Reagent for treating vitiligo and preparation method thereof

Abstract

The invention discloses a reagent for treating vitiligo and a preparation method thereof, and belongs to the technical field of vitiligo treatment agents.The reagent is prepared from itaconic acid, diethylhexyl carbonate, silicone oil, glycerin, an emulsifier, polyvinyl alcohol, sodium chloride, oleic acid, abietic acid and spruce volatile oil. The problems that at present, a vitiligo medicament prepared by taking itaconic acid as an effective component is poor in stability, difficult to store conveniently and high in irritation are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of vitiligo treatment agents, and in particular to a vitiligo treatment agent and a preparation method thereof. Background Art

[0002] Vitiligo is an autoimmune skin disease caused by the destruction of melanocytes. Its clinical manifestations are clearly demarcated white spots, which seriously endanger the patient's physical and mental health. Existing treatments are mainly topical glucocorticoids / calcineurin inhibitors and phototherapy, but there are still shortcomings such as low overall efficacy, long treatment cycle, and easy recurrence after drug withdrawal. The cause of this disease is complex and is caused by the combined effects of multiple factors such as genetics, oxidative stress, and autoimmunity. Therefore, it is crucial to develop new clinical drugs that are effective in treating vitiligo based on the pathogenesis of vitiligo.

[0003] Itaconic acid is an endogenous immune cell metabolite produced by activated macrophages under inflammatory stimulation, has anti-inflammatory activity, and belongs to unsaturated organic dicarboxylic acid. Early studies reported that itaconic acid has antibacterial and antiviral effects and mechanisms. For example, itaconic acid can inhibit the expression and activity of isocitrate lyase, preventing bacteria from using other carbon sources for energy metabolism and biosynthesis when glucose is scarce, thereby exerting an antibacterial effect. However, when itaconic acid is used as an effective ingredient to make a conventional vitiligo reagent, it is found that it denatures or self-polymerizes under high temperature, high humidity, and light conditions, resulting in a serious decrease in drug efficacy, and itaconic acid generates hydroxy acids in aqueous solution that are easy to react with other raw materials, thereby causing its storage conditions to be more harsh, which is not conducive to the production and preservation of the agent. Based on this, the present invention is to disclose a novel agent for treating vitiligo to solve the above problems. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a reagent for treating vitiligo and a preparation method thereof, so as to solve the problems that the current vitiligo medicines prepared with itaconic acid as an active ingredient have poor stability, are difficult to store conveniently, and have strong irritation.

[0005] The present invention solves the above technical problems by the following technical means:

[0006] A reagent for treating vitiligo, comprising the following ingredients:

[0007] Itaconic acid, diethylhexyl carbonate, silicone oil, glycerin, emulsifier, polyvinyl alcohol, sodium chloride, oleic acid, rosin acid, spruce essential oil.

[0008] Itaconic acid inhibits the immune response of vitiligo and exerts an anti-inflammatory therapeutic effect, delaying the progression of white spots of vitiligo and thus treating vitiligo. Therefore, the main active ingredient of the reagent for treating vitiligo of the present invention is itaconic acid. However, the physicochemical properties of itaconic acid are relatively special. It is usually used immediately after preparation in the laboratory. Therefore, it is still necessary to explore the direction of preparing a therapeutic agent. At the same time, the skin of vitiligo patients is fragile. Based on this, the present invention adds auxiliary materials to form a cream for easy use, while reducing production and storage costs and irritation, which is helpful for the clinical treatment of vitiligo patients.

[0009] Furthermore, the reagent is made into an ointment or cream and applied to the affected area.

[0010] Furthermore, the concentration of itaconic acid in the reagent is (40-50) mg / g.

[0011] Further, the reagent comprises the following raw materials in parts by weight:

[0012] 3-4 parts by mass of diethylhexyl carbonate, 2-3 parts by mass of silicone oil, 8-10 parts by mass of glycerin, 1.5-2 parts by mass of emulsifier, 10-15 parts by mass of polyvinyl alcohol, 1-1.5 parts by mass of sodium chloride, 1-2 parts by mass of oleic acid, 0.5-0.7 parts by mass of rosin acid, and 0.5-1 parts by mass of spruce volatile oil.

