A low-isomer tacrolimus ointment and a method for preparing the same

Abstract

The application discloses a tacrolimus ointment with low isomer content and a preparation method thereof. Raw materials and weight parts of the ointment are as follows: tacrolimus 3-10 parts, polyoxyethylene fatty acid ester 20-50 parts, triethanolamine 10-30 parts, liquid paraffin 1200-1800 parts, beeswax 200-500 parts, white vaseline 7500-8000 parts; sample preparation is carried out at 60±5 DEG C under vacuum. The isomer content of tacrolimus in the ointment is controlled within 2.0% by improving emulsifiers, adjuvants and the like added in the ointment and combining with process improvement in the ointment preparation process, and the isomer content is still controlled at a better level after an accelerated test, which has very important practical significance for isomer content control of the tacrolimus ointment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine preparation, in particular to a tacrolimus ointment with low isomer content and a preparation method thereof. BACKGROUND

[0002] Tacrolimus, also known as FK506, is a macrolide antibiotic with high immunosuppressive effect. It was first isolated from Streptomyces tsukubaensis by Fujisawa Pharmaceutical Industries Co., Ltd. in 1984. Tacrolimus mainly inhibits the release of interleukin-2 (IL-2) and the function of T lymphocytes. Clinical experiments show that it has good effect in heart, lung, intestinal and bone marrow transplantation. At the same time, tacrolimus also plays a positive role in the treatment of atopic dermatitis, systemic lupus erythematosus, autoimmune eye disease and other autoimmune diseases. Tacrolimus, molecular formula C 44 H 69 NO 12 , molecular weight 804.018, CAS registration number: 104987-11-3, structural formula as shown below.

[0003]

[0004] Tacrolimus is derived from microbial fermentation, and multiple tacrolimus analogues such as ascomycin and dihydro tacrolimus are produced during fermentation. Different degradation products and tautomers may also be produced during production. Due to the existence of tacrolimus tautomerism, related isomer impurities will inevitably be produced during preparation. The United States Pharmacopoeia USP 37 edition tacrolimus capsule standard collects tacrolimus known impurity tacrolimus positional isomer. Many patents in the prior art are related to isomer detection, purification and preparation technology, such as CN201410057153.X discloses a preparation method of tacrolimus known impurity tacrolimus positional isomer, CN201410057192.X discloses a preparation method of tacrolimus known impurity dihydro tacrolimus, and CN201310534184.5 discloses a process for preparing high-purity tacrolimus.

[0005] Tacrolimus ointment is a kind of skin external medicine, which has the effect of inhibiting immunity, and is mainly used for treating atopic dermatitis. The main component of tacrolimus ointment is tacrolimus, and the base, emulsifier, penetrant, antioxidant are also added to make it easy to disperse, absorb and antioxidant. Some patents related to the detection of impurities and isomers in tacrolimus ointment are disclosed in the prior art, such as CN202111620546.3 discloses a method for detecting the content of impurity XV in tacrolimus ointment by reverse phase chromatography; 2021106240117 discloses a pretreatment method for determining the isomer of tacrolimus ointment; 2021106234366 discloses a detection method for related substances in tacrolimus ointment.

[0006] However, there are few reports on the content control of isomers in tacrolimus ointment in the prior art. The content of tacrolimus isomer is a main quality control index in the quality control of tacrolimus ointment, therefore, it is necessary to develop a tacrolimus ointment preparation process to control the content of isomers in tacrolimus ointment. Tacrolimus 19-epimer, also known as tacrolimus 19, tacrolimus tautomer II, tacrolimus EP impurity G, CAS registration number 144490-63-1. The content of tacrolimus 19-epimer is the highest among the isomers (tacrolimus is prone to isomerization to tacrolimus 19-epimer), and its content control is particularly important. SUMMARY

[0007] In view of the above problems, the present application provides a tacrolimus ointment with low isomer content and a preparation method thereof. By improving the emulsifier, excipient and the like added in the ointment, and combining with the process improvement in the preparation process of the ointment, the content of tacrolimus 19-epimer in the ointment is controlled within 2.0%, and after accelerated test, it is still controlled at a better level, which has very important practical significance for the content control of isomers in tacrolimus ointment.

