CN118121534A - Composition for treating acne and preparation method and application thereof - Google Patents
Composition for treating acne and preparation method and application thereof Download PDFInfo
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- CN118121534A CN118121534A CN202410194696.XA CN202410194696A CN118121534A CN 118121534 A CN118121534 A CN 118121534A CN 202410194696 A CN202410194696 A CN 202410194696A CN 118121534 A CN118121534 A CN 118121534A
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- ionic liquid
- azelaic acid
- acid
- choline
- fusidic acid
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Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to a composition for treating acne as well as a preparation method and application thereof. The composition for treating acne comprises an ionic liquid, a water phase and an oil phase; the ionic liquid comprises choline, azelaic acid and fusidic acid. The ionic liquid is prepared from choline, azelaic acid and fusidic acid, and the combined action of the choline, the azelaic acid and the fusidic acid can effectively solve the problem of indissolvable property of the azelaic acid and the fusidic acid, and improve bioavailability. In addition, the ionic liquid, the water phase and the oil phase interact to obtain biphasic gel, and azelaic acid and fusidic acid are co-delivered to focus positions by loading the ionic liquid into the biphasic gel, so that the release of medicines from the ionic liquid is promoted, the transdermal penetration and retention of azelaic acid and fusidic acid are further promoted, and the biphasic gel has a good synergistic effect on treating acne by combining azelaic acid and fusidic acid, and further has good bioavailability and acne treatment effect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a composition for treating acne as well as a preparation method and application thereof.
Background
Acne is a common, chronic inflammatory disorder of the sebaceous glands. Most traditional topical formulations for treating acne generally have a high incidence of side effects such as skin irritation, erythema, and itching, and low patient compliance.
Azelaic acid is a saturated dicarboxylic acid naturally produced by malassezia and has anti-inflammatory, antibacterial and keratolytic activities; azelaic acid competitively inhibits tyrosinase, DNA synthesis and mitochondrial enzymes, and is therefore an effective drug for the topical treatment of mild to moderate acne and post-inflammatory pigmentation, but azelaic acid has the problem of poor solubility, low bioavailability and limited effectiveness in treating acne. Fusidic acid is an antibiotic with a steroid skeleton, is effective against staphylococcus aureus and other gram positive bacteria (e.g., propionibacterium acnes), and can be used topically for treating skin or wound infections. The fusidic acid plays an antibacterial role by inhibiting the transfer of the elongation factor G from ribosome in the synthesis process of bacterial proteins, and simultaneously can inhibit chloramphenicol acetyl transferase, so that the action mechanism effectively avoids the cross drug resistance of the fusidic acid and other antibacterial drugs, and the current drug resistance rate is lower. However, the fusidic acid also has the problem of extremely low water solubility and almost no water solubility, which limits the bioavailability of the fusidic acid and the effect of treating acne, and challenges the application of the fusidic acid preparation in the field of treating acne.
Therefore, there is a need to provide a composition which has good transdermal effect, high skin retention, and thus good bioavailability and acne treatment effect.
Disclosure of Invention
The present invention is directed to solving one or more of the problems of the prior art described above, and providing at least one of a beneficial choice or creation. In particular, the invention provides a composition for treating acne, which has good transdermal effect and high skin retention, and further has good bioavailability and acne treatment effect.
The invention is characterized in that: the composition comprises an ionic liquid, a water phase and an oil phase; the ionic liquid comprises choline, azelaic acid and fusidic acid. The ionic liquid is an active ingredient and is prepared from choline, azelaic acid and fusidic acid, the ionic liquid containing the active pharmaceutical ingredient can improve the solubility of the medicine in an aqueous medium, the problem of indissolvable azelaic acid and fusidic acid can be effectively solved, and the bioavailability is improved. In addition, the ionic liquid, the water phase and the oil phase interact to obtain biphasic gel, the biphasic gel combines the properties of hydrogel and organic gel, and meanwhile, the limitation of the two types of gel is overcome, and azelaic acid and fusidic acid are co-delivered to a focus part by loading the ionic liquid into the biphasic gel, so that the release of medicines from the ionic liquid is promoted, the transdermal penetration and the retention of the azelaic acid and the fusidic acid are further promoted, and the biphasic gel has good synergistic effect on the treatment of acne by combining the azelaic acid and the fusidic acid, and further has good bioavailability and acne treatment effect.
Accordingly, in a first aspect the present invention provides a composition for the treatment of acne.
Specifically, a composition for treating acne comprises an ionic liquid, an aqueous phase and an oil phase; the ionic liquid comprises choline, azelaic acid and fusidic acid.
Preferably, the molar ratio of choline, azelaic acid, fusidic acid is 1: (0.8-1.2): (0.040-0.057); further preferably, the molar ratio of choline, azelaic acid, fusidic acid is 1: (0.9-1.1): (0.044-0.052); still more preferably, the molar ratio of choline, azelaic acid, fusidic acid is 1:1:0.048.
Specifically, the ionic liquid has a permeation promotion effect, and the permeation promotion mechanism comprises the following two mechanisms: firstly, the ionic liquid can open tight connection in the stratum corneum and promote the paracellular transport of the medicine; secondly, the ionic liquid can extract lipid components of cell membranes and promote the transcellular transport of medicines. The medicine active ingredient is designed into medicine ionic liquid, which can solve the problems of low solubility, low bioavailability and polymorphism of the traditional medicine.
