A reagent for treating vitiligo and a preparation method thereof

Abstract

The application discloses a reagent for treating vitiligo and a preparation method thereof, and belongs to the technical field of the reagent for treating vitiligo. The reagent comprises the following components: itaconic acid, diethylhexyl carbonate, silicone oil, glycerol, an emulsifier, polyvinyl alcohol, sodium chloride, oleic acid, abietic acid and spruce volatile oil. The reagent solves the problems of poor stability, inconvenient storage and strong irritation of the reagent for treating vitiligo prepared by taking itaconic acid as an effective component.

Description

TECHNICAL FIELD

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

[0002] Vitiligo is an autoimmune skin disease caused by the destruction of melanocytes, and its clinical manifestations are clear boundary white spots, which seriously endanger the physical and mental health of patients. The existing treatment mainly uses topical glucocorticoids / calcineurin inhibitors and phototherapy, but there are still some deficiencies such as low overall efficiency, long treatment cycle, and easy recurrence after drug withdrawal. The disease has a complex etiology, which is caused by the combined action of genetic, oxidative stress, and autoimmune factors. Therefore, it is crucial to develop new clinical drugs for 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 have reported that itaconic acid has antibacterial and antiviral effects and mechanisms. For example, itaconic acid can inhibit the expression and activity of isocitrate lyase, prevent bacteria from using other carbon sources for energy metabolism and biosynthesis when glucose is scarce, and thus exert antibacterial effects. However, when itaconic acid is used as an effective ingredient to make conventional vitiligo agents, it is found that it will denature or self-aggregate under high temperature, high humidity, and light conditions, resulting in a serious decrease in drug efficacy. Moreover, itaconic acid can generate hydroxy acid in aqueous solution, which is easy to react with other raw materials, thus making the storage conditions more demanding, which is not conducive to the production and storage of the agent. Therefore, the present application discloses a new type of vitiligo treatment agent to solve the above problems. SUMMARY

[0004] In view of this, the purpose of the present application is to provide a vitiligo treatment agent and a preparation method thereof, which solves the problems of poor stability, difficult convenient storage, and strong irritation of the vitiligo agent prepared by using itaconic acid as an effective ingredient.

[0005] The present application solves the above technical problems through the following technical means:

[0006] A vitiligo treatment agent, which comprises the following ingredients:

[0007] Itaconic acid, diethylhexyl carbonate, silicone oil, glycerol, emulsifier, polyvinyl alcohol, sodium chloride, oleic acid, abietic acid, spruce volatile oil.

[0008] The itaconic acid inhibits the immune response of vitiligo to play an anti-inflammatory therapeutic effect, delays the progress of vitiligo white spot and then treats vitiligo, so the main effective component of the reagent for treating vitiligo of the present application is itaconic acid, but the physicochemical properties of itaconic acid are relatively special, and the laboratory usually uses it immediately after preparation, so it still needs to be explored in the direction of preparing a therapeutic agent, and the skin of vitiligo patients is fragile, based on which the present application adds adjuvants to prepare a cream for easy use, reduces the production and storage cost, reduces the irritation, and is helpful for the clinical treatment of vitiligo patients.

[0009] Further, the reagent is prepared into a cream or a cream type and is wiped on the affected area.

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

[0011] Further, the reagent contains the following mass parts of raw materials:

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

[0013] Since the suitable concentration of the external use of itaconic acid is (40-50) mg / g, in order to reduce the dose while achieving the treatment purpose, it is necessary to achieve high-efficiency transdermal absorption effect, solve the problem of poor bioavailability of itaconic acid through the skin, and therefore the prepared reagent is in the form of cream or cream, which is coated on the affected area and gently rubbed to scatter when used, at which time the water phase and the oil phase are separated, the water phase is formed, the water is evaporated, the effective component is mixed with the oil phase, and the skin is more easily absorbed, and the rapid treatment purpose is achieved.

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

[0015] Further, the silicone oil is a silicone oil with a viscosity of 200.

[0016] The present application also discloses a preparation method of a reagent for treating vitiligo.