[0013] Since the suitable external concentration of itaconic acid is (40-50) mg / g, in order to reduce the dosage while achieving the therapeutic purpose, it is necessary to achieve a highly efficient transdermal absorption effect and solve the problem of poor bioavailability of itaconic acid through skin absorption. Therefore, the reagent prepared by the present invention is in the form of an ointment or cream. When used, it is applied to the affected area and gently rubbed to spread. At this time, the water phase and the oil phase are separated, forming a demulsification phenomenon, which promotes the rapid evaporation of the water phase, and the active ingredient is more easily absorbed by the skin after mixing with the oil phase, thereby achieving the purpose of rapid treatment.

[0014] Of course, an appropriate amount of phenoxyethanol can also be added to the aqueous phase of the prepared reagent as a preservative.

[0015] Furthermore, the silicone oil has a viscosity of 200.

[0016] The present invention also discloses a method for preparing a reagent for treating vitiligo, and the method for preparing the reagent is as follows:

[0017] (1) diethylhexyl carbonate, glycerol, diethylhexyl carbonate, and an emulsifier are stirred uniformly to obtain an oil phase;

[0018] (2) dissolving sodium chloride in water, adding rosin acid after heating, and stirring evenly to obtain an aqueous phase;

[0019] (3) dissolving polyvinyl alcohol in hot water, cooling to room temperature to obtain a polyvinyl alcohol solution, then adding spruce volatile oil and itaconic acid, stirring evenly, freezing at -10°C for 5-6 hours, thawing at room temperature for 10-12 hours, repeating twice, and then crushing to obtain a solid phase;

[0020] (4) After the solid phase and the oil phase are mixed, oleic acid is added, and then the aqueous phase is slowly added. After high-speed stirring, the pH of the reagent is adjusted to 4.5 to obtain the reagent.

[0021] The reagent of the present invention is composed of an aqueous phase, an oil phase, and a solid phase, wherein the solid phase is mainly a gel formed by polyvinyl alcohol to wrap itaconic acid, so as to avoid reducing the drug efficacy of itaconic acid during preparation, storage, and transportation. In addition, the solid phase added can improve the stability in the process of mixing with the aqueous phase and the oil phase and increase the adhesion to the skin during use. At the same time, in order to improve the stability of the reagent, in the oil phase, the present invention selects diethylhexyl carbonate and a silicone oil with a viscosity value of 200 to mix and form a light oil phase component, and sodium chloride and abietic acid are added to the aqueous phase to form a salt solution with a certain viscosity, and a critical system with a low freezing point is formed in the process of making the reagent by high-speed stirring in combination with components such as glycerol, so that it will not condense under low temperature conditions, and will not form a demulsification phenomenon after restoring room temperature, thereby improving the stability of the reagent. When the reagent is used, water evaporates, and the spruce volatile oil in the solid phase and itaconic acid are twisted and mixed with the oil phase to adhere to the skin surface, increasing the fluidity of the intercellular lipid region and improving the bioavailability of the drug. At the same time, because the reagent contains salt ions, the drug will also be prompted to enter the skin due to mechanical pressing and osmotic pressure difference during the massage and rubbing process. In addition, since spruce volatile oil has certain antioxidant, anti-inflammatory and bactericidal effects, it can not only improve the stability of the reagent, but also play a certain auxiliary therapeutic role in vitiligo. During the high-speed stirring process, adding oleic acid can reduce the phase transition temperature of the reagent, promote the separation of water and oil phases during the kneading process, and reduce the difficulty of using the reagent.

[0022] Furthermore, in the water phase, the mass of water added is 7-8 times the total mass of the oil phase.

[0023] Furthermore, the mass concentration of the polyvinyl alcohol solution is 5-8%.

[0024] Furthermore, in step (4), the rotation speed is 950-1000 r / min.

[0025] Since the reagent of the present invention is an ointment or cream in a critical stable state, the raw material component ratio and the preparation process are particularly critical.