Specifically, in practical application, azelaic acid and fusidic acid have the problem of indissolvable property, have low solubility in water, are difficult to reach effective concentration, are difficult to be absorbed by organisms through skin, and have low bioavailability and poor treatment effect. The invention determines the types of anions and cations to be choline, azelaic acid and fusidic acid by limiting the proportion of anions and cations in the ionic liquid and screening the prescription, and limits the feeding mole ratio of anions and cations, namely the mole ratio of choline, azelaic acid and fusidic acid. The ionic liquid prepared by taking choline as cation has good biodegradability and low toxicity, and the problem of indissolvable azelaic acid and fusidic acid can be solved by preparing the choline, the choline and the fusidic acid into the ionic liquid and limiting the specific dosage ratio, so that the bioavailability is improved, and the effect of treating acne is further improved.
Preferably, the choline comprises choline bicarbonate.
Preferably, the aqueous phase comprises at least one of poloxamer, water.
Preferably, the oil phase comprises at least one of tea tree oil and soybean phospholipids.
In particular, the composition for treating acne of the present invention is a biphasic gel (dual gel) having limited ability to cross the lipophilic barrier of the skin, and the organogel has low patient compliance due to its residual viscosity and oiliness, and the dual gel combines the properties of both gels while overcoming the limitations of both types of gels. The ionic liquid is loaded into the double gel, so that the percutaneous permeation and detention of the medicine can be promoted, the medicine is completely released from the ionic liquid, the effect of treating acne is improved to the greatest extent, the safety is good, and the use is convenient.
Preferably, the mass ratio of the ionic liquid to the water phase to the oil phase is (0.30-0.55): 1: (0.05-0.50); further preferably, the mass ratio of the ionic liquid, the water phase and the oil phase is (0.33-0.50): 1: (0.06-0.46).
Preferably, the composition further comprises an antioxidant.
Preferably, the antioxidant comprises butylated hydroxyanisole.
In a second aspect, the invention provides a method for preparing a composition for treating acne according to the first aspect of the invention.
Specifically, the preparation method of the composition for treating acne comprises the following steps:
mixing the raw material components to prepare the composition for treating acne.
Preferably, the preparation method of the composition for treating acne comprises the following steps:
Mixing the water phase and the oil phase, adding the ionic liquid, the antioxidant and heating to obtain the composition for treating acne.
Preferably, the preparation method of the composition for treating acne can further comprise the following steps:
Mixing the water phase and the ionic liquid, adding the oil phase, the antioxidant and heating to obtain the composition for treating acne.
Preferably, the heating temperature is 70-90 ℃, and the heating time is 8-12min; further preferably, the heating temperature is 75-85 ℃, and the heating time is 9-11min; still more preferably, the temperature of the heating is 80℃and the time of the heating is 10 minutes.
Preferably, stirring is performed during the heating.
Preferably, the preparation method of the water phase comprises the step of fully swelling poloxamer in water at a certain temperature to prepare the water phase.
Preferably, the temperature is 2-6 ℃; further preferably, the temperature is 3-5 ℃; still more preferably, the temperature is 4 ℃.
Specifically, the water phase is colorless and transparent semi-solid gel.
Preferably, the oil phase is prepared by mixing soybean phospholipid and tea tree oil, and heating.
Preferably, the temperature of the heating is 70-90 ℃; further preferably, the temperature of the heating is 75-85 ℃; still more preferably, the temperature of the heating is 80 ℃.
Preferably, the oil phase is prepared by stirring during heating until the soybean phospholipids are completely dissolved and then cooling to room temperature.
Specifically, the oil phase is yellow transparent liquid.
In particular, the composition for treating acne is a biphasic gel comprising an aqueous phase and an oily phase.
Preferably, the preparation method of the ionic liquid comprises the following steps:
mixing choline, azelaic acid and fusidic acid, and drying to obtain the ionic liquid.
Preferably, the preparation method of the ionic liquid comprises the following steps:
(1) Mixing azelaic acid, fusidic acid and alcohol, then adding choline, and stirring to obtain a mixture;
(2) And (3) drying the mixture obtained in the step (1) to obtain the ionic liquid.
Preferably, in step (1), the alcohol comprises absolute ethanol.
Preferably, in the step (1), the stirring temperature is normal temperature, and the stirring time is 7-9h; further preferably, the stirring time is 7.5-8.5 hours; still more preferably, the stirring time is 8 hours.
Preferably, in the step (2), the drying temperature is 25-35 ℃, and the drying time is 65-80 hours; further preferably, the drying temperature is 27-33 ℃, and the drying time is 70-75 hours; still more preferably, the temperature of the drying is 30 ℃, and the time of the drying is 72 hours.
Preferably, in step (2), the mixture is first subjected to rotary evaporation and then dried.
Preferably, the temperature of the rotary evaporation is 25-35 ℃; further preferably, the temperature of the rotary evaporation is 27-33 ℃; still more preferably, the temperature of the rotary evaporation is 30 ℃.
In particular, the rotary evaporation serves to remove water and ethanol.
Preferably, in step (2), the drying is vacuum drying.
Preferably, the preparation method of the ionic liquid may further be:
Mixing azelaic acid with choline, adding fusidic acid to obtain liquid, and vacuum drying the liquid to obtain the ionic liquid.
Preferably, the temperature of the vacuum drying is 25-35 ℃, and the time of the vacuum drying is 65-80h; further preferably, the temperature of the vacuum drying is 27-33 ℃, and the time of the vacuum drying is 70-75 hours; still more preferably, the temperature of the vacuum drying is 30 ℃, and the time of the vacuum drying is 72 hours.
A third aspect of the invention provides a medicament.
In particular, the medicament comprises a composition for treating acne according to the first aspect of the present invention.
