A cyclosporine self - microemulsifying preparation, its preparation method and application

Abstract

The present invention aims to provide a cyclosporine self-microemulsion preparation, its preparation method and application. The preparation is composed of cyclosporine, α-tocopherol, amino acids, a solvent, a surfactant, and a co-surfactant. This cyclosporine preparation has good stability and palatability. While effectively improving the bioavailability of the active ingredient, it also improves the stability and palatability of the cyclosporine preparation. After oral administration, under the action of the gastrointestinal tract, the preparation is first dispersed and diluted by gastric juice to form a microemulsion, and then digested by digestive enzymes, etc., and has obvious effects in the treatment of canine and feline atopic dermatitis or allergic dermatitis, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary chemical preparations, and specifically relates to a cyclosporine self-microemulsification preparation and a preparation method and application thereof. Background Art

[0002] Cyclosporine is a calcineurin inhibitor whose primary mode of action is to inhibit T cell activation. Cyclosporine achieves its immunosuppressive activity by binding to the intracellular receptor protein cyclosporine-1. The resulting cyclosporine-cyclophilin complex inhibits calcineurin, thereby preventing the dephosphorylation and activation of the transcription factor, nuclear factor AT (NE-AT), of activated T cells. NF-AT helps regulate the production of several important proinflammatory cytokines, including interleukin (IL)-2, IL-4, interferon-γ, and tumor necrosis factor-α. The atopic inhibition of IL-2 plays a key role, and the activation and proliferation of T cells is the main immunosuppressive mechanism of cyclosporine. Therefore, cyclosporine affects both innate and adaptive immune responses. There are an increasing number of other cells involved in inflammation and immune responses, including B cells, antigen-presenting cells, keratinocytes, endothelial cells, mast cells, basophils, and eosinophils. The overall effect of cyclosporine is to reduce the number and activity of proinflammatory cells at the site of inflammation.

[0003] However, cyclosporine is a poorly soluble drug. After being prepared into a self-microemulsifying preparation, under the gastrointestinal environment, even if it is emulsified quickly, there are problems such as poor stability and easy precipitation. In addition, a large amount of oily solvents are used in the prescription, which makes the drug palatability worse. In order to prevent the oxidation problem of the oily solvent, the step of filling the headspace with nitrogen has to be increased in the process, which reduces the production efficiency. At the same time, when the drug is used, nitrogen will leak after the package is opened, resulting in a shorter shelf life of the product after the package is opened. Therefore, preparing a cyclosporine self-microemulsifying preparation with a simple production process, a long shelf life and good palatability is a major problem to be solved in this field. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a cyclosporine self-microemulsification preparation and a preparation method and application thereof. The specific technical scheme is as follows:

[0005] In one aspect, the present invention provides a cyclosporine self-microemulsification preparation, which is composed of the following components: cyclosporine, α-tocopherol, amino acid, solvent, surfactant, and co-surfactant.

[0006] Preferably, the mass percentages of the components of the cyclosporine self-microemulsion preparation of the present invention are: cyclosporine 5% to 20%, α-tocopherol 0.02% to 10%, amino acid 0.02% to 6%, solvent 30% to 50%, surfactant 20% to 60%, and co-surfactant 10% to 30%.

[0007] Further preferably, the cyclosporine self-microemulsion preparation of the present invention is in terms of mass percentage: cyclosporine 10% - 15%, α-tocopherol 0.05% - 6%, amino acid 0.05% - 2%, solvent 30% - 35%, surfactant 30% - 40%, co-surfactant 15% - 25%.

[0008] More preferably, the mass percentages of the components of the cyclosporine self-microemulsion preparation of the present invention are: cyclosporine 10.07%, α-tocopherol 0.05%, amino acid 0.05%, solvent 32.11%, surfactant 38.64%, co-surfactant 19.08%.