[0026] Furthermore, the emulsifier is EM90 emulsifier.

[0027] Beneficial effects:

[0028] Itaconic acid can be used for the treatment of vitiligo, and since vitiligo patients have fragile skin, based on this, the present invention adds auxiliary materials to make a cream that is easy to use, reduces production and storage costs, reduces irritation, and helps the clinical treatment of vitiligo patients. In addition, the prepared cream has good stability, is easy to store, and is convenient to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 :Mouse experiment diagram and epidermal melanocytes / CD8 + T cell immunofluorescence images. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to specific embodiments:

[0031] Embodiment 1:

[0032] Weigh the following raw materials:

[0033] 3g diethylhexyl carbonate, 2g silicone oil (viscosity 200mPa.s), 8g glycerin, 1.5g EM90 emulsifier, 10g polyvinyl alcohol, 1g sodium chloride, 1g oleic acid, 0.5g rosin acid, 0.5g spruce volatile oil, 13.65g itaconic acid.

[0034] (1) adding diethylhexyl carbonate, glycerin, silicone oil and emulsifier and stirring to obtain an oil phase;

[0035] (2) Dissolve 110 g of sodium chloride in water, heat to about 80° C., add rosin acid, and stir evenly to obtain an aqueous phase;

[0036] (3) adding polyvinyl alcohol to 90° C. hot water, dissolving it and cooling it to room temperature to obtain a 5 wt% polyvinyl alcohol solution, then adding spruce volatile oil and itaconic acid, stirring evenly, freezing at -10° C. for 5 h, thawing at room temperature for 10 h, repeating this process twice, and then crushing or mashing to obtain a solid phase;

[0037] (4) After the solid phase and the oil phase are mixed, oleic acid is added, and then the aqueous phase is slowly added. After high-speed stirring at 950 r / min to form a cream, the pH value of the reagent is adjusted to 4.5 to obtain the reagent.

[0038] Embodiment 2:

[0039] 4g diethylhexyl carbonate, 3g silicone oil (viscosity 200mPa.s), 10g glycerin, 2g EM90 emulsifier, 15g polyvinyl alcohol, 1.5g sodium chloride, 2g oleic acid, 0.7g rosin acid, 1g spruce volatile oil, and 19g itaconic acid.

[0040] (1) adding diethylhexyl carbonate, glycerin, silicone oil and emulsifier and stirring evenly to obtain an oil phase;

[0041] (2) Dissolve 110 g of sodium chloride in water, heat to about 80° C., add rosin acid, and stir evenly to obtain an aqueous phase;

[0042] (3) adding polyvinyl alcohol to 90° C. hot water, dissolving it and cooling it to room temperature to obtain a 5 wt% polyvinyl alcohol solution, then adding spruce volatile oil and itaconic acid, stirring evenly, freezing at -10° C. for 6 h, thawing at room temperature for 12 h, repeating this process twice, and then crushing or mashing to obtain a solid phase;

[0043] (4) After the solid phase and the oil phase are mixed, oleic acid is added, and then the aqueous phase is slowly added. After high-speed stirring at 950 r / min to form a cream, the pH value of the reagent is adjusted to 4.5 to obtain the reagent.

[0044] Comparative Example 1:

[0045] Itaconic acid cream was also prepared by a traditional method, and the specific preparation method is as follows:

[0046] (1) Aqueous phase: 4.4 g of itaconic acid was dissolved in 100 g of water to prepare an aqueous phase;

[0047] (2) Oil phase: 3 g of diethylhexyl carbonate, 2 g of silicone oil, and 1.5 g of EM90 emulsifier were mixed to prepare the oil phase;

[0048] (3) Add the oil phase to an emulsifier, and slowly add the water phase while stirring at a stirring rate of 50 r / min, and stir until the cream is formed to obtain the reagent.

[0049] Comparative Example 2:

[0050] A control reagent of Example 1 was also prepared, without adding glycerol to the oil phase, and the amount of itaconic acid added was adjusted to keep the concentration consistent with that of Example 1, and the remaining preparation steps were the same.