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:
(1) The composition for treating acne comprises an ionic liquid, a water phase and an oil phase; the ionic liquid comprises choline, azelaic acid and fusidic acid. The ionic liquid is an active ingredient and is prepared from choline, azelaic acid and fusidic acid, so that the problem of indissolvable property of azelaic acid and fusidic acid can be solved, and the bioavailability is improved. In addition, the water phase and the oil phase interact to obtain the biphasic gel, the biphasic gel combines the properties of hydrogel and organic gel, and simultaneously overcomes the limitation of the two types of gel, and by loading the ionic liquid into the biphasic gel, azelaic acid and fusidic acid are co-delivered to the focus part, the release of the medicine from the ionic liquid is promoted, the transdermal penetration and retention of azelaic acid and fusidic acid are further promoted, and the biphasic gel has good synergistic effect on the combined azelaic acid and fusidic acid for treating acne, and further has good bioavailability and acne treatment effect.
(2) The composition for treating acne is ionic liquid biphasic gel, has simple components and good safety, can be directly smeared on skin for transdermal administration, is convenient for administration, and provides a new thought for the design of treating acne.
(3) The invention has simple and convenient instrument and equipment, and the preparation process is efficient, simple and controllable, and is suitable for industrialized mass production.
Drawings
FIG. 1 is a graph showing the evaluation of cytotoxicity of choline-azelaic acid-fusidic acid ionic liquids on human immortalized keratinocytes according to example 1 of the present invention;
FIG. 2 is a graph showing cytotoxicity evaluation of choline-azelaic acid-fusidic acid ionic liquid on human fibroblasts according to example 1 of the present invention;
FIG. 3 is a graph showing the evaluation of cytotoxicity of choline-azelaic acid-fusidic acid ionic liquids on human sebaceous cells according to example 1 of the present invention;
FIG. 4 is a diagram showing the appearance of rabbit skin at various times after the subject is removed according to the present invention;
FIG. 5 is a graph showing the cumulative release profile of azelaic acid in the choline-azelaic acid-fusidic acid ionic liquid double gel of the composition of example 1 of the present invention;
FIG. 6 is a graph showing the cumulative release profile of fusidic acid in the choline-azelaic acid-fusidic acid ionic liquid double gel of the composition of example 1 of the present invention;
FIG. 7 is a chart showing the morphology of acne in mice skin following different prescribed administrations;
Fig. 8 is a graph showing the change in relative volume of skin acne on the back of mice during different prescribed dosing.
FIG. 9 is a graph of acne relative volume on day 7 of the back skin of mice following different prescribed administrations;
FIG. 10 is a plot of plated colonies of skin bacteria from mice following different prescribed administrations;
FIG. 11 is a graph showing the skin bacteria content of mice following different prescribed administrations;
FIG. 12 is a graph of hematoxylin-eosin staining observations of acne skin from each group of mice;
FIG. 13 is a graph of relative thickness of skin epidermis in each set of hematoxylin-eosin stained sections;
FIG. 14 is a graph of IL 1. Beta. Immunohistochemistry results for acne skin of mice in each group;
FIG. 15 is a graph of results of oil red O staining of acne skin of each group of mice;
FIG. 16 is a graph showing the results of the relative expression amounts of 5. Alpha. -reductase mRNAs in each group;
FIG. 17 is a graph showing the results of the relative expression amounts of TNFα mRNA in each group;
FIG. 18 is a graph showing the results of the relative expression amounts of K16 mRNA in each group.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Example 1
A composition for treating acne comprises choline-azelaic acid ionic liquid 2.47g, poloxamer 4071.46g, tea tree oil 1.79g, soybean lecithin 0.45g, water 3.83g, and butyl hydroxy anisole 0.04mg.
A method of preparing a composition for treating acne comprising the steps of:
Fully swelling poloxamer P407 in water at the temperature of 4 ℃ and recovering to room temperature to obtain colorless transparent semi-solid gel (water phase); dissolving soybean phospholipids in tea tree oil, heating and stirring at 80deg.C to dissolve completely, and cooling to room temperature to obtain yellow transparent liquid (oil phase); mixing the water phase and the oil phase, adding choline-azelaic acid-fusidic acid ionic liquid and butyl hydroxy anisole, heating and stirring for 10min at 80 ℃, and cooling to room temperature to obtain choline-azelaic acid-fusidic acid ionic liquid double gel, namely the composition for treating acne.
Wherein, the raw materials of the choline-azelaic acid-fusidic acid ionic liquid comprise 4.37g of choline bicarbonate, 4.97g of azelaic acid, 0.66g of fusidic acid and 4mL of absolute ethyl alcohol.
The preparation method of the choline-azelaic acid-fusidic acid ionic liquid comprises the following steps:
Adding azelaic acid and fusidic acid into absolute ethyl alcohol, dispersing uniformly, then slowly dropwise adding choline bicarbonate, stirring at room temperature for 8 hours, rotationally evaporating most of water and ethanol at 30 ℃ to obtain liquid, and then vacuum drying the liquid at 30 ℃ for 72 hours to obtain light yellow viscous liquid, namely choline-azelaic acid-fusidic acid ionic liquid.
Example 2
A composition for treating acne comprises choline-azelaic acid ionic liquid 2.47g, poloxamer 4071.46g, tea tree oil 1.79g, soybean lecithin 0.45g, water 3.83g, and butyl hydroxy anisole 0.04mg.
A method of preparing a composition for treating acne comprising the steps of:
Fully swelling poloxamer P407 in water at the temperature of 4 ℃ and recovering to room temperature to obtain colorless transparent semi-solid gel (water phase); dissolving soybean phospholipids in tea tree oil, heating at 80deg.C, stirring to dissolve completely, and cooling to room temperature to obtain yellow transparent liquid (oil phase); adding choline-azelaic acid-fusidic acid ionic liquid into an aqueous phase, fully mixing, adding an oil phase, then adding butyl hydroxy anisole, heating and stirring for 10min at 80 ℃, and cooling to room temperature to obtain choline-azelaic acid-fusidic acid ionic liquid double gel, namely the composition for treating acne.