[0008] The technical scheme of the present application is: a tacrolimus ointment with low isomer content, characterized in that the raw materials and weight parts are: tacrolimus 3-10 parts, polyoxyethylene fatty acid ester 20-50 parts, triethanolamine 10-30 parts, liquid paraffin 1200-1800 parts, beeswax 200-500 parts, white vaseline 7500-8000 parts.

[0009] The preferred ratio is: tacrolimus 10 parts, polyoxyethylene fatty acid ester 50 parts, triethanolamine 30 parts, liquid paraffin 1200 parts, beeswax 200 parts, white vaseline 8000 parts.

[0010] Preparation method:

[0011] (1) heating and dissolving tacrolimus and polyoxyethylene fatty acid ester, triethanolamine under vacuum condition, stirring to obtain a mixed solution;

[0012] (2) mixing liquid paraffin and beeswax, heating and dissolving, stirring to obtain an oil phase solution;

[0013] (3) adding the mixed solution of step (1) into the oil phase solution of step (2), stirring under vacuum condition, finally adding white petrolatum, stirring under vacuum condition, to obtain the tacrolimus ointment.

[0014] The heating in steps (1)-(3) is preferably at a temperature of 60±5℃.

[0015] The tacrolimus ointment prepared by using the above raw materials and preparation method has a 19-epi-tacrolimus content of less than 2.0%.

[0016] The isomer of tacrolimus is a degradation product, and water is the main factor accelerating the degradation of tacrolimus into isomer. The emulsifier of the present application uses polyoxyethylene fatty acid ester and triethanolamine, which have good emulsifying effect and good water absorption effect, and can more strictly control the water content in the sample. The sample preparation condition of the present application is selected at 60±5℃ under vacuum, which can effectively remove the water in the auxiliary materials liquid paraffin, beeswax and white petrolatum, and further reduce the isomer content.

[0017] Technical effects of the present application:

[0018] 1. The emulsifier of the present application uses polyoxyethylene fatty acid ester and triethanolamine. According to experimental data, the 19-epi-tacrolimus content in the ointment can be controlled to less than 1% after compounding. Compared with the single emulsifier and the existing common tacrolimus ointment formula, the formula containing the above compounded emulsifier greatly reduces the 19-epi-tacrolimus content in the ointment.

[0019] 2. The ointment of the present application still has a good level (less than 4.2%) of 19-epi-tacrolimus content after accelerated testing (placed at a temperature of 30℃±2℃ and a relative humidity of 65%±5% for 6 months).

[0020] In summary, the present application controls the 19-epi-tacrolimus content in the ointment to less than 2.0% by improving the emulsifier and auxiliary materials in the ointment and combining with the process improvement in the preparation of the ointment. The ointment still has a good level (less than 4.2%) of 19-epi-tacrolimus content after accelerated testing, which has very important practical significance for controlling the isomer content in the tacrolimus ointment. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 HPLC chromatogram of the content of the 19-epi-isomer of tacrolimus in the tacrolimus ointment prepared in Example 1;

[0022] Figure 2 HPLC chromatogram of the content of the 19-epi-isomer of tacrolimus in the tacrolimus ointment prepared in Example 2;

[0023] Figure 3 HPLC chromatogram of the content of the 19-epi-isomer of tacrolimus in the tacrolimus ointment prepared in Example 3;

[0024] Figure 4 HPLC chromatogram of the content of the 19-epi-isomer of tacrolimus in the tacrolimus ointment prepared in Comparative Example 1;

[0025] Figure 5 HPLC chromatogram of the content of the 19-epi-isomer of tacrolimus in the tacrolimus ointment prepared in Comparative Example 2;

[0026] Figure 6 HPLC chromatogram of the content of the 19-epi-isomer of tacrolimus in the tacrolimus ointment prepared in Comparative Example 3. DETAILED DESCRIPTION

[0027] The following examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the examples, and any changes, modifications, combinations, substitutions, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement ways, and all are included in the protection scope of the present application. The polyoxyethylene fatty acid ester in the present example is polyoxyethylene stearate (PEG-10 stearate, CAS registration number: 9004-99-3).