[0009] The dosage ratio of α-tocopherol to amino acid in the cyclosporine preparation is 0.8:1 - 5:1; more preferably 1:1.

[0010] The amino acid is preferably one of glycine, cysteine, glutathione, or any two of them.

[0011] Further preferably, the amino acid is glutathione.

[0012] The solvent is preferably one of soybean oil, corn oil, cottonseed oil, glycerol monolaurate, medium-chain triglycerides, oleic acid, or any two of them.

[0013] Further preferably, the solvent is glycerol monolaurate.

[0014] The surfactant is preferably one of polyethylene glycol 32 stearate, stearoyl polyoxyethylene glycerol ester, polyoxyethylene hydrogenated castor oil, diethylene glycol monoethyl ether, or any two of them.

[0015] Further preferably, the surfactant is polyoxyethylene 40 hydrogenated castor oil.

[0016] The co-surfactant is preferably one of propylene glycol, polyethylene glycol, ethanol, glycerol, or any two of them.

[0017] Further preferably, the co-surfactant is one or two of propylene glycol and ethanol.

[0018] In the second aspect of the present invention, a preparation method of a cyclosporine self-microemulsion preparation is provided, and the steps are as follows:

[0019] (1) Mix the prescribed amount of co-surfactant, α-tocopherol, and amino acid evenly to obtain a solution for standby;

[0020] (2) Add the prescribed amount of cyclosporine to the solution in step (1), start stirring at room temperature, and dissolve the cyclosporine completely to obtain a solution for standby;

[0021] (3) Add the prescribed amount of solvent and surfactant to the solution obtained in step (2), and mix evenly to obtain the cyclosporine preparation;

[0022] (4) Directly fill the cyclosporine preparation to obtain the finished product.

[0023] In the third aspect of the present invention, there is provided the use of the cyclosporine self-microemulsion preparation in the preparation of a drug for preventing and treating atopic dermatitis or allergic dermatitis in dogs and cats.

[0024] Based on the technical solution of this application, since cyclosporine is a cyclic polypeptide with powerful immunosuppressive and immunomodulatory properties, it was first used in human medicine to prevent rejection of transplanted organs and later used in animal treatment of atopic dermatitis. However, cyclosporine is a poorly soluble lipophilic drug, and its low solubility results in low effective concentration and poor stability in the preparation of this drug. The oral preparations of cyclosporine in the prior art contain oily solvents, which not only have poor palatability but are also easily oxidized, further affecting the bioavailability. The design idea of the present invention is to add surfactants and co-surfactants to the prescription to improve the drug solubility; add α-tocopherol and glutathione as antioxidants to synergistically improve the drug stability and prevent the oxidation of the oily solvent in the prescription, eliminating the cumbersome step of headspace nitrogen filling in the process; on the other hand, glutathione can act as a flavoring agent to improve the problem of poor palatability of oral liquid.

[0025] Beneficial effects:

[0026] 1. The cyclosporine preparation prepared by the present invention has a relatively simple preparation process. Under the action of the synergistic antioxidant, the product does not require headspace nitrogen filling protection, simplifies the production process, and has higher production efficiency.

[0027] 2. The α-tocopherol and glutathione added to the cyclosporine preparation prepared by the present invention not only act as a synergistic antioxidant to prevent the oxidation of the oily solvent, but glutathione also has the function of a flavoring agent, improving the disadvantage of poor palatability of oral drugs, and has the function of enhancing the immunity of dogs and cats, contributing to the therapeutic effect of diseases.

[0028] 3. The cyclosporine preparation prepared by the present invention forms a self-microemulsion drug delivery system under the action of the solvent, surfactant, and co-surfactant. After oral administration, the drug is first dispersed and diluted by gastric juice to form a microemulsion under the action of the gastrointestinal tract, and then digested by digestive enzymes, etc., improving the solubility and oral bioavailability of the poorly soluble drug.