[0051] Comparative Example 3:

[0052] A control reagent of Example 1 was also prepared, without adding sodium chloride to the aqueous phase, and the amount of itaconic acid added was adjusted to keep the concentration consistent with that of Example 1. The remaining preparation steps were the same. The specific preparation method is as follows:

[0053] (2) After heating 110 g to about 80° C., add rosin acid and stir evenly to obtain an aqueous phase; the subsequent preparation method is the same.

[0054] Comparative Example 4:

[0055] A control reagent of Example 1 was also prepared, without adding rosin acid to the aqueous phase, and the amount of itaconic acid added was adjusted to keep the concentration consistent with that of Example 1, and the remaining preparation steps were the same.

[0056] Comparative Example 5:

[0057] A control reagent of Example 1 was also prepared, without adding spruce volatile oil to the solid phase, and the amount of itaconic acid added was adjusted to keep the concentration consistent with that of Example 1, and the remaining preparation steps were the same.

[0058] Comparative Example 6:

[0059] A control reagent of Example 1 was also prepared, without adding oleic acid in step (4), and adjusting the amount of itaconic acid added to keep the concentration consistent with that of Example 1. The remaining preparation steps were the same.

[0060] Comparative Example 7:

[0061] A control reagent of Example 1 was also prepared. In the preparation of the polyvinyl alcohol solid phase, the freezing operation conditions were changed to "freeze at -25°C for 10 hours, thaw at room temperature for 24 hours, and then crush or mash to obtain a solid phase", and the remaining preparation steps were the same.

[0062] Comparative Example 8:

[0063] A control reagent of Example 1 was also prepared, and the addition ratio of the emulsifier in the oil phase was changed to 3 g of diethylhexyl carbonate and 2 g of silicone oil (viscosity value 200 mPa.s), the addition amount of EM90 emulsifier was changed to 5 g, and the addition amount of itaconic acid was adjusted to keep it consistent with the concentration in Example 1.

[0064] Comparative Example 9:

[0065] A control reagent of Example 1 was also prepared, in which polyvinyl alcohol was not prepared into a solid phase. The specific preparation method was as follows:

[0066] (3) adding polyvinyl alcohol to 90° C. hot water, dissolving the polyvinyl alcohol, and cooling the solution to room temperature to obtain a 5 wt % polyvinyl alcohol solution, then adding spruce volatile oil and itaconic acid, stirring the solution to obtain a polyvinyl alcohol solution;

[0067] (4) Oleic acid was added to the oil phase, and then the aqueous phase and the polyvinyl alcohol solution were slowly added. After high-speed stirring at 950 r / min, the pH value of the reagent was adjusted to 4.5 to obtain the reagent.

[0068] Comparative Example 10:

[0069] The control reagent of Example 1 was also prepared. During the stirring process in step (4), low-speed stirring was adopted, with a speed of 50 r / min.

[0070] 1. Treatment effect of vitiligo model

[0071] 1. Mouse modeling: The vitiligo mouse model was induced by intradermal injection of melanoma B16F10 in C57BL / 6J mice combined with CD4 antibody to eliminate Treg.

[0072] On day 0, melanoma cells B16F10 (2×10 5 The injection needle was almost completely parallel to the skin, and a round skin bump appeared on the skin immediately after injection.

[0073] On the 4th and 10th days, CD4 neutralizing antibodies were injected intraperitoneally. The weight of 8-10 week old mice was about 18-20 g, and 10 μg of CD4 neutralizing antibodies was injected per gram of mouse.

[0074] The tumor was removed on the 12th day and sutured with a stapler. About 2-3 months after inducing vitiligo in mice, significant white spots appeared on the tail of the mice.

[0075] 2. Treatment process:

[0076] After 5 weeks of induction of vitiligo mice, they were treated with topical itaconic acid reagent 40 mg / g (itaconic acid + vaseline) for 5 consecutive weeks, and the changes of white spots on the tails of vitiligo mice were recorded by taking photos. At the end of the treatment, the tails of mice were collected, and whole-mount immunofluorescence was used to detect CD8 in the tail epidermis of mice (vitiligo mice, mice treated with topical itaconic acid). + T cell infiltration and melanocyte number are used to clarify the therapeutic effect of itaconic acid on vitiligo.