Wherein, the raw materials of the choline-azelaic acid-fusidic acid ionic liquid comprise 4.37g of choline bicarbonate, 4.97g of azelaic acid and 0.66g of fusidic acid.
The preparation method of the choline-azelaic acid-fusidic acid ionic liquid comprises the following steps:
Slowly dripping choline bicarbonate into azelaic acid, stirring for 2 hours at room temperature, adding fusidic acid, stirring for 8 hours at room temperature to obtain liquid, and then vacuum drying the liquid at 30 ℃ for 72 hours to obtain yellowish viscous liquid, namely choline-azelaic acid-fusidic acid ionic liquid.
Example 3
A composition for treating acne comprises choline-azelaic acid ionic liquid 2.47g, poloxamer 4071.97g, tea tree oil 0.4g, soybean lecithin 0.1g, water 5.04g, and butyl hydroxy anisole 0.04mg.
A method of preparing a composition for treating acne comprising the steps of:
Fully swelling poloxamer P407 in water at the temperature of 4 ℃ and recovering to room temperature to obtain colorless transparent semi-solid gel (water phase); dissolving soybean phospholipids in tea tree oil, heating at 80deg.C, stirring to dissolve completely, and cooling to room temperature to obtain yellow transparent liquid (oil phase); mixing the water phase and the oil phase, adding choline-azelaic acid-fusidic acid ionic liquid and butyl hydroxy anisole, heating and stirring for 10min at 80 ℃, and cooling to room temperature to obtain choline-azelaic acid-fusidic acid ionic liquid double gel, namely the composition for treating acne.
Wherein, the raw materials of the choline-azelaic acid-fusidic acid ionic liquid comprise 4.37g of choline bicarbonate, 4.97g of azelaic acid and 0.66g of fusidic acid.
The preparation method of the choline-azelaic acid-fusidic acid ionic liquid comprises the following steps:
Slowly dripping choline bicarbonate into azelaic acid, stirring for 2 hours at room temperature, adding fusidic acid, stirring for 8 hours at room temperature to obtain liquid, and then vacuum drying the liquid at 30 ℃ for 72 hours to obtain yellowish viscous liquid, namely choline-azelaic acid-fusidic acid ionic liquid.
Performance testing
1. Safety evaluation
Cytotoxicity test
Considering the safety problem of ionic liquid for treating acne vulgaris, the safety of the choline-azelaic acid-fusidic acid ionic liquid in the sebaceous gland cells, the immortalized keratinocytes and the fibroblasts of the human in the example 1 is evaluated by adopting a cytotoxicity experiment, and the specific method is as follows: human immortalized keratinocytes in the logarithmic growth phase were taken, after digestion with pancreatin and resuspension with DMEM complete medium, 10 μl was pipetted into a blood cell counting plate, the cells were counted under a microscope, and diluted with DMEM complete medium to a cell concentration of about 1×10 5/mL. 100 μl of cell suspension was added to each well of a 96-well plate, so that the number of cells per well was about 1×10 4. The 96-well plate was incubated overnight at 37℃in a 5% CO 2 incubator to allow cells to adhere. The culture medium in each well was removed, 100. Mu.L of choline-azelaic acid-fusidic acid ionic liquid DMEM solution with the concentration of 0.1, 1, 10 and 100. Mu.M was added respectively, and the 96-well plate was further placed in an incubator for 24 hours for cultivation, and the toxicity of choline-azelaic acid-fusidic acid ionic liquid solutions with different concentrations to cells was examined. 5mg/mL of MTT solution was prepared with sterilized water, and the MTT solution was prepared in the following manner in 1:5 ratio dilution. Taking out the 96-well plate from the incubator, removing the original culture medium, adding 120 mu L of the diluted MTT solution, incubating for 4 hours in a dark place, removing the culture medium in the well, adding 150 mu L of DMSO in each well, and shaking for 15 minutes in a dark place to enable the crystals to fully develop. The absorbance of each well in a 96-well plate was measured at a wavelength of 490nm using an enzyme-labeled instrument. Cell viability was calculated as follows, with cytotoxicity reflected by cell viability:
Wherein, the administration group is a group which is inoculated with cells and administered, the control group is a group which is inoculated with cells and not administered, and the zeroing group is a group which is not inoculated with cells and not administered.
The cytotoxicity evaluation of human fibroblasts and human sebaceous cells was the same as the evaluation method of the above-mentioned human immortalized keratinocytes.
Example 1 evaluation of cytotoxicity of choline-azelaic acid-fusidic acid ionic liquids on human immortalized keratinocytes is shown in figure 1.
Example 1 evaluation of cytotoxicity of choline-azelaic acid-fusidic acid ionic liquid on human fibroblasts is shown in figure 2.
Example 1 results of cytotoxicity evaluation of choline-azelaic acid-fusidic acid ionic liquid on human sebaceous cells are shown in fig. 3.
As is clear from FIGS. 1, 2 and 3, the choline-azelaic acid-fusidic acid ionic liquid of example 1 was diluted to 0.1. Mu.M, 1. Mu.M, 10. Mu.M, and 100. Mu.M, and then was allowed to act on human immortalized keratinocytes, human fibroblasts, and human sebaceous gland cells, and the survival rates of the cells were all 80% or more, indicating that the safety of the choline-azelaic acid-fusidic acid ionic liquid of 0.1. Mu.M, 1. Mu.M, 10. Mu.M, and 100. Mu.M was good on these three cells.
