Pharmaceutical composition containing lysine modified amino tetraphenylporphyrin and carboxymethyl chitosan and application

Abstract

The invention discloses a pharmaceutical composition containing lysine modified amino tetraphenyl porphyrin and carboxymethyl chitosan, which is prepared by mixing lysine modified amino tetraphenyl porphyrin and carboxymethyl chitosan according to a mass ratio of 1: 1-1: 50, or adding a pharmaceutically acceptable carrier to prepare a clinically acceptable external dosage form. The invention also provides an application of the pharmaceutical composition in preparation of acne treatment drugs. The pharmaceutical composition disclosed by the invention has a synergistic effect of resisting propionibacterium acnes and treating acnes, and is good in biocompatibility.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a medicine composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan and application, in particular to a medicine composition prepared from a photosensitizer lysine-modified amino tetraphenyl porphyrin and a water-soluble polymer compound carboxymethyl chitosan, and application of the medicine composition in preparation of a medicine for treating acne. BACKGROUND

[0002] Acne is a chronic inflammatory disease of the pilosebaceous unit, with a global prevalence of 9-10%. As the most common skin disease in the world, more than 85% of patients are concentrated in the age of 12-25. In most cases, acne will disappear when the patient is in his or her twenties, and sometimes it will last into adulthood, but this is usually seen in women. Acne can have a very negative impact on adolescents. For example: psychological discomfort, emotional stress, and even permanent scarring on the skin. In addition, acne can cause anxiety and embarrassment in patients, reducing their physical and social well-being. With more and more research on acne, the pathogenesis of acne has gradually become clear, and more and more physical treatment methods have been used to treat acne. Among them, the commonly used physical treatment is photodynamic therapy (PDT). Photodynamic therapy was early used to treat tumor-related diseases. In recent years, researchers have found that photodynamic therapy can also treat bacterial infectious diseases, not only with good effect, small side effects, but also without drug resistance. In addition, PDT uses a low-power light source, which is low in cost and affordable, so it has great application potential in the treatment of acne. Recently, PDT has been applied to the treatment of dermatological diseases. Studies have found that PDT has three basic elements: photosensitizer (PS), light source and tissue oxygen. The main mechanism of PDT for killing bacteria is that the photosensitizer transitions from the ground state (singlet state) to the excited singlet state under the irradiation of visible light of a certain wavelength. The excited singlet state with a short lifetime generates an excited triplet state through a fast inter-system crossing, and then undergoes electron transfer or energy transfer reactions with the molecular oxygen in the environment to produce reactive oxygen species (ROS) with cytotoxicity. ROS can effectively kill sensitive and drug-resistant pathogens in a non-specific manner and will not cause drug resistance in bacteria.

[0003] Porphyrin is a kind of conjugated cyclic compound formed by the methine bridge (=CH-) of the alpha-carbon atom of four pyrrole subunits. Different porphyrin derivatives (hematoporphyrin, tetraphenylporphyrin) have certain antibacterial activity under light conditions, for example: 0.1-1 μg / ml of hematoporphyrin, under white light for 10 minutes, more than 99.9% of the Streptococcus faecalis in the culture medium is inactivated. Porphyrin is first applied to clinical treatment as a photosensitizer by Dougherty et al. due to the advantages of ultraviolet absorption wavelength in the near-infrared region, high singlet oxygen yield, and convenient synthesis. However, the porphyrin photosensitizer has the problems of poor targeting, easy aggregation between molecules, and weak killing ability to gram-negative bacteria. In order to solve the above problems, Meng Shuai et al. use the basic amino acids (lysine, arginine and histidine) in natural amino acids as modification groups to improve the water solubility, biocompatibility and bacterial targeting of porphyrin, and design and synthesize a series of basic amino acid modified tetraphenylporphyrin compounds, which all show good physicochemical properties. Among them, compound 5,10,15,20 tetra {4-[(S)-2,6-diamino-hexylamino] phenyl} porphyrin (LD4) has good water solubility, low toxicity and targeting, and has a unique effect of promoting wound healing and immunoregulation in the treatment of wound infection, as well as inhibiting the growth of pathogenic microorganisms. Xu Zengping et al. found that LD4 has the best photo-inactivation effect on MRSA, Escherichia coli and Pseudomonas aeruginosa, and has lower toxicity to mouse fibroblast L-929. In addition, in vivo tests prove that LD4 mediated PDT can also promote the healing of wounds infected with multi-drug resistant strains. The above studies show that LD4 has great therapeutic potential in photosensitive antibacterial and anti-inflammatory aspects, but the related strains in previous studies are aerobic bacteria, and the antibacterial and anti-inflammatory effects on anaerobic bacteria have not been studied. Therefore, carrying out photodynamic experiments of LD4 on anaerobic bacteria can better verify the photosensitive antibacterial and anti-inflammatory effects of LD4. SUMMARY