[0029] 4. The cyclosporine preparation prepared by the present invention has good stability, high bioavailability, good palatability, and a simple preparation process, and has obvious effects in the treatment of allergic dermatitis in dogs and cats. Specific embodiments

[0030] Example 1

[0031] A cyclosporine self - microemulsion preparation is prepared by the following method:

[0032] Ingredients Single dose (100 g / bottle) Cyclosporine 10.07％ α-Tocopherol 0.05％ Glutathione 0.05％ Monoolein 32.11％ Polyoxyethylene 40 Hydrogenated Castor Oil 38.64％ Propylene Glycol 9.54％ Ethanol 9.54％ Total 100％

[0033] Under stirring conditions, first mix α - tocopherol, glutathione, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, and then add glyceryl mono - linoleate and polyoxyethylene 40 hydrogenated castor oil and mix evenly to obtain it.

[0034] Example 2

[0035] A cyclosporine self - microemulsion preparation is prepared by the following method:

[0036]

[0037]

[0038] Under stirring conditions, first mix α - tocopherol, glutathione, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, and then add glyceryl mono - linoleate and polyoxyethylene 40 hydrogenated castor oil and mix evenly to obtain it.

[0039] Example 3

[0040] A cyclosporine self - microemulsion preparation is prepared by the following method:

[0041] Ingredients Single dose (100 g / bottle) Cyclosporine 15％ α-Tocopherol 2.5％ Glutathione 0.5％ Monoolein 35％ Polyoxyethylene 40 Hydrogenated Castor Oil 31％ Propylene Glycol 8％ Ethanol 8％ Total 100％

[0042] Under stirring conditions, first mix α - tocopherol, glutathione, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, and then add glyceryl mono - linoleate and polyoxyethylene 40 hydrogenated castor oil and mix evenly to obtain it.

[0043] Example 4

[0044] A cyclosporine self - microemulsion preparation is prepared by the following method:

[0045] Ingredients Single dose (100 g / bottle) Cyclosporine 10％ α-Tocopherol 5.1％ Glutathione 0.9％ Monoolein 30％ Polyoxyethylene 40 Hydrogenated Castor Oil 30％ Propylene Glycol 12％ Ethanol 12％ Total 100％

[0046] Under stirring conditions, first mix α - tocopherol, glutathione, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, and then add glyceryl mono - linoleate and polyoxyethylene 40 hydrogenated castor oil and mix evenly to obtain it.

[0047] Example 5

[0048] A cyclosporine self - microemulsion preparation is prepared by the following method:

[0049]

[0050]

[0051] Under stirring conditions, first mix α-tocopherol, glutathione, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, then add glyceryl monolaurate and polyoxyl 40 hydrogenated castor oil, and mix evenly to obtain.

[0052] Comparative Example 1

[0053] Ingredients Single dose (100 g / bottle) Cyclosporine 10.07％ α-Tocopherol 0.1％ Monoolein 32.11％ Polyoxyethylene 40 Hydrogenated Castor Oil 38.64％ Propylene Glycol 9.54％ Ethanol 9.54％ Total 100％

[0054] Under stirring conditions, first mix α-tocopherol, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, then add glyceryl monolaurate and polyoxyl 40 hydrogenated castor oil, and mix evenly to obtain.

[0055] Comparative Example 2

[0056] A cyclosporine preparation is prepared by the following method:

[0057] Ingredients Single dose (100 g / bottle) Cyclosporine 10.07％ α-Tocopherol 0.05％ Butylated Hydroxyanisole 0.05％ Monoolein 32.11％ Polyoxyethylene 40 Hydrogenated Castor Oil 38.64％ Propylene Glycol 9.54％ Ethanol 9.54％ Total 100％

[0058] Under stirring conditions, first mix α-tocopherol, butylated hydroxyanisole, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, then add glyceryl monolaurate and polyoxyl 40 hydrogenated castor oil, and mix evenly to obtain.