Acute irritation test
The hairs on both sides of the spine of the back of the rabbit are removed by using a shaver, and after 24 hours of recovery, the subsequent experiment is carried out. About 0.5g of the test object is smeared on the skin, covered by two layers of gauze and then fixed by using non-irritating adhesive tape. The test article included normal saline, commercially available azelaic acid (Finacea @), commercially available fusidic acid (Fusidin @), a blank double gel matrix (no choline-azelaic acid-fusidic acid ionic liquid as compared to example 1, otherwise identical to example 1), choline-azelaic acid-fusidic acid ionic liquid of example 1, choline-azelaic acid-fusidic acid ionic liquid double gel of example 1, and a positive control 10% sodium lauryl sulfate solution. The application time was 4 hours using the blocking test, and the skin reaction at the application site was observed at 1, 24, 48 and 72 hours after removal of the test substance.
The appearance of the skin of the rabbits after each test object is removed is shown in figure 4, and as can be seen from figure 4, the skin of the 10% sodium dodecyl sulfate group has obvious erythema and shows severe irritation; the commercial formulation group and the blank double gel matrix group showed slight irritation; the skin of the choline-azelaic acid-fusidic acid ionic liquid and ionic liquid double gel group was similar to the physiological saline group, showing no irritation. The choline-azelaic acid-fusidic acid ionic liquid and ionic liquid double gel are mild, have no stimulation and have good safety.
2. In vitro Release test
The composition for treating acne prepared in example 1, i.e., choline-azelaic acid-fusidic acid ionic liquid double gel, was subjected to a release test to examine the release characteristics of azelaic acid and fusidic acid from choline-azelaic acid-fusidic acid ionic liquid double gel.
The specific method comprises the following steps:
Screening of the receiving solution: determining the saturated solubility of azelaic acid and fusidic acid in different media, wherein the saturated solubility of azelaic acid in water is 1.53+ -0.08 mg/mL and the saturated solubility of azelaic acid in PBS solution containing 0.1% oleyl alcohol polyether-20 (Oleth-20) is 3.24+ -0.18 mg/mL; the saturated solubility of fusidic acid in water was 0.18.+ -. 0.12. Mu.g/mL and in PBS containing 0.1% Oleth-20 was 0.66.+ -. 0.01mg/mL. In this experiment 50mg of the composition choline-azelaic acid-fusidic acid ionic liquid double gel of example 1 was administered in the supply tank, i.e. the amount of azelaic acid administered was 7.5mg, the amount of fusidic acid administered was 1mg, the volumes required for total dissolution of azelaic acid and fusidic acid in PBS solution containing 0.1% of Oleth-20 were about 2.31mL and 1.52mL, respectively, while the volume of the receiving tank was 7.5mL, satisfying the sink conditions.
In this experiment, a 0.45 μm organic nylon filter membrane was used as a support and was fixed between a supply cell and a receiving cell of a Franz diffusion cell, and commercially available azelaic acid, commercially available fusidic acid, and choline-azelaic acid-fusidic acid ionic liquid double gel of example 1 were added to the supply cell, respectively. The Franz diffusion cell had an effective release area of 3.14cm 2 and a receiving cell volume of 7.5mL, and the release medium was a PBS solution containing 0.1% Oleth-20. Injecting release medium into a receiving tank, placing the diffusion tank in a diffusion instrument, placing the diffusion tank in a constant-temperature water bath at 32 ℃ and rotating at 250rpm, taking 1mL of release medium at 5min, 15min, 30min, 1h, 2h, 4h, 6h and 12h respectively, and supplementing the blank release medium with the same volume. Filtering the obtained sample with a 0.22 μm microporous filter membrane, measuring the content of azelaic acid and fusidic acid by adopting high performance liquid chromatography, calculating the accumulated release amount according to the following formula, and calculating the accumulated release (%) according to the ratio of the accumulated release amount to the total drug loading of the ionic liquid;
where Cn represents the drug concentration measured in the nth sample, ci is the drug concentration measured in each sample, V represents the volume of the release medium, vi represents the volume of each sample, and Qn (μg) is the cumulative release amount of the drug.
Chromatographic conditions for azelaic acid: the chromatographic column was a C18 column (Kromasil 100-5C18 column, 250X 4.6mm,5 μm); the mobile phase was 50mM NaH 2PO4 solution at pH 3.5 and acetonitrile, ratio 75:25 (v/v); the flow rate is 1.2mL/min; the detection wavelength is 206nm; the detection temperature is 25 ℃; the sample loading was 20. Mu.L.
Chromatographic conditions of fusidic acid: the chromatographic column is XB-C18 column250X 4.6mm,5 μm); the mobile phase is methanol, acetonitrile and 0.05mol/L phosphoric acid solution, and the ratio is 10:60:30 (v/v); the flow rate is 1.0mL/min; the detection wavelength is 235nm; the detection temperature is 35 ℃; the sample loading was 20. Mu.L.
Example 1 cumulative release profiles of azelaic acid and fusidic acid in choline-azelaic acid-fusidic acid ionic liquid double gels are shown in figures 5 and 6, respectively. As can be seen from fig. 5 and 6, azelaic acid in the choline-azelaic acid-fusidic acid ionic liquid double gel is completely released in 2 hours, and the accumulated release amount is more than 90%; azelaic acid in the commercial formulation reached a maximum release at 8 hours, with a cumulative release of about 70%; the release of the fusidic acid in the choline-azelaic acid-fusidic acid ionic liquid double gel is complete in 6 hours, and the accumulated release amount is more than 90%; the cumulative release of fusidic acid in the commercial formulation for 24 hours was less than 70%. Example 1 in vitro release of azelaic acid in choline-azelaic acid-fusidic acid ionic liquid double gel was rapid, fusidic acid exhibited a slow release trend.