[0004] In view of the fact that the photosensitizer lysine-modified amino tetraphenylporphyrin (LD4) in the prior art has good killing effect on aerobic bacteria, but the treatment effect on anaerobic bacteria has not been studied. The present application provides a pharmaceutical composition containing lysine-modified amino tetraphenylporphyrin (LD4) and carboxymethyl chitosan (CMCS), which exhibits synergistic anti-C. acnes activity in the field of anti-infection, and has better treatment effect on mouse acne model, which is better than the positive control drug 5-aminolevulinic acid (5-ALA).

[0005] The present application aims to overcome the shortcomings of the prior art, and provides a pharmaceutical composition containing lysine-modified amino tetraphenylporphyrin (LD4) and carboxymethyl chitosan (CMCS) for treating acne.

[0006] The second object of the present application is to provide an application of the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) in preparation of a medicament for treating acne.

[0007] The object of the present application is mainly achieved by the following technical solutions.

[0008] The pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) for treating acne of the present application can mix lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) according to a mass ratio of 1:1-1:50, or add a pharmaceutically acceptable carrier to prepare a clinically acceptable external dosage form, including ointments, gels, patches, and microneedles, wherein the pharmaceutically acceptable carrier includes at least one of conventional diluents (such as water for injection, microcrystalline cellulose, etc.), fillers (such as mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, vaseline, stearic acid, glycerol monostearate, lanolin, mineral oil, DMSO, etc.), binders (such as carbomer, acacia, starch, cellulose, gelatin, polyvinylpyrrolidone, polyacrylamide, etc.), disintegrants (such as sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, low-substituted hydroxypropyl cellulose, etc.), lubricants (such as talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, micro-powder silica, etc.), humectants (such as propylene glycol, glycerol, ethanol, etc.), stabilizers (such as disodium ethylenediaminetetraacetate, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, nicotinamide, vitamin C, etc.), osmotic pressure regulators (such as sodium chloride, glucose, etc.), pH regulators (such as triethanolamine, sodium hydroxide, sodium citrate, etc.), preservatives (such as chlorobutanol, nipagin, hydroxyphenyl ethyl ester, benzalkonium bromide, etc.), and the above excipients can be a common dosage, mixed with lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) in a common ratio, and the ratio between the pharmaceutical excipients can be adjusted as needed after the amount of lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) is determined, and the chemical structure of the lysine-modified amino tetraphenyl porphyrin (LD4) is

[0009] The pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) for treating acne of the present application is applied in preparation of a medicament for treating acne.

[0010] The beneficial effects of the present application are as follows.

[0011] (1) The pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) has good therapeutic effect on in vitro cultured Propionibacterium acnes and mouse acne model.

[0012] (2) The pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) has good inhibitory effect on the expression of inflammatory factors caused by acne.

[0013] (3) The pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) is expected to become a therapeutic drug for completely eradicating acne with less side effects. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the lysine-modified amino tetraphenyl porphyrin (LD4) of Example 4 of the present application.

[0015] Figure 2 The nuclear magnetic resonance carbon spectrum of the lysine-modified amino tetraphenyl porphyrin (LD4) of Example 4 of the present application.

[0016] Figure 3 The mass spectrum of the lysine-modified amino tetraphenyl porphyrin (LD4) of Example 4 of the present application.