[0028] Example 1:

[0029] Tacrolimus ointment formula (weight parts): tacrolimus 3 parts, polyoxyethylene stearate 20 parts, triethanolamine 10 parts, liquid paraffin 1800 parts, beeswax 500 parts, white vaseline 7500 parts.

[0030] The preparation method of the tacrolimus ointment is as follows:

[0031] (1) The tacrolimus and polyoxyethylene stearate, triethanolamine are heated and dissolved under vacuum conditions at 60±5℃, and stirred uniformly to obtain a mixed solution;

[0032] (2) The liquid paraffin and beeswax are mixed and heated and dissolved at 60±5℃, and stirred uniformly to obtain an oil phase liquid;

[0033] (3) Add the mixed solution of step (1) into the oil phase solution of step (2), heat and stir under vacuum at 60±5℃ until uniform, then add white petrolatum, heat and stir under vacuum at 60±5℃ until uniform, and obtain the product.

[0034] Isomer content detection of the tacrolimus ointment: Take about 2 g of the product [containing about 3 mg of tacrolimus] and place it in a 10-ml glass tube with a stopper. Add 4.0 ml of n-hexane and dissolve in a 60℃ water bath. Precisely take 4.0 ml of acetonitrile and place it in the same glass tube with a stopper. Allow it to stand and separate the layers. Place it in a freezer (-20℃) for 20 minutes. Immediately carefully take the acetonitrile layer as the test sample solution. According to the chromatographic conditions (see the chromatographic conditions of tacrolimus in USP 40) under the tacrolimus content determination, precisely take 20 μl of the test sample solution and inject it into the liquid chromatograph. Record the chromatogram and calculate the content. After detection, the 19-epi-isomer content of tacrolimus in the ointment is 1.2%, see Figure 1 .

[0035] Place the sample under the condition of temperature 30℃±2℃ and relative humidity 65%±5% for 6 months. Take samples at 0, 3, and 6 months for detection. The data of the 19-epi-isomer content of tacrolimus obtained at each time point are shown in Table 1 below.

[0036] Table 1: The 19-epi-isomer content of tacrolimus obtained at each time point

[0037]

[0038] Example 2:

[0039] Tacrolimus ointment formula (weight parts): tacrolimus 10 parts, polyoxyethylene stearate 50 parts, triethanolamine 30 parts, liquid paraffin 1200 parts, beeswax 200 parts, and white petrolatum 8000 parts.

[0040] The preparation method and isomer content detection method are the same as in Example 1. After detection, the 19-epi-isomer content of tacrolimus in the ointment is 1.0%, see Figure 2 .

[0041] Place the sample under the condition of temperature 30℃±2℃ and relative humidity 65%±5% for 6 months. Take samples at 0, 3, and 6 months for detection. The data of the 19-epi-isomer content of tacrolimus obtained at each time point are shown in Table 2 below.

[0042] Table 2: The 19-epi-isomer content of tacrolimus obtained at each time point

[0043]

[0044] Example 3:

[0045] Tacrolimus ointment formulation (parts by weight): tacrolimus 3 parts, polyoxyethylene stearate 25 parts, triethanolamine 15 parts, liquid paraffin 1800 parts, beeswax 500 parts, white vaseline 7500 parts.

[0046] The preparation method and isomer content detection method are the same as in Example 1. After detection, the 19-epi-isomer content of tacrolimus in the ointment is 1.8%, see Figure 3 .