[0059] Comparative Example 3

[0060] A cyclosporine preparation is prepared by the following method:

[0061]

[0062]

[0063] Under stirring conditions, first mix α-tocopherol, dibutylhydroxytoluene, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, then add glyceryl monolaurate and polyoxyl 40 hydrogenated castor oil, and mix evenly to obtain.

[0064] Comparative Example 4

[0065] The prescription and preparation method approved for marketing abroad are as follows:

[0066] Ingredients Single dose (100 g / bottle) Cyclosporine 10.07％ α-Tocopherol 0.1％ Monoolein 32.11％ Polyoxyethylene 40 Hydrogenated Castor Oil 38.64％ Propylene Glycol 9.54％ Ethanol 9.54％ Total 100％

[0067] Under stirring conditions, first mix α-tocopherol, propylene glycol and ethanol evenly, add cyclosporine, stir at room temperature until cyclosporine is completely dissolved, then add glyceryl monolaurate and polyoxyl 40 hydrogenated castor oil, and mix evenly to obtain. Nitrogen is filled throughout the preparation process, and nitrogen is filled into the headspace during the filling of the finished product.

[0068] Example 6 Investigation of Antioxidant Effect

[0069] Test method: Using the related substances detection method, the antioxidant effects of the following prescriptions were investigated in groups. Group 1: Including Example 1, Example 2, Example 3, Example 4, Example 5; Group 2: Including Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4. Examine the growth trend of the peaks of oily solvent excipients in the samples at 0 hour, 10 days at high temperature (60 °C) and 6 months at accelerated (40 °C).

[0070] Related substances detection method: Refer to the related substances detection method under cyclosporine in the second part of the Chinese Pharmacopoeia (2020 edition).

[0071] The test results of Group 1 are as follows in Table 1:

[0072] Table 1 Comparison of Antioxidant Effects of the Products of the Invention

[0073]

[0074]

[0075] In Group 1, the formulations and processes of Example 1, Example 2, Example 3, Example 4, and Example 5 are the same, but the dosage ratios of α-tocopherol and glutathione are different. It can be seen from the experimental results in Table 1 that when the ratio of α-tocopherol to glutathione in Example 5 is 2:3, the antioxidant effect is significantly poor, and when the ratio of α-tocopherol to glutathione in Example 4 is 5.6:1, the antioxidant effect is slightly poor. The antioxidant effects of the products of the other ratios of the examples are obvious.

[0076] The test results of Group 2 are as follows in Table 2:

[0077] Table 2 Comparative Test of Antioxidant Effects of Different Products

[0078]

[0079] In Example 1, Comparative Example 2, and Comparative Example 3 of Group 2, the preparation processes are the same, but the prescriptions are different. Glutathione, butylated hydroxyanisole, and dibutylhydroxytoluene are used in combination with α-tocopherol as compound antioxidants in sequence; while in Comparative Example 4, only α-tocopherol is used as an antioxidant, and a nitrogen filling process is added during the preparation process. The test results show that there are fewer impurity peaks in the excipients of Example 1, indicating that the synergistic antioxidant effect of glutathione and α-tocopherol is better; by comparing Example 1 and Comparative Example 4, it can be seen that the synergistic effect of adding the antioxidants glutathione and α-tocopherol can completely replace the process operations of nitrogen filling during the preparation process and filling in Comparative Example 4, simplify the preparation process, and improve production efficiency.

[0080] In addition, it was also found during the experiment that the samples of Example 5, Comparative Example 1, Comparative Example 2, and Comparative Example 3 had peculiar smells after being placed at high temperature for 10 days. The reason was that there was a large amount of oil solvent in the excipients, and the oil solvent became rancid, producing peculiar smells. While Example 1, Example 2, Example 3, Example 4, and Comparative Example 4 did not produce peculiar smells under the same placement conditions, indicating that the combined use of glutathione and α-tocopherol has almost the same antioxidant effect as nitrogen filling in the