[0017] Figure 4 The ultraviolet absorption spectrum of the lysine-modified amino tetraphenyl porphyrin (LD4) of Example 4 of the present application.

[0018] Figure 5 The body weight change of the mouse acne model treated with the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) of Example 13 of the present application.

[0019] Figure 6 The appearance of the ear skin of the mouse acne model treated with the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) of Example 13 of the present application.

[0020] Figure 7 The degree of inflammatory reaction in the lesion area of the ear of the mouse acne model treated with the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) of Example 13 of the present application.

[0021] Figure 8 The HE staining of the ear skin of the mouse acne model treated with the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) of Example 13 of the present application. DETAILED DESCRIPTION

[0022] The present application provides a kind of light dynamic treatment acne medicine composition containing lysine modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS).

[0023] The specific embodiments of the present application are further described in detail below with reference to Examples and accompanying drawings, which purpose is only to better understand the content of the present application and not to limit the protection scope of the present application. The reagents without source provided in the present application are all commercially available reagents, and the methods not described in detail are all conventional and well-known experimental methods.

[0024] Example 1 Synthesis of 5,10,15,20-tetra(4-nitrophenyl)porphyrin

[0025] P-nitrobenzaldehyde (11.3 g, 75 mmol) was placed in a 500 mL three-necked reaction flask with a thermometer, condenser and stirrer, and propionic acid (200 mL) and acetic anhydride (14 mL, 151 mmol) were added. The mixture was mechanically stirred and heated to reflux, and pyrrole (5.2 mL, 75 mmol) was added dropwise from a constant-pressure dropping funnel. The reaction was carried out for 30 min. The mixture was cooled to room temperature and allowed to stand for 24 h. The product was filtered, washed with a small amount of DMF and hot water, and recrystallized from pyrrole to obtain 5.10,15,20-tetra(4-nitrophenyl)porphyrin as a purple solid (12.1 g, 24%).

[0026] Example 2 Synthesis of 5,10,15,20-tetra(4-aminophenyl)porphyrin (TAPP)

[0027] Compound 5.10,15,20-tetra(4-nitrophenyl)porphyrin (12.1 g, 75 mmol) was placed in 600 mL of concentrated hydrochloric acid and stirred until dissolved. SnCl2.2H2O (51.5 g, 228 mmol) was added, and the mixture was quickly heated to 80°C and stirred for 30 min. The reaction was monitored by thin layer chromatography until completion. 200 mL of water was added, and the mixture was cooled to room temperature and allowed to stand for 24 h. A green precipitate was formed, which was filtered. The precipitate was dissolved in water, and ammonia was added until the color turned purple. The product was filtered, washed with water, and recrystallized from methanol to obtain 5.10,15,20-tetra(4-aminophenyl)porphyrin as a purple solid (9.65 g, 94%).

[0028] Example 3 Synthesis of 5,10,15,20-tetra{4-[(S)-2,6-di-tert-butoxycarbonylaminohexanoylamino]phenyl}porphyrin (Boc-LD4)

[0029] tert-butyloxycarbonyl amino protected lysine (L-Boc-Lys(Boc)-OH, 511.9 mg, 1.479 mmol), 5,10,15,20-tetra(4-aminophenyl)porphyrin (200 mg, 0.296 mmol), 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 674.6 mg, 1.774 mmol), N,N-diisopropylethylamine (DIPEA, 229.3 mg, 1.774 mmol) were placed in a 100 mL reaction bottle, 10 mL of anhydrous DMF was added, stirred until completely dissolved, reacted at room temperature for 14 h, thin layer chromatography was monitored until the reaction was complete (developing agent: dichloromethane / methanol / ammonia water = 20 / 1 / 0.15). After the reaction was completed, 40 mL of deionized water was added to precipitate, vacuum filtration, the filter cake was washed with deionized water three times, vacuum dried, column chromatography separation (eluent: dichloromethane / methanol / ammonia water = 30 / 1 / 0.15), to obtain 5,10,15,20-tetra{4-[(S)-2,6-di-tert-butyloxycarbonyl amino hexanoylamino]phenyl}porphyrin (529.60 mg, 90%).