The choline-azelaic acid-fusidic acid ionic liquid double gel prepared in example 1 of the present invention is illustrated to rapidly release the drug and rapidly reach high levels of concentration, and then provide stable and sustained inhibitory concentration, which is advantageous for improving the therapeutic effect of acne.
3. In vivo and in vitro transdermal experiments
The composition of example 1, choline-azelaic acid-fusidic acid ionic liquid double gel, was subjected to in vivo and in vitro transdermal experiments to examine the transdermal effect.
(1) In vitro transdermal experiments
Skin treatment: about 220g SD rats were anesthetized with 20% (w/v) uratam, sacrificed by dislocation of cervical vertebrae, abdominal hairs were removed from the rats, abdominal skin was separated with a scalpel, and subcutaneous tissues, blood vessels, and fat layers were removed. The skin was thoroughly cleaned with physiological saline and the filter paper was blotted to remove excess water from the skin surface. Finally, the skin was encapsulated with aluminum foil paper and stored at-20 ℃. The skin is carefully inspected to ensure its integrity prior to transdermal experiments using the skin.
In vitro transdermal test: a piece of skin is placed in normal saline, thawed at room temperature, and then the moisture on the skin surface is sucked up by filter paper, and the skin is mounted between a supply tank and a receiving tank of a diffusion tank, and the skin horny layer faces the side of the supply tank. The effective permeation area of the diffusion cell used in this experiment was 3.14cm 2, the volume of the receiving cell was 7.5mL, and commercially available azelaic acid, commercially available fusidic acid, and the choline-azelaic acid-fusidic acid ionic liquid double gel composition of example 1 were added to the supply cell, respectively, the release medium was PBS solution containing 0.1% Oleth-20, and the temperature was constant water bath at 32℃and the rotational speed was 250rpm. The obtained sample is filtered by a microporous membrane with the diameter of 0.22 mu m, and the content of azelaic acid and fusidic acid in the sample is measured by a high performance liquid chromatography, and the accumulated permeation quantity of azelaic acid and fusidic acid in unit area is calculated according to the following formula;
Wherein C n represents the concentration measured at the nth sampling point, ci represents the concentration measured at the ith sampling point, V 0 represents the volume of the receiving tank, V represents the volume of each sampling, A represents the effective permeation area, and Qn (μg/cm 2) is the cumulative permeation quantity per unit area.
Chromatographic conditions for azelaic acid: the chromatographic column was a C18 column (Kromasil 100-5C18 column, 250X 4.6mm,5 μm); the mobile phase was 50mM NaH 2PO4 solution at pH 3.5 and acetonitrile, ratio 75:25 (v/v); the flow rate is 1.2mL/min; the detection wavelength is 206nm; the detection temperature is 25 ℃; the sample loading was 20. Mu.L.
Chromatographic conditions of fusidic acid: the chromatographic column is XB-C18 column250X 4.6mm,5 μm); the mobile phase is methanol, acetonitrile and 0.05mol/L phosphoric acid solution, and the ratio is 10:60:30 (v/v); the flow rate is 1.0mL/min; the detection wavelength is 235nm; the detection temperature is 35 ℃; the sample loading was 20. Mu.L.
The results of the in vitro transdermal test of azelaic acid and fusidic acid are shown in table 1.
Table 1: results of in vitro transdermal test of azelaic acid and fusidic acid
Wherein, there is a statistical difference (P < 0.001) from the commercial azelaic acid group; ### Is statistically different from the commercial fusidic acid group (P < 0.001).
As can be seen from Table 1, compared with commercial azelaic acid and commercial fusidic acid, the choline-azelaic acid-fusidic acid ionic liquid double gel composition obtained in example 1 can well enhance skin permeation of azelaic acid and fusidic acid, is beneficial to exerting drug effects of azelaic acid and fusidic acid and improves effect of treating acne.
(2) In vivo transdermal experiments
About 200g SD rats were anesthetized with 20% (w/v) Ulatan, and after shaving the abdominal hair with a shaver, depilation was performed using a depilatory cream, and after skin recovery was performed for 24 hours, a subsequent experiment was performed. Rats were anesthetized with 20% (w/v) uratam and then fixed to a rat plate, a supply well (administration area: 3.14cm 2) of a diffusion well was stuck to the abdominal skin of the rats with 502 glue, 50mg of commercially available azelaic acid, commercially available fusidic acid, and the composition choline-azelaic acid-fusidic acid ionic liquid double gel prepared in example 1 were respectively administered to the supply well, the upper end of the supply well was sealed with a sealing film, and an in vivo transdermal test was performed for 24 hours. After 24h, the rats were sacrificed by cervical dislocation, the skin at the administration site was rubbed 3 times with cotton balls stained with water, methanol and water, the skin was rapidly removed, the skin was crushed in an EP tube, 1mL of methanol was added and sonicated for 30min to extract the drug, the mixture was allowed to stand at room temperature for 2h, and the supernatant was filtered through a 0.22 μm microporous filter membrane and the drug content was measured by HPLC.
Chromatographic conditions for azelaic acid: the chromatographic column was a C18 column (Kromasil 100-5C18 column, 250X 4.6mm,5 μm); the mobile phase was 50mM NaH 2PO4 solution at pH 3.5 and acetonitrile, ratio 75:25 (v/v); the flow rate is 1.2mL/min; the detection wavelength is 206nm; the detection temperature is 25 ℃; the sample loading was 20. Mu.L.
