3 show that the antibacterial activity of quaternized hyperbranched polylysine against the superficial fungi tested, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton schrenckii, Microsporum canis, Epidermophyton floccosum, and Candida albicans, Candida parapsilosis, and Candida glabrata, is enhanced as the degree of quaternization substitution in the quaternized hyperbranched polylysine and the carbon chain length of the quaternary ammonium salt increase. When the carbon chain length is less than 8 and the degree of quaternary ammonium substitution in the quaternary hyperbranched polylysine is less than 3, its antibacterial activity against Trichophyton rubrum, Trichophyton tonsurans, Trichophyton schrenkiana, Microsporum canis, Candida albicans, Candida parapsilosis and Candida glabrata is poor; for other strains in the experiment, when the carbon chain length is greater than 10 and the degree of quaternary ammonium substitution in the quaternary hyperbranched polylysine is greater than 3, the MIC has basically good antibacterial properties for all strains in the experiment, and the MIC values ​​are generally in the range of 0.5-96 μg / mL.

[0104] Test Example 2

[0105] Skin irritation test of quaternized hyperbranched poly-lysine

[0106] Sterile physiological saline was used as negative control, the quaternized hyperbranched polylysine solution (HPL-QAS6) prepared in Example 1 was used as the test drug, the test concentration was 1000 μg / mL, and New Zealand rabbits were selected as experimental animals for skin irritation experiments. 12 New Zealand rabbits, half male and half female, were randomly divided into a control group, a quaternized hyperbranched polylysine group 1, and a quaternized hyperbranched polylysine group 2, with 4 rabbits in each group. 4 to 24 hours before the experiment, the hair on both sides of the rabbit's back spine (about 8cm×8cm) was removed for use as the experimental and observation site, the experimental drug was infiltrated into a 2.5cm×2.5cm gauze block and then covered to the experimental area, and fixed with a breathable patch for at least 4 hours. Repeat once a day, and apply continuously for 14 days. During the test, in order to facilitate observation or re-experimentation, hair removal is required repeatedly.

[0107] The results showed that during the experiment, no acute or cumulative irritation reaction was observed on the skin surface of the rabbits in the experimental group and the control group under natural light, and no inflammatory reaction such as erythema or edema occurred, indicating that quaternary ammonium hyperbranched polylysine had no obvious skin irritation.

[0108] Test Example 3

[0109] Drug sensitivity test of gel

[0110] The antibacterial activity of the gel preparation was determined by the punching method. First, a sterile cotton swab was dipped into an appropriate amount of the bacterial suspension to be tested and evenly coated on the surface of the PDA agar medium to prepare a bacterial plate. Then, a sterile puncher (outer diameter 16 mm) was used to punch holes in the bacterial plate medium, and the agar in the holes was removed and the gel prepared in Example 6 was filled into the holes. The plate was placed in a constant temperature incubator to culture and observe the growth of Trichophyton rubrum around the gel holes.

[0111] The results showed that after 7 days of culture on the agar plate inoculated with Trichophyton rubrum, an obvious inhibition zone was observed to be formed around the gel on the agar surface, and the width of the inhibition zone was 11 mm, indicating that the antibacterial gel containing the quaternary ammonium hyperbranched polylysine active ingredient prepared in Example 6 had a good antibacterial effect on Trichophyton rubrum.

[0112] Test Example 4

[0113] Therapeutic effect of gel preparation on animal model of superficial fungal infection with Trichophyton rubrum

[0114] Twenty healthy guinea pigs weighing about 450-550g were selected, regardless of gender, and randomly divided into two groups, 10 in each group, the control group and the experimental group. Inoculation of bacterial solution in the experimental group: The hair on the abdomen of the guinea pigs was carefully shaved with an electric shaver (area 2.5cm×2.5cm), and gently polished with fine sandpaper until punctate bleeding was observed on the skin surface. A sterile cotton swab was dipped in a fresh suspension of Trichophyton rubrum spores (2×10 7 CFU / mL) was applied to the wound, and the bacterial solution was applied continuously for 10 times (once a day) to complete the inoculation. Subsequently, the skin squamous cells of the experimental area were picked for microscopic observation to determine whether the infection model was established. When the model was successfully established, an appropriate amount of the gel ointment containing the present invention was applied to the infected part twice a day, with an application amount of about 0.5g each time, and sterile gauze was used for bandaging and fixing after each administration. The blank control group was coated with sterile saline.

[0115] Effect evaluation: After four days of treatment, a small amount of dandruff from the experimental site was scraped every three days, and then digested with 10% KOH and examined under a microscope. If there is hyphae, it is positive, otherwise it is negative. At the same time, the scraped dandruff was inoculated on the surface of the PDA agar medium containing chloramphenicol to observe whether hyphae were generated. After 14 days of continuous drug administration, the skin condition of the experimental site of the test animal was observed. 0 means no skin damage; 1 is punctate erythema; 2 is range erythema; 3 is redness, swelling, and scaling; 4 is erythema and scabs beyond the range. At the end of the experiment, the number of guinea pig skins that have been cured is used as an indicator of treatment. The standard of recovery is: the symptoms of erythema, redness, swelling, scaling, itching, etc. completely disappear, and the fungal microscopic examination result is negative.

[0116] The results are shown in Table 4. Compared with the control group, the symptoms of 9 out of 10 guinea pigs with skin infection in the gel treatment group subsided, and the microscopic examination results showed that there were no hyphae and spores and were negative. Although there was still a small amount of punctate erythema at the infected site of the other guinea pig with skin infection, the symptoms were significantly improved compared with those of the control group. The results showed that quaternary ammonium hyperbranched polylysine gel has a significant therapeutic effect on the guinea pig skin model infected with Trichophyton rubrum.