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

[0018] (2) Dissolve sodium chloride in water, add abietic acid after heating, and stir uniformly to obtain a water phase;

[0019] (3) Dissolving polyvinyl alcohol in hot water and 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 this process twice, and then crushing to obtain a solid phase;

[0020] (4) After mixing the solid phase and the oil phase, 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 aqueous phase, oil phase and solid phase, wherein the solid phase is mainly the gel formed by polyvinyl alcohol that itaconic acid is wrapped, avoids causing itaconic acid drug effect to reduce in preparation, storage and transportation process, and the solid phase added in addition can improve the stability in the aqueous-oil phase mixing process and increase the adhesion with skin when using. Simultaneously, in order to improve reagent stability, in the oil phase, the present invention selects diethylhexyl carbonate and 200 viscosity values ​​of silicone oil to be mixed to form a light oil phase component, in the aqueous phase, sodium chloride and rosin acid are added to form a saline solution with a certain viscosity, and simultaneously in combination with glycerol and other components, a critical system of low freezing point is formed in the process of making the reagent in high-speed stirring, so as to promote not to condense under low temperature conditions, and also not to form demulsification phenomenon after recovering 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 mobility in the intercellular lipid region and improving the bioavailability of the drug, and simultaneously because the reagent contains salt ions, in the massage and wiping process, mechanical pressing and osmotic pressure difference also can promote the drug to enter the skin. 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, the addition of oleic acid can reduce the phase transition temperature of the reagent, promote the separation of the water and oil phases during the kneading process, and reduce the difficulty of using the reagent.

[0022] Furthermore, in the aqueous 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] The itaconic acid can be used for the treatment of vitiligo, and because the skin of the vitiligo patient is fragile, based on this, the auxiliary material is added to prepare the cream, so that the cream is convenient to use, the production and storage cost is reduced, the irritation is reduced, and the clinical treatment of the vitiligo patient is helped. In addition, the prepared cream has good stability, is convenient to store, and is convenient to use. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 : mouse experiment figure and epidermal melanocyte / CD8 + T cell immunofluorescence figure. DETAILED DESCRIPTION

[0030] The application will be described in detail below in combination with specific examples:

[0031] Example 1:

[0032] The raw materials are weighed according to the following quality:

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

[0034] (1) After mixing and adding diethylhexyl carbonate, glycerol, silicone oil and emulsifier, stirring is uniformly carried out to obtain an oil phase;

[0035] (2) Sodium chloride is dissolved in 110g of water, heated to about 80℃, then rosin acid is added, and stirring is uniformly carried out to obtain an aqueous phase;

[0036] (3) Polyvinyl alcohol is added to 90℃ hot water, dissolved, then cooled to room temperature to obtain a 5wt% polyvinyl alcohol solution, then spruce volatile oil and itaconic acid are added, stirring is uniformly carried out, freezing is carried out at-10℃ for 5h, melting is carried out at room temperature for 10h, and the operation is repeated twice, then stirring or smashing is carried out to obtain a solid phase;

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

[0038] Example 2:

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

[0040] (1) After mixing and adding diethylhexyl carbonate, glycerol, silicone oil and emulsifier, stirring is uniformly carried out to obtain an oil phase;

[0041] (2) 110 g of sodium chloride was dissolved in water and heated to about 80°C, then rosin acid was added, and stirred to obtain an aqueous phase;

[0042] (3) Polyvinyl alcohol was added to 90°C hot water, dissolved, and then cooled to room temperature to obtain a 5wt% polyvinyl alcohol solution, then spruce volatile oil and itaconic acid were added, stirred to uniform, frozen at -10°C for 6h, thawed at room temperature for 12h, repeated twice, then stirred or crushed to obtain a solid phase;

[0043] (4) The solid phase was mixed with the oil phase, then the aqueous phase was slowly added, stirred at 950r / min to form a cream, then the pH of the reagent was 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 was as follows:

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

[0048] (3) The oil phase was added to the emulsifier, and the aqueous phase was slowly added while stirring, the stirring rate was 50r / min, and the cream was stirred to form, to obtain the reagent.