[0047] The sample is placed under the condition of temperature 30°C±2°C and relative humidity 65%±5% for 6 months, and sampled and detected at 0, 3, and 6 months. The data of the 19-epi-isomer content of tacrolimus detected at each time point are shown in Table 3 below.

[0048] Table 3: 19-epi-isomer content of tacrolimus detected at each time point

[0049]

[0050] Comparative Example 1:

[0051] Tacrolimus ointment formulation (parts by weight): tacrolimus 10 parts, propylene carbonate 50 parts, liquid paraffin 1200 parts, beeswax 200 parts, paraffin 100 parts, white vaseline 8000 parts

[0052] The preparation method is as follows:

[0053] (1) Dissolve tacrolimus and propylene carbonate at 60±5°C under vacuum, and stir uniformly to obtain a mixed solution;

[0054] (2) Mix liquid paraffin, beeswax, and paraffin, heat and dissolve at 60±5°C, and stir uniformly to obtain an oil phase liquid;

[0055] (3) Add the mixed solution of step (1) to the oil phase liquid of step (2), heat and stir uniformly at 60±5°C under vacuum, add white vaseline, heat and stir uniformly at 60±5°C under vacuum, and obtain the product.

[0056] The detection method is the same as in Example 1. After detection, the 19-epi-isomer content of tacrolimus in the ointment is 5.0%, see Figure 4 .

[0057] The sample is placed under the condition of temperature 30°C±2°C and relative humidity 65%±5% for 6 months, and sampled and detected at 0, 3, and 6 months. The data of the 19-epi-isomer content of tacrolimus detected at each time point are shown in Table 4 below.

[0058] Table 4: 19-epi-isomer content of tacrolimus detected at each time point

[0059]

[0060] Comparative Example 2

[0061] Tacrolimus ointment formulation (parts by weight): tacrolimus 10 parts, polyoxyethylene stearate 70 parts, liquid paraffin 1200 parts, beeswax 200 parts, white petrolatum 8000 parts.

[0062] Preparation method (without adding triethanolamine) and isomer content detection method are the same as Example 1. After detection, the content of tacrolimus 19-epimer in the ointment is 4.5%, see Figure 5 .

[0063] The sample was placed under the condition of temperature 30°C ± 2°C and relative humidity 65% ± 5% for 6 months, and samples were taken at 0, 3, 6 months for detection. The data of tacrolimus 19-epimer content obtained at each time point is shown in Table 5 below.

[0064] Table 5: Tacrolimus 19-epimer content obtained at each time point

[0065]

[0066] Comparative Example 3

[0067] Tacrolimus ointment formulation (parts by weight): tacrolimus 10 parts, triethanolamine 70 parts, liquid paraffin 1200 parts, beeswax 200 parts, white petrolatum 8000 parts.

[0068] Preparation method (without adding polyoxyethylene stearate) and isomer content detection method are the same as Example 1. After detection, the content of tacrolimus 19-epimer in the ointment is 4.4%, see Figure 6 .

[0069] The sample was placed under the condition of temperature 30°C ± 2°C and relative humidity 65% ± 5% for 6 months, and samples were taken at 0, 3, 6 months for detection. The data of tacrolimus 19-epimer content obtained at each time point is shown in Table 6 below.

[0070] Table 6: Tacrolimus 19-epimer content obtained at each time point

[0071]

[0072] In addition, the tacrolimus ointments prepared in Examples 1-3 and Comparative Examples 1-3 were placed under the conditions of a temperature of 30℃±2℃ and a relative humidity of 65%±5% for 6 months, sampled at time points of 0, 3 and 6 months, and the viscosity, related substance 1 (diene tacrolimus, lactone isomer, remaining known maximum single impurity, maximum unknown single impurity), related substance 2 (8-position isomer) and tacrolimus content were detected, and the content detection method of the related substances was referred to CN2021106234366 (Invention title: A detection method of related substances of tacrolimus ointment), and the detection results are shown in Tables 7 and 8, respectively.