Chromatographic conditions of fusidic acid: the chromatographic column is XB-C18 column250X 4.6mm,5 μm); the mobile phase is methanol, acetonitrile and 0.05mol/L phosphoric acid solution, and the ratio is 10:60:30 (v/v); the flow rate is 1.0mL/min; the detection wavelength is 235nm; the detection temperature is 35 ℃; the sample loading was 20. Mu.L.
The results of the in vivo transdermal experiments of azelaic acid and fusidic acid are shown in table 2.
Table 2: results of in vivo transdermal test of azelaic acid and fusidic acid
Wherein, there is a statistical difference (P < 0.001) from the commercial azelaic acid group; ### Is statistically different from the commercial fusidic acid group (P < 0.001).
As can be seen from Table 2, compared with the commercial azelaic acid and the commercial fusidic acid, the choline-azelaic acid-fusidic acid ionic liquid double gel prepared in the example 1 can well enhance the skin retention condition of the azelaic acid and the fusidic acid, is beneficial to the treatment of the azelaic acid and the fusidic acid, and further improves the treatment effect of acne.
4. Acne treatment efficacy test
Example 1 the therapeutic effect of choline-azelaic acid-fusidic acid ionic liquid double gel on acne model mice was observed.
Establishment of acne vulgaris model: this experiment was performed on BALB/c mice subcutaneously injected with P.acnes to model the acne model of the mice. Specifically, after shaving the back of the mice and dehairing with depilatory cream, skin was recovered for 1 day, 50. Mu.L of Propionibacterium acnes (P.acnes) bacteria solution (PBS solution containing 2X 10 9 CFU/mL P.acne) was subcutaneously injected into the back of the mice except for the control group, and modeling of the mouse acne model was performed. The period of time when the back of the mouse swells (after two days of injection of the bacterial liquid) was recorded as day 1. The control group was normal BALB/c mice without molding. The acne mice after molding were divided into seven groups (n=6): the model set (model set), the commercial formulation set (commercial azelaic acid, commercial azelaic acid + commercial fusidic acid), the example 1 choline-azelaic acid-fusidic acid ionic liquid set (ionic liquid set), the example 1 choline-azelaic acid-fusidic acid ionic liquid double gel set (ionic liquid double gel set), the blank double gel matrix set (no choline-azelaic acid-fusidic acid ionic liquid compared to example 1, otherwise identical to example 1). Each group was dosed daily on time, with the amounts of azelaic acid and fusidic acid remaining consistent. The back acne sites of the mice were photographed on days 1,3,5, 7 for each group. After the experiment is finished, the mice are killed by cervical dislocation, the back skin is removed, a part of the skin is added with a sterile culture medium and ground for bacterial culture, a part of the skin is put into 4% paraformaldehyde for fixation, hematoxylin-eosin staining, oil red O staining and immunohistochemistry are carried out after paraffin section, a part of the skin is preserved for polymerase chain reaction experiment (PCR experiment), and the expression of mRNA of 5 alpha-reductase, TNF-alpha and K16 closely related to the development of acne vulgaris is measured by adopting the PCR experiment.
The acne morphology of the skin of each group of mice after administration is shown in fig. 7; the change in relative volume of skin acne on the back of mice during each group of dosing is shown in figure 8; the relative acne on day 7 of the back skin of each group of mice after dosing is shown in fig. 9, in which, # indicates a statistical difference (P < 0.01) from the ionic liquid double gel group, # indicates a statistical difference (P < 0.05) from the ionic liquid double gel group, # indicates a statistical difference (P < 0.001) from the ionic liquid double gel group, and # indicates a statistical difference (P < 0.05) from the model group. As can be seen in fig. 7-9, the first day, the acne sizes of the groups are similar. In the model group, the back skin acne of the mice has the tendency of volume increase in the whole process, the back skin acne of the mice in all administration groups has a certain degree of reduction in size, and the acne treatment effect is as follows: choline-azelaic acid-fusidic acid ionic liquid double gel group > Choline-azelaic acid-fusidic acid ionic liquid group > commercial co-administration group (commercial azelaic acid + commercial fusidic acid) > commercial azelaic acid group > commercial fusidic acid group > blank double gel matrix group.
A plot of the plated colonies of skin bacteria from mice after each group of dosing is shown in fig. 10; the skin bacteria content of mice after each group administration is shown in FIG. 11, # represents a statistical difference (P < 0.001) from the ionic liquid double gel group, a statistical difference (P < 0.01) from the model group, and a statistical difference (P < 0.001) from the model group. As can be seen from fig. 10 and 11, the in vivo antibacterial effect was that the group containing ionic liquid (choline-azelaic acid-fusidic acid ionic liquid double gel group, choline-azelaic acid-fusidic acid ionic liquid group) > the commercial co-administration group (commercial azelaic acid + commercial fusidic acid) > the commercial single group (commercial azelaic acid group, commercial fusidic acid group) > the blank double gel matrix group.
The hematoxylin-eosin staining result diagram of the acne skin of each group of mice is shown in fig. 12, each group has two pictures with different scales, and the scales of each group of pictures are the same as those of the control group; the relative thickness of the epidermis of the skin in each hematoxylin-eosin stained section is shown in FIG. 13, where # in FIG. 13 indicates a statistical difference (P < 0.01) from the ionic liquid double gel group and # in FIG. 13 indicates a statistical difference (P < 0.001) from the model group. As can be seen from fig. 12 and 13, the control mice had a skin thickness of 20±4 μm, a compact skin structure, and after p.acnes subcutaneous injection mice were subjected to acne molding, the overall skin thickness was greatly increased, the skin thickness became 3.9 times that of the normal group, and inflammatory cell infiltration occurred. The epidermis thickness of the administration group is obviously reduced, wherein the epidermis thickness is reduced by the range of ion-containing liquid group (choline-azelaic acid-fusidic acid ionic liquid double gel group, choline-azelaic acid-fusidic acid ionic liquid group) > commercial administration group (commercial combined administration group, commercial azelaic acid group, commercial fusidic acid group) > blank double gel matrix group.