[0049] Comparative Example 2:

[0050] A control reagent of Example 1 was also prepared, glycerol was not added in the oil phase, the amount of itaconic acid was adjusted to keep the same concentration as that of Example 1, and the rest of the preparation steps were the same.

[0051] Comparative Example 3:

[0052] A control reagent of Example 1 was also prepared, sodium chloride was not added in the aqueous phase, the amount of itaconic acid was adjusted to keep the same concentration as that of Example 1, and the rest of the preparation steps were the same, and the specific preparation method was as follows:

[0053] (2) 110 g of sodium chloride was dissolved in water and heated to about 80°C, then rosin acid was added, and stirred to obtain an aqueous phase; the subsequent preparation method was the same.

[0054] Comparative Example 4:

[0055] A control reagent of Example 1 was also prepared, rosin acid was not added in the aqueous phase, the amount of itaconic acid was adjusted to keep the same concentration as that of Example 1, and the rest of the preparation steps were the same.

[0056] Comparative Example 5:

[0057] A control reagent of Example 1 was also prepared, without adding spruce volatile oil in the solid phase, while adjusting the amount of itaconic acid to keep the same concentration as that of Example 1, and the rest of the 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), while adjusting the amount of itaconic acid to keep the same concentration as that of Example 1, and the rest of the preparation steps were the same.

[0060] Comparative Example 7:

[0061] A control reagent of Example 1 was also prepared, in the preparation of polyvinyl alcohol solid phase, the freezing operation condition was changed to "frozen at -25°C for 10h, and then thawed at room temperature for 24h, and then crushed or pounded to obtain the solid phase", and the rest of the preparation steps were the same.

[0062] Comparative Example 8:

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

[0064] Comparative Example 9:

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

[0066] (3) The polyvinyl alcohol was added to hot water at 90°C, dissolved, and then cooled to room temperature to obtain a 5wt% polyvinyl alcohol solution, and then spruce volatile oil and itaconic acid were added and stirred uniformly 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, and after high-speed stirring at 950r / min, the pH of the reagent was adjusted to 4.5 to obtain the reagent.

[0068] Comparative Example 10:

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

[0070] I. Treatment effect on vitiligo model

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

[0072] On day 0, melanoma cells B16F10 (2×10 5 The injection needle was almost completely parallel to the skin, and a round 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 CD4 neutralizing antibodies were injected per gram of mouse.

[0074] On day 12, the tumor was removed and sutured with a suture device. About 2-3 months after induction of 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. The changes of white spots on the tails of vitiligo mice were recorded by taking photos. At the end of 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 greater than that of vitiligo mice without drug treatment, and CD8 + T cell skin infiltration was significantly reduced (Figure C). It has a certain therapeutic effect on vitiligo.

[0079] 2. Cream stability test

[0080] The creams prepared in Example 1 and Comparative Examples 1-10 were subjected to a high temperature test, a cold resistance test, and a 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 phenomena.

[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) Room temperature standing: randomly take 10 g of each prepared cream, stand at room temperature, observe oil-water separation and delamination phenomenon at 1, 2, 4 months.

[0084] "-" cream has no change, "+" cream appears thickening phenomenon, "++" cream appears delamination phenomenon.

[0085] The test is repeated three times, and the results are shown in Table 1.

[0086] Table 1

[0087]

[0088] III. Transdermal experiment

[0089] 1. Male Panama pig skin was used as transdermal penetration test material. The Panama pig was sacrificed, and the back skin was removed, and the subcutaneous tissue and fat were removed. After washing with physiological saline and wiping with alcohol, it was stored at -20°C. Before use, it was thawed;

[0090] 2. The reagent prepared in Example 1 was used for test in experimental group 1, and the reagent prepared in Comparative Example 1-10 was used for test in comparative group 1-10. The concentration of itaconic acid in the reagent was 40 mg / g.