[0073] Table 7: Sampling detection results of the tacrolimus ointments of Examples 1-3 at time points of 0, 3 and 6 months

[0074]

[0075] Table 8: Sampling detection results of the tacrolimus ointments of Comparative Examples 1-3 at time points of 0, 3 and 6 months

[0076]

[0077]

[0078] From the experimental results in Tables 1-8, it can be seen that:

[0079] 1. Compared with Example 2, the content of tacrolimus 19-epimer in the ointment of Comparative Example 1 using the commonly used tacrolimus ointment formula (emulsifier using propylene carbonate) was as high as 5.0%, while the content of tacrolimus 19-epimer in the ointment using the formula of the present application (emulsifier using polyoxyethylene stearate and triethanolamine) was controlled at 1%, which fully shows that the content of tacrolimus 19-epimer in the ointment is greatly reduced after using the formula of the present application.

[0080] 2. Compared with Example 2, the contents of tacrolimus 19-epimer in the ointments of Comparative Examples 2-3 using single emulsifiers (polyoxyethylene stearate or triethanolamine) were 4.5% and 4.4%, respectively, while the content of tacrolimus 19-epimer in the ointment using the complex emulsifier of the present application was controlled at 1%, which fully shows that the content of tacrolimus 19-epimer is greatly reduced after using the complex emulsifier of the present application.

[0081] 3. The ointment of the present application is proved by accelerated test that the sample is still controlled at a better level (the ointment of the present application is controlled within 4.2%, while the content of the ointment of the comparative example is as high as 10.5%) after being placed under the conditions of a temperature of 30℃±2℃ and a relative humidity of 65%±5% for 6 months, which has very important practical significance for the control of the isomer content in the tacrolimus ointment.

[0082] 4. From the results of the tests in Tables 7 and 8, there were no significant differences in viscosity, related substance 1, related substance 2, and tacrolimus content between the tacrolimus ointments prepared in Examples 1-3 and Comparative Examples 1-3.

Claims

1. A tacrolimus ointment characterized in that, The raw materials and weight parts of the tacrolimus ointment are: tacrolimus 3-10 parts, polyoxyethylene fatty acid ester 20-50 parts, triethanolamine 10-30 parts, liquid paraffin 1200-1800 parts, beeswax 200-500 parts and white vaseline 7500-8000 parts.

2. The tacrolimus ointment of claim 1, wherein the amount of tacrolimus is about 0.1% to about 1.0% by weight of the ointment. The raw materials and weight parts of the tacrolimus ointment are: tacrolimus 10 parts, polyoxyethylene fatty acid ester 50 parts, triethanolamine 30 parts, liquid paraffin 1200 parts, beeswax 200 parts and white vaseline 8000 parts.

3. The method of producing the tacrolimus ointment as claimed in claim 1 or 2, characterized by, The steps include: (1) heating and dissolving tacrolimus and polyoxyethylene fatty acid ester, triethanolamine under vacuum condition, stirring uniformly to obtain a mixed solution; (2) mixing liquid paraffin and beeswax, heating and dissolving, stirring uniformly to obtain an oil phase liquid; (3) adding the mixed solution of step (1) into the oil phase liquid of step (2), heating and stirring uniformly under vacuum condition, finally adding white vaseline, heating and stirring uniformly under vacuum condition, and obtaining the tacrolimus ointment.

4. The production method according to claim 3, wherein The heating temperature of steps (1)-(3) is 60±5℃.

5. The production method according to claim 4, wherein The content of tacrolimus 19-epi-isomer in the prepared ointment is controlled within 2.0%.

6. The production method according to claim 4, wherein The prepared ointment is placed under the condition of temperature 30℃±2℃ and relative humidity 65%±5% for 6 months, and the content of tacrolimus 19-epi-isomer is controlled within 4.2%.

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