[0077] 3. Evaluation of treatment effect:

[0078] After 10 weeks of treatment, topical itaconic acid reagent inhibited the progression of vitiligo (Figure B). Immunofluorescence showed that the number of melanocytes was higher than that of vitiligo mice not treated with drugs, and CD8 + T cell skin infiltration was significantly reduced (Figure C). It has a certain effect in treating vitiligo.

[0079] 2. Cream stability test

[0080] The creams prepared in Example 1 and Comparative Examples 1-10 were subjected to high temperature test, cold resistance test and room temperature standing test. The specific experimental methods are as follows:

[0081] (1) High temperature test: 10 g of each prepared cream was randomly taken and placed in a constant temperature and humidity chamber at 40°C for 10 days to observe the oil-water separation and stratification phenomenon.

[0082] (2) Cold resistance test: 10 g of each prepared cream was randomly taken and placed in a constant temperature and humidity chamber at -15°C for 24 hours to observe the oil-water separation and stratification phenomenon.

[0083] (3) Standing at room temperature: Randomly take 10 g of each prepared cream and stand it at room temperature. Observe the oil-water separation and stratification phenomena once every 1, 2, and 4 months.

[0084] “-” means no change in the cream, “+” means the cream becomes thicker, and “++” means the cream becomes stratified.

[0085] The experiment was repeated three times and the results are shown in Table 1.

[0086] Table 1

[0087]

[0088] 3. Transdermal Experiment

[0089] 1. Male Panamanian miniature pig skin was used as the transdermal permeation test material. The Panamanian miniature pig was killed, the back skin was cut, the subcutaneous tissue and fat were removed, the skin was rinsed with saline, wiped with alcohol and stored in a frozen state at -20℃, and thawed before use.

[0090] 2. Experimental group 1 was tested using the reagent prepared in Example 1, and comparative groups 1-10 were tested using the reagents prepared in Comparative Examples 1-10. The concentration of itaconic acid in the reagents was 40 mg / g.

[0091] 3. The transdermal test was performed using a modified Franz diffusion cell. The pig skin was fixed between the sample chamber and the receiving chamber, with the stratum corneum facing upward and the dermis in contact with the receiving solution, and all bubbles were removed. The transdermal area was 3.8 cm 2 , the receiving pool area is 15ml. The receiving solution is 0.9% physiological buffered saline solution, and the amount of reagent sample added is 1g, which is evenly applied on the pig skin surface. During the test, it is operated under constant temperature (37℃) and constant speed (300r / min). 2ml of receiving solution is taken at 0.5h, 1h, 3h, 6h, and 12h, respectively, and 3 parallel portions are added, and 2ml of isothermal receiving solution is added. The cumulative penetration percentage Q=P is calculated. i / P0×100%(Q is the cumulative transdermal percentage at different times; P i is the cumulative release of itaconic acid in the receiving fluid measured at different times, and P0 is the total amount of itaconic acid added to the supply pool)

[0092] Table 2

[0093] 0.5h 1h 3h 6h 12h Example 1 3.167 4.258 5.487 6.954 10.129 Comparative Example 1 2.051 2.839 3.941 4.861 7.482 Comparative Example 2 2.924 3.921 5.203 6.361 9.682 Comparative Example 3 1.832 2.677 2.994 3.128 4.845 Comparative Example 4 2.876 3.832 5.128 4.489 7.023 Comparative Example 5 3.208 4.189 5.338 6.781 9.67 Comparative Example 6 2.156 2.42 3.117 3.933 6.896 Comparative Example 7 1.923 2.629 3.778 4.53 7.114 Comparative Example 8 2.871 3.814 4.788 5.894 9.146 Comparative Example 9 2.156 3.133 4.063 5.149 7.998 Comparative Example 10 2.484 2.991 3.925 4.969 7.894

[0094] Analyzing the data in Table 1 and Table 2, we can see that:

[0095] 1. The stability of itaconic acid cream prepared by the present invention is still good at high and low temperatures, and there is no oil-water separation or roughness, and it is also stable under long-term room temperature standing conditions. And what comparative example 1 adopts is the traditional itaconic acid ointment preparation method, and its stability is poor, and it can only meet the laboratory medication needs, and it is difficult to make a medicament for use. Compared with other comparative examples, the present invention uses polyvinyl alcohol gel to wrap itaconic acid, uses light oil components as oil phase, and simultaneously mixes salt solution to make a critical system, has higher stability, and the ratio of diethylhexyl carbonate, silicone oil, and EM90 emulsifier in the preparation process is also more important for the preparation of the cream system, and it is difficult to form a stable critical system at low speed stirring, and the emulsifier addition is too high and the oil phase can be made into an emulsifiable paste, but it is very easy to break emulsion and stratify during high or low temperature. In addition, in the polyvinyl alcohol solid phase preparation process, freezing and dissolving temperature during freeze-thaw process can significantly affect the cross-linking effect of polyvinyl alcohol and then affect the preparation of emulsifiable paste.

[0096] 2. After itaconic acid was prepared into a traditional cream, its 12h permeability was 7.482%, which was significantly lower than that in Example 1. This is because the mechanical force used in the rubbing process of the cream of the present invention will cause the ointment to break, and after the water phase evaporates, itaconic acid adheres to the skin surface, increasing the fluidity of the intercellular lipid region and improving the bioavailability of the drug, while also promoting an increase in transdermal performance.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A reagent for treating vitiligo, characterized in that: The reagents include the following ingredients: Itaconic acid, diethylhexyl carbonate, silicone oil, glycerin, emulsifier, polyvinyl alcohol, sodium chloride, oleic acid, rosin acid, spruce essential oil.

2. The agent for treating vitiligo according to claim 1, characterized in that: The reagent is made into an ointment or cream and applied to the affected area.

3. The agent for treating vitiligo according to claim 1, characterized in that: The reagent comprises the following raw materials in parts by weight: 3-4 parts by mass of diethylhexyl carbonate, 2-3 parts by mass of silicone oil, 8-10 parts by mass of glycerin, 1.5-2 parts by mass of emulsifier, 10-15 parts by mass of polyvinyl alcohol, 1-1.5 parts by mass of sodium chloride, 1-2 parts by mass of oleic acid, 0.5-0.7 parts by mass of rosin acid, and 0.5-1 parts by mass of spruce volatile oil.

4. The method for preparing a reagent for treating vitiligo according to claim 1, characterized in that: The concentration of itaconic acid in the reagent is (40-50) mg / g.

5. A method for preparing a reagent for treating vitiligo, characterized in that: The preparation method of the reagent is as follows: (1) diethylhexyl carbonate, glycerin, silicone oil, and emulsifier are stirred to obtain an oil phase; (2) dissolving sodium chloride in water, adding rosin acid after heating, and stirring evenly to obtain an aqueous phase; (3) dissolving polyvinyl alcohol in hot water, cooling to room temperature to obtain a polyvinyl alcohol solution, then adding spruce volatile oil and itaconic acid, stirring evenly, freezing at -10°C for 5-6 hours, thawing at room temperature for 10-12 hours, repeating twice, and then crushing to obtain a solid phase; (4) After the solid phase and the oil phase are mixed, oleic acid is added, and then the aqueous phase is slowly added. After high-speed stirring, the pH of the reagent is adjusted to 4.5 to obtain the reagent.

6. The method for preparing a reagent for treating vitiligo according to claim 5, characterized in that: In the water phase, the mass of water added is 7-8 times the total mass of the oil phase.

7. The method for preparing a reagent for treating vitiligo according to claim 6, characterized in that: The mass concentration of the polyvinyl alcohol solution is 5-8%.

8. The method for preparing a reagent for treating vitiligo according to claim 7, characterized in that: In the step (4), the rotation speed is 950-1000 r / min.

9. The method for preparing a reagent for treating vitiligo according to claim 8, characterized in that: The emulsifier is EM90 emulsifier.

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