[0030] Example 4 Synthesis of lysine-modified amino tetraphenyl porphyrin (LD4)

[0031] 5,10,15,20-tetra{4-[(S)-2,6-di-tert-butyloxycarbonyl amino hexanoylamino]phenyl}porphyrin (Boc-LD4) was dissolved with dry CH2Cl2, trifluoroacetic acid (v / v, 10 mL) was slowly added dropwise, and reacted at room temperature for 30 min. The solvent was removed by rotary evaporation, washed with anhydrous diethyl ether, and the organic phase was discarded to obtain a light green precipitate, which was washed with anhydrous dichloromethane and anhydrous diethyl ether three times. The green precipitate was dissolved in distilled water, the pH of the solution was adjusted to 7-8 with ammonia water, and a purple precipitate was precipitated. The precipitate was filtered and washed with water 2-3 times to obtain lysine-modified amino tetraphenyl porphyrin (275.03 mg, 87%).

[0032] The characterization spectrum is shown in Figures 1-4 . The results show that the maximum absorption wavelength of the compound is 421 nm, and there is also ultraviolet absorption at wavelengths of 525 nm, 590 nm, and 652 nm. HRMS (ESI-TOF): m / z [M+H] + 1187.8769; 1 H NMR (300 MHz, DMSO-d6) b-2.00 (s, 2H), 1.56-1.69 (m, 20H), 1.87 (m, 4H), 2.51-2.50 (m, 8H), 2.88 (br, 16H), 3.67 (t, J = 60 Hz, 4H), 8.12-8.20 (m, 16H), 8.89 (s, 8H). The compound was confirmed to be the target compound LD4 by mass spectrometry and nuclear magnetic resonance hydrogen spectrum.

[0033] Example 5 Pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan

[0034] Take 2 mg of lysine-modified amino tetraphenyl porphyrin (LD4) and 98 mg of carboxymethyl chitosan (CMCS), dissolve in PBS (1.9 mL), and you get a pharmaceutical composition with a mass concentration of 0.1% lysine-modified amino tetraphenyl porphyrin and 4.9% carboxymethyl chitosan. Take 6 mg of lysine-modified amino tetraphenyl porphyrin (LD4) and 94 mg of carboxymethyl chitosan (CMCS), dissolve in PBS (1.9 mL), and you get a pharmaceutical composition with a mass concentration of 0.3% lysine-modified amino tetraphenyl porphyrin and 4.7% carboxymethyl chitosan. Take 10 mg of lysine-modified amino tetraphenyl porphyrin (LD4) and 90 mg of carboxymethyl chitosan (CMCS), dissolve in PBS (1.9 mL), and you get a pharmaceutical composition with a mass concentration of 0.5% lysine-modified amino tetraphenyl porphyrin and 4.5% carboxymethyl chitosan.

[0035] Example 6 Pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan

[0036] Mix lysine-modified amino tetraphenyl porphyrin (LD4) with carboxymethyl chitosan (CMCS) in a mass ratio of 1:50, 1:25, 2:25, 1:9, 4:25, 8:25, 16:25, and 1:1, respectively.

[0037] Example 7 Ointment (oily base)

[0038] Melt and filter 5 g of vaseline and 4 g of liquid paraffin separately while hot, remove impurities, then heat to 150°C for about 1 h to sterilize and remove moisture, and cool naturally. Take 62.50 mg of lysine-modified amino tetraphenyl porphyrin and 3.06 g of carboxymethyl chitosan and place them in a mortar, add an appropriate amount of liquid paraffin to make a paste, and then add vaseline in portions and grind until uniform.