[0091] 3. The modified Franz diffusion cell was used for transdermal test. The pig skin was fixed between the sample chamber and the receiving chamber, with the horny layer facing up and the dermis layer contacting the receiving liquid. The transdermal area was 3.8 cm 2 , and the receiving pool area was 15 ml. The receiving liquid was 0.9% physiological buffered saline solution, and the amount of reagent sample added was 1 g, which was evenly spread on the surface of the pig skin. The test was run under constant temperature (37°C) and constant speed (300 r / min) conditions. 2 ml of receiving liquid was taken at 0.5 h, 1 h, 3 h, 6 h, and 12 h, in triplicate, and 2 ml of isothermal receiving liquid was supplemented. The cumulative penetration percentage Q = P i / P0 x 100% (Q is the cumulative transdermal percentage at different times; P i is the cumulative release amount of itaconic acid in the receiving liquid 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] Analysis of the data in Table 1 and Table 2 shows that:

[0095] 1、The itaconic acid cream prepared by the application still has good stability at high temperature and low temperature, and no oil-water separation or roughness phenomenon occurs, and the cream is stable under long-time normal temperature standing conditions. The comparative example 1 uses the traditional itaconic acid ointment preparation method, and the stability is poor, and only the laboratory drug demand can be met, and it is difficult to be made into a medicament for use. Compared with other comparative examples, the application uses polyvinyl alcohol gel to wrap itaconic acid, uses light oil component as oil phase, and mixes salt solution to prepare a critical system, has high stability, and the ratio of carbonic acid diethylhexyl ester, silicone oil and EM90 emulsifier and the stirring rate in the preparation process are also important for the preparation of the cream system. It is difficult to form a stable critical system under low-speed stirring, and the emulsifier is too high, and although the oil phase can be made into a cream, it is easy to break the emulsion and separate in the process of high temperature or low temperature. In addition, in the polyvinyl alcohol solid phase preparation process, the freezing and dissolution temperature in the freeze-thaw process will significantly affect the crosslinking effect of polyvinyl alcohol and then affect the preparation of the cream.

[0096] 2、The 12h permeability of itaconic acid prepared into a traditional cream is 7.482%, which is significantly reduced compared with example 1, because the mechanical force used in the kneading process of the cream of the application can make the ointment demulsification, and the itaconic acid is attached to the skin surface after water phase evaporation, which increases the flowability of the intercellular lipid area and improves the bioavailability of the drug, and also promotes the increase of transdermal performance.

[0097] The above examples are only used to illustrate the technical solutions of the application and not to limit, although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the application can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the application, which should be covered in the scope of the claims of the application. The technical, shape and structure parts not described in detail in the application are well-known technologies.

Claims

1. A reagent for treating vitiligo, characterized in that: The reagent consists of the following components: Itaconic acid, diethylhexyl carbonate, silicone oil, glycerin, emulsifier, polyvinyl alcohol, sodium chloride, oleic acid, rosin acid, spruce essential oil; The concentration of itaconic acid in the reagent is (40-50) mg / g, and the other raw materials are as follows: 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; The preparation method of the reagent is as follows: (1) Diethylhexyl carbonate, glycerin, silicone oil, and emulsifier are stirred uniformly to obtain an oil phase; (2) Dissolve sodium chloride in water, add rosin acid after heating, and stir evenly to obtain an aqueous phase; (3) Dissolve polyvinyl alcohol in hot water, cool to room temperature to obtain a polyvinyl alcohol solution, then add spruce volatile oil and itaconic acid, stir evenly, freeze at -10°C for 5-6 hours, thaw at room temperature for 10-12 hours, repeat twice, and then crush to obtain a solid phase; (4) After mixing the solid phase and the oil phase, 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.

2. The agent for treating vitiligo according to claim 1, characterized in that The agent is prepared into an ointment or cream.

3. The agent for treating vitiligo according to claim 2, characterized in that: In the water phase, the mass of water added is 7-8 times the total mass of the oil phase.

4. The agent for treating vitiligo according to claim 3, characterized in that: The mass concentration of the polyvinyl alcohol solution is 5-8%.

5. The agent for treating vitiligo according to claim 4, characterized in that: In the step (4), the rotation speed is 950-1000 r / min.

6. The agent for treating vitiligo according to claim 5, characterized in that: The emulsifier is EM90 emulsifier.

Images