IL 1. Beta. Immunohistochemistry of acne skin of each group of mice is shown in FIG. 14, and the scale of each group of pictures is the same as that of the control group. As can be seen from fig. 14, IL 1 β is an inflammatory factor highly expressed in acne, and is closely related to the inflammatory response of acne, and the results show that there is a small amount of IL 1 β expression in the skin of mice in the control group, and the expression of IL 1 β in the skin is obvious after injection molding by p.acnes, and the administration group significantly improves the inflammatory response of skin, wherein the ionic liquid double gel group is most significantly improved.
The oil red O staining of acne skin of each group of mice is shown in fig. 15, and the scale of each group of pictures is the same as that of the control group. As can be seen from fig. 15, the skin of the control mice had a low fat content and concentrated distribution in the pilo-sebaceous gland region, and after the acne molding of the mice using p.acnes subcutaneous injection, there was a large lipid distribution in the dermis and subcutaneous layers of the skin, and the skin lipid was significantly reduced after administration, with the ionic liquid double gel group improving most significantly.
As can be seen from fig. 7 to 15, the choline-azelaic acid-fusidic acid ionic liquid double gel group prepared in example 1 of the present invention has a better improvement effect on acne mice and has a good effect of treating acne.
The relative expression levels of the 5. Alpha. -reductase mRNAs in each set are shown in FIG. 16, where # in FIG. 16 indicates a statistical difference (P < 0.01) between the ionic liquid double gel set, P < 0.05) between the model set and P <0.001 between the model set. As can be seen from FIG. 16, the increased expression of 5α -reductase after molding compared to the control group means that the skin of the model group had an increased androgen sensitivity, and that the commercial azelaic acid group, the commercial co-administration group (commercial azelaic acid+commercial fusidic acid), the ionic liquid group, and the ionic liquid dual gel group were all statistically different from the model group, and the specific regulatory effect on the mRNA expression of 5α -reductase was that the ionic liquid dual gel group > the ionic liquid group > the commercial co-administration group > the commercial azelaic acid group, and that the commercial fusidic acid group had a certain down-regulating effect on the mRNA expression of 5α -reductase after molding, but not statistically different from the model group.
The relative expression levels of TNFα mRNA in each group are shown in FIG. 17, where in FIG. 17 there is a statistical difference (P < 0.001) between the groups and the model. As can be seen from fig. 17, the expression of tnfα mRNA after molding was increased compared to the control group, and the administration group and the model group were both statistically different, and the specific regulatory effect on tnfα mRNA expression was ionic liquid double gel group > ionic liquid group > commercial combined administration group > commercial azelaic acid group > commercial fusidic acid group.
The relative expression levels of K16 mRNA from each group are shown in FIG. 18, where in FIG. 18 there is a statistical difference (P < 0.001) between the groups and the model. As can be seen from fig. 18, the expression of K16 after molding was improved compared with the control group, and the commercial azelaic acid group, the commercial co-administration group, the ionic liquid group, and the ionic liquid double gel group were statistically different from the model group, and the specific regulatory effect on the expression of K16 mRNA was equivalent to that of the ionic liquid double gel group and the ionic liquid group, and the ionic liquid-containing group (choline-azelaic acid-fusidic acid ionic liquid double gel group, choline-azelaic acid-fusidic acid ionic liquid group) > the commercial co-administration group > the commercial azelaic acid group.
The compositions prepared in examples 2 and 3 of the present invention have an effect of treating acne comparable to that of the composition of example 1.
In summary, azelaic acid and fusidic acid are prepared into ionic liquid, the water phase and the oil phase interact to obtain biphasic gel, the biphasic gel combines the properties of hydrogel and organic gel, meanwhile, the limitation of the two types of gel is overcome, the problem of indissolvable property of azelaic acid and fusidic acid can be solved, the bioavailability is improved, the percutaneous permeation and retention of azelaic acid and fusidic acid are promoted, and the biphasic gel has good synergistic effect on treating acne by combining azelaic acid and fusidic acid and has good acne treatment effect; in addition, the composition can be directly smeared on skin for transdermal administration, is convenient to use, has no skin irritation, enhances the compliance of patients, and provides a new thought for the design of external preparations for treating acne.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A composition, which is characterized by comprising an ionic liquid, an aqueous phase and an oil phase; the ionic liquid comprises choline, azelaic acid and fusidic acid.
2. The composition according to claim 1, wherein the molar ratio of choline, azelaic acid, fusidic acid is 1: (0.8-1.2): (0.040-0.057).
3. The composition of claim 1, wherein the choline comprises choline bicarbonate.
4. The composition of claim 1, wherein the aqueous phase comprises at least one of poloxamer, water.
5. The composition of claim 1, wherein the oil phase comprises at least one of tea tree oil, soybean phospholipids.
6. The composition according to any one of claims 1 to 5, wherein the mass ratio of ionic liquid, aqueous phase, oil phase is (0.30-0.55): 1: (0.05-0.50).
7. The composition of any one of claims 1-5, wherein the composition further comprises an antioxidant.
8. A process for the preparation of a composition as claimed in any one of claims 1 to 7, comprising the steps of:
the raw material components are mixed to prepare the composition.
9. The preparation method of claim 8, wherein the preparation method of the ionic liquid comprises the following steps:
mixing choline, azelaic acid and fusidic acid, and drying to obtain the ionic liquid.
10. A medicament comprising a composition according to any one of claims 1to 7.
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