[0039] Example 8 Ointment (o / w emulsifier base)

[0040] Take stearic acid 1 g, glycerin monostearate 700 mg, white vaseline 700 mg and liquid paraffin 600 mg in a beaker, heated to about 80℃ on a water bath, stirring to melt, take Tween 80 5 mg and distilled water 6 mL in another small beaker, heated to about 80℃ on a water bath, stirring evenly. At the same temperature, the water phase is added to the oil phase in a thin stream, and the water bath is continuously stirred in a clockwise direction to form a milky white semi-solid, and then stirred at room temperature until near condensation. Take 30.00 mg of lysine-modified amino tetraphenyl porphyrin and 470 mg of carboxymethyl chitosan and place them in an ointment plate and a mortar. Add the prepared O / W emulsion base in portions, and grind evenly. Then, it is obtained.

[0041] Example 9 gel

[0042] Take carbomer 934 1 g, and add it to an appropriate amount of distilled water in portions to make it swell slowly. Add glycerin 10 g, and stir to make a transparent gel base. Dissolve menthol in ethanol, and dissolve boric acid 1 g in an appropriate amount of water. Combine the above, and stir evenly. While stirring, add 20 mg of lysine-modified amino tetraphenyl porphyrin and 180 mg of carboxymethyl chitosan. Adjust the pH to 4.5-5.5 with triethanolamine, and add water to 100 mL. Stir evenly, and then dispense.

[0043] Example 10 patch

[0044] Weigh carbomer 4 g, propylene glycol, Tween-80 and distilled water according to the ratio of 10:20:3:12, heat to 60℃ and stir for 20 min. Add 10 mg of lysine-modified amino tetraphenyl porphyrin and 500 mg of carboxymethyl chitosan, and stir evenly. Apply it to the backing in portions until the desired thickness is reached. Dry or dry in an oven, and then cut according to the specifications of the preparation.

[0045] Example 11 micro-needle

[0046] Wrap 500.00 mg of lysine-modified amino tetraphenyl porphyrin and 500 mg of carboxymethyl chitosan in a 5 g / mL micro-needle tip matrix material to obtain a needle tip solution. Then, dissolve the matrix material of the micro-needle backing in a solvent to obtain a backing solution with a concentration of 5 g / mL. Add the needle tip solution to the mold, vacuum dry, then add the backing solution, and dry and demold.

[0047] Example 12 in vitro photodynamic therapy of acne with lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS)

[0048] Evaluation of the in vitro antibacterial effect of a pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) on Propionibacterium acnes, including the following steps:

[0049] Cutibacterium acnes standard strain ATCC6919 was selected as the research object, which was provided by the Dermatology Institute of Chinese Academy of Medical Sciences (Nanjing).

[0050] Preparation of bacterial suspension: Cutibacterium acnes frozen in a-20℃ refrigerator was recovered to 37℃, 20μL of bacterial solution was inoculated on BHI solid agar medium by plate streaking method, placed in an anaerobic culture box, and added with anaerobic gas bubbles, and then incubated in a 37℃ bacterial incubator for 24h. A single colony was picked up with a sterile inoculation loop and inoculated in 10mL of BHI liquid medium, placed in an anaerobic culture box, and added with anaerobic gas bubbles, and then cultured in a 37℃ bacterial incubator until the bacteria were in the logarithmic growth phase, and the bacterial solution was diluted to 10 5 CFU / mL for standby.

[0051] Drug solution preparation: LD4 was dissolved in sterile phosphate buffer (PBS) to prepare a stock solution with a concentration of 2 mg / mL, and then the drug was diluted with PBS to prepare solutions with concentrations of 1000.00, 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, and 1.95 μg / mL by the two-fold dilution method. CMCS was dissolved in sterile PBS to prepare a stock solution with a concentration of 18 mg / mL, and then diluted with PBS to prepare solutions with concentrations of 9000.00, 4500.00, 2250.00, 1125.00, 562.50, 281.25, 140.63, 70.31, 35.16, and 17.58 μg / mL by the two-fold dilution method. Equal volumes of the LD4 solution with a concentration of 1000.00 μg / mL and the CMCS solution with a concentration of 9000.00 μg / mL were mixed directly to obtain a drug solution containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan (LD4: 500.00 μg / mL, CMCS: 4500.00 μg / mL); drug solutions containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan were obtained in sequence according to the above method (LD4: 250.00 μg / mL, CMCS: 2250.00 μg / mL; LD4: 125.00 μg / mL, CMCS: 1125.00 μg / mL; LD4: 62.50 μg / mL, CMCS: 562.50 μg / mL; LD4: 31.25 μg / mL, CMCS: 281.25 μg / mL; LD4: 15.63 μg / mL, CMCS: 140.63 μg / mL; LD4: 7.81 μg / mL, CMCS: 70.31 μg / mL; LD4: 3.91 μg / mL, CMCS: 35.16 μg / mL; LD4: 1.95 μg / mL, CMCS: 17.58 μg / mL). The positive control drug 5-aminolevulinic acid (5-ALA) was dissolved in sterile PBS to prepare a stock solution with a concentration of 800 mg / mL, and then diluted with PBS to prepare solutions with concentrations of 400.00, 200.00, 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, and 0.02 mg / mL.

[0052] MIC and MBC determination: The experiment was performed using a 96-well plate, and negative control, positive control and experimental groups were set up, with three independent repeated experiments for each concentration. 200 μL of BHI liquid medium was added to the negative control wells, 100 μL of BHI liquid medium and 100 μL of bacterial suspension were added to the positive control wells, and 100 μL of bacterial suspension and 100 μL of corresponding concentration of drug solution were added to the experimental group wells, so that the final concentration of LD4 drug solution was 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.98 μg / mL, respectively. After mixing uniformly, they were placed in a 37°C bacterial incubator for 30 min of dark incubation, then taken out and irradiated under a 650 nm laser for 30 min, with a light dose of 6 J / cm 2, after light irradiation, placed in a 37°C incubator for 48h, and then taken out to observe the growth of the strains in each well. The first well in which the turbidity changed to clarity corresponded to the minimum inhibitory concentration (MIC). 20μL of the bacterial solution in each well with a concentration equal to or greater than the MIC was taken and inoculated on BHI solid medium, and incubated in the dark for 48h. The growth of the bacteria in each culture dish was observed, and the concentration of the drug solution in which the number of colonies was less than or equal to 5 was the minimum bactericidal concentration (MBC). The MIC and MBC determination methods for carboxymethyl chitosan were the same as above. The final concentrations of the CMCS drug solution were 4500.00, 2250.00, 1125.00, 562.50, 281.25, 140.63, 70.31, 35.16, 17.58 and 8.79μg / mL. The MIC and MBC determination methods for the drug composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) were the same as above. The final concentrations of the drug composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan were LD4: 250.00μg / mL, CMCS: 2250.00μg / mL; LD4: 125.00μg / mL, CMCS: 1125.00μg / mL; LD4: 62.50μg / mL, CMCS: 562.50μg / mL; LD4: 31.25μg / mL, CMCS: 281.25μg / mL; LD4: 15.63μg / mL, CMCS: 140.63μg / mL; LD4: 7.81μg / mL, CMCS: 70.31μg / mL; LD4: 3.91μg / mL, CMCS: 35.16μg / mL; LD4: 1.95μg / mL, CMCS: 17.58μg / mL; LD4: 0.98μg / mL, CMCS: 8.79μg / mL. The MIC and MBC determination methods for 5-aminolevulinic acid were the same as above. The final concentrations of the 5-ALA drug solution were 200.00, 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, 0.02 and 0.01mg / mL. The MIC and MBC determination methods for the dark reaction group were the same as above, but without laser irradiation.

[0053] The results are shown in Table 1. Under the light reaction condition, the MIC and MBC of LD4 against P. acnes were both 15.63 μg / mL; under the dark reaction condition, the MIC and MBC of LD4 against P. acnes were both greater than 500.00 μg / mL. Under the light reaction and dark reaction conditions, the MIC and MBC of CMCS against P. acnes were both greater than 4500.00 μg / mL. Under the light reaction condition, the MIC and MBC of the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan against P. acnes were LD4: 7.81 μg / mL and CMCS: 70.31 μg / mL, respectively; under the dark reaction condition, the MIC and MBC of the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan against P. acnes were LD4: 250.00 μg / mL and CMCS: 2250.00 μg / mL, respectively. The results show that the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan has synergistic antibacterial activity, and the carboxymethyl chitosan can enhance the photodynamic antibacterial activity of lysine-modified amino tetraphenyl porphyrin. Under the light reaction condition, the MIC and MBC of 5-ALA against P. acnes were both 12.50 mg / mL; under the dark reaction condition, the MIC and MBC of 5-ALA against P. acnes were both greater than 200.00 mg / mL.

[0054] Table 1 MIC and MBC of the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan against P. acnes

[0055]

[0056] Example 13 In vivo photodynamic treatment of acne with the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS)

[0057] The in vivo experiment of treating the mouse acne model with the pharmaceutical composition containing lysine-modified amino tetraphenyl porphyrin (LD4) and carboxymethyl chitosan (CMCS) prepared in Example 5 comprises the following steps:

[0058] Balb / c male mice aged 6-8 weeks and weighing 20-24 g were selected. On the experimental day, the mice were fasted and anesthetized by intraperitoneal injection of 4% chloral hydrate normal saline solution at a dose of 400 mg / kg. After entering the anesthetized state, the mice were placed in a fixed plate in a prone position, the ear tissue was treated with 75% ethanol disinfection, the hair on the lateral auricle was shaved, and 20 μL of P. acnes bacterial suspension with a concentration of 10 8 CFU / mL was subcutaneously injected with an insulin needle. After the mice woke up, they were normally fed for 1 day to observe the construction of the mouse acne model.

[0059] Experimental grouping and administration: The mice were randomly divided into 6 groups, normal group (Normal, non-molded, non-treatment), model group (Model, given PBS smear), carboxymethyl chitosan group (CMCS, given 4.5% carboxymethyl chitosan aqueous solution smear), lysine-modified amino tetraphenyl porphyrin group (LD4, given 0.5% LD4 aqueous solution smear), lysine-modified amino tetraphenyl porphyrin and carboxymethyl chitosan drug composition group (LD4+CMCS, given the drug composition prepared in Example 5 with a mass concentration of 0.5% lysine-modified amino tetraphenyl porphyrin and 4.5% carboxymethyl chitosan), 5-aminolevulinic acid group (5-ALA, given 5% 5-aminolevulinic acid aqueous solution smear). Each group of mice was smeared with 50 μL of drug solution of corresponding concentration at the affected site. The LD4, LD4+CMCS and 5-ALA experimental groups were dark incubated for 4 h, and then irradiated with a 650 nm laser at a dose of 100 J / cm 2 for 10 min. After treatment, the mice were kept in the dark for 10 days.

[0060] Data collection: ① Weigh each group of experimental animals, compare the weight changes of each group of experimental animals; ② Record the appearance changes of the ear skin, measure the ear thickness of the lesion area with a vernier caliper, and calculate the degree of inflammatory reaction: inflammatory reaction degree = (thickness of the molded ear - thickness of the non-molded ear) / thickness of the molded ear. ③ HE staining to observe the pathological changes of the ear tissue of each group of experimental animals.

[0061] The results are shown in Figures 5-8 . The weight results show that Figure 5 there is no statistical difference in the body weight of mice in each group before and after modeling and during treatment. The appearance of the ear skin shows that Figure 6 the normal group: the auricle tissue of the normal group of mice is soft, the surface is smooth and delicate, and is white pink, the subcutaneous capillary is clear, there is no expansion, and there is no obvious change between the left and right ears. Model group and CMCS group: one day after modeling, the mouse ear at the modeling site swelled obviously, the temperature touched was higher, the auricle tissue was hard, the tissue was obviously hyperplastic and hypertrophic, the epidermis was rough and covered with a large number of scabs, and during the treatment period, except that some of the keratin was shed due to the mice scratching the ear for a long time due to pain and itching, the rest showed no obvious improvement. After photodynamic therapy for 10 days, the auricle of the CMCS+LD4 group of mice gradually recovered to white pink, the auricle hyperplasia and hypertrophy were obviously improved, the ear was obviously not swollen, and there was no obvious keratinization, papule and swelling, the auricle tissue became soft, the skin temperature was normal, and the effect was obviously better than that of the LD4 group, showing a synergistic treatment effect; the LD4 group and the 5-ALA group also had obvious improvement, but there were still slight scabs on the ear, and the auricle was slightly red. The inflammatory reaction degree of the ear lesion area shows that Figure 7), compared with the normal group, the degree of inflammation of the auricle of the model group mice was significantly increased (P<0.01), and compared with the model group, the degree of inflammation of the auricle of the CMCS+LD4 group mice was significantly reduced (P<0.01). The results of ear skin HE staining showed that Figure 8 ), the normal group mice ( Figure 8 a), no obvious abnormality was observed at the auricle site, the boundaries of each skin layer were clear, and no inflammatory cell infiltration was observed; the model group and the CMCS group mice ( Figure 8 b / c), the auricle site and its epidermis were significantly thickened, the hair follicle wall was thickened, the blood vessels were congested, the boundaries of each skin layer became very blurred, a large number of inflammatory cells were observed to infiltrate the dermis layer, and the capillary vessels were obviously dilated. The CMCS+LD4 group mice ( Figure 8 e), no inflammatory cell infiltration was observed in the dermis layer, the thickness of the hair follicle wall returned to normal, the capillary vessels were not dilated, and the boundaries of each skin layer returned to clear. Figure 8 d / f), the boundaries of each skin layer gradually became clear, but the epidermis was significantly thickened, the blood vessels were congested, and a large number of inflammatory cells were still observed to infiltrate the dermis layer.

[0062] It is apparent for those skilled in the art that the present application is not limited to the details of the foregoing exemplary embodiments, and the present application can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all aspects as illustrative and not restrictive. In addition, it should be understood that although the present specification is described in terms of embodiments, it does not contain only one technical solution, and the present specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that those skilled in the art can understand. It should be noted that, for those skilled in the art, a number of modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present patent application should be subject to the appended claims.

Claims

1. A pharmaceutical composition for treating acne containing lysine-modified aminotetraphenylporphyrin (LD4) and carboxymethyl chitosan (CMCS), wherein the lysine-modified aminotetraphenylporphyrin (LD4) and carboxymethyl chitosan (CMCS) are mixed in a mass ratio of 1:1-1:50, or a pharmaceutically acceptable carrier is added to prepare a clinically acceptable external dosage form, including an ointment, a gel, a patch, and a microneedle.

2. The pharmaceutical composition according to claim 1, wherein The chemical structure of the lysine-modified aminotetraphenylporphyrin is 3. The pharmaceutical composition according to claim 1, wherein The pharmaceutically acceptable carrier includes a conventional diluent (such as at least one of water for injection and microcrystalline cellulose), a filler (such as at least one of mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), a binder (such as at least one of carbomer, gum arabic, starch, cellulose, gelatin, polyvinyl pyrrolidone, polyacrylamide, etc.), a disintegrant (such as sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, etc.), a lubricant (such as talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, micro-powdered silica, etc.), a wetting agent (such as propylene glycol, glycerol, ethanol, etc.). at least one of the following: a stabilizer (such as disodium edetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, niacinamide, vitamin C, etc.), an osmotic pressure regulator (such as at least one of sodium chloride and glucose), a pH regulator (such as at least one of triethanolamine, sodium hydroxide, sodium citrate, etc.), a preservative (such as at least one of chlorobutanol, paraben, ethylparaben, benzalkonium bromide, etc.). The above excipients can be mixed with lysine-modified aminotetraphenylporphyrin (LD4) and carboxymethyl chitosan (CMCS) in commonly used dosages and ratios. Once the dosages of lysine-modified aminotetraphenylporphyrin (LD4) and carboxymethyl chitosan (CMCS) are determined, the ratios between the various pharmaceutical excipients can be appropriately adjusted as needed.

4. Use of the pharmaceutical composition comprising lysine-modified aminotetraphenylporphyrin (LD4) and carboxymethyl chitosan (CMCS) for treating acne according to claim 1, 2 or 3 in the preparation of a drug for treating acne.

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