[0117] Table 4 The therapeutic effect of the gel preparation on the guinea pig skin model of superficial fungal infection

[0118] Group Number of animals Number of recoveries Control group 10 0 Gel preparation group 10 9

[0119] Test Example 5

[0120] Antimicrobial fabric susceptibility test

[0121] First, a sterile cotton swab was dipped into an appropriate amount of Trichophyton rubrum suspension and evenly spread on the surface of PDA agar medium to prepare a bacterial plate. Then, the antibacterial fabric that had been sterilized at high temperature and the blank fabric of the control group (2cm×2cm) were attached to the surface of the bacterial plate and placed in a constant temperature incubator, and the growth of Trichophyton rubrum on the surface and around the fabric was observed and photographed.

[0122] The results are as follows Figure 8 As shown in the figure: On the 5th day of the experiment, a large number of white Trichophyton rubrum colonies began to form on the surface of the control group fabric, while no colonies appeared on the surface of the experimental group antibacterial fabric; at a longer observation time, on the 14th day of the experiment, we can observe that the surface of the control group fabric has been completely covered with white hyphae of Trichophyton rubrum, while no colonies appeared on the surface of the experimental group antibacterial fabric. The experimental results show that the antibacterial fabric treated with quaternary ammonium hyperbranched polylysine also has a good antibacterial effect on superficial fungi represented by Trichophyton rubrum.

[0123] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a quaternized hyperbranched polylysine in the preparation of a drug for preventing and treating superficial fungal infections, characterized in that: The quaternized hyperbranched polylysine has a general structural formula shown in Formula I or Formula II; Wherein, x1, y1, z1, m1 and n1 are the degrees of polymerization, selected from integers of 1 to 100, and 10≤x1+y1+z1+m1+n1≤200; R1 and R2 are selected from substituted or unsubstituted alkyl groups having 1 to 10 carbon atoms; a is selected from integers of 2 to 20; Wherein, x2, y2, z2, m2, n2 and o2 are the degrees of polymerization, selected from integers of 1 to 100, and 10≤x2+y2+z2+m2+n2+o2≤200; R3 and R4 are selected from or hydrogen, and are not hydrogen at the same time; R5 is selected from -CH2-, -CH2CH2-, -CH2CO-, -CO-, -CH2CH2CO-, -CH2CH2CH2CO-, -OCO- or -CH2CH(OH)-; R6 and R7 are selected from substituted or unsubstituted alkyl groups having 1 to 10 carbon atoms; b is selected from an integer of 2 to 20.

2. The use according to claim 1, characterized in that: The quaternization degree of the quaternized hyperbranched polylysine is 0.5%-50%; a and b are independently selected from integers of 2-20.

3. The use according to claim 2, characterized in that: The quaternization degree of the quaternized hyperbranched polylysine is 1.0%-30%, and a and b are independently selected from integers of 4-14.

4. The use according to claim 3, characterized in that: The anion group of the quaternized hyperbranched polylysine is selected from at least one of a sulfonic acid group, a carboxylic acid group, a phosphoric acid group, a chloride ion, a bromide ion, and an iodide ion.

5. The use according to any one of claims 1 to 4, characterized in that: The superficial fungi are one or more of Trichophyton species, Microsporum species, Epidermophyton species and Candida species.

6. The use according to claim 5, characterized in that: The superficial fungi are one or more of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton schoenleinii, Microsporum canis, Epidermophyton floccosum, Candida albicans, Candida parapsilosis, and Candida glabrata.

7. The use according to claim 5, characterized in that: The effective concentration of the quaternized hyperbranched polylysine is 0.1 wt‰ to 10 wt%.

8. The use according to claim 7, characterized in that: The quaternized hyperbranched polylysine is compounded with one or more pharmaceutically acceptable auxiliary agents, wherein the auxiliary agents include one or more of diluents, excipients, moisturizers, preservatives, antioxidants, skin penetration enhancers, and pH regulators.

9. The use according to claim 8, characterized in that: The diluent is selected from one or more of water, ethanol, glycerol, dimethyl sulfoxide, and propylene glycol; The excipient is selected from one or more of natural or semi-synthetic polymers such as alginate, gelatin, pectin, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, starch and its derivatives; synthetic polymers such as polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone and sodium polyacrylate; The moisturizing agent is selected from one or more of glycerin, propylene glycol, hyaluronic acid, urea, mannitol, polyethylene glycol, sodium lactate and panthenol; The preservative is selected from one or more of benzyl alcohol, parabens, benzalkonium chloride, sorbic acid and its salts, benzoic acid and its salts, and ethanol; The antioxidant is selected from one or more of ascorbic acid and its derivatives, ethylenediaminetetraacetic acid and its salts, sodium thiosulfate, sulfites, citric acid and its salts, and phenol; The skin penetration enhancer is selected from one or more of glycerol, propylene glycol, ethanol, dimethyl sulfoxide, phospholipids and urea; The pH regulator is selected from one or more of citric acid, sodium citrate, phosphoric acid, phosphate, lactic acid, acetic acid, sodium bicarbonate, sodium hydroxide, potassium hydroxide and triethanolamine.

10. The use according to any one of claims 1 to 4, characterized in that: Application of quaternized hyperbranched polylysine as active ingredient in fabrics and shoe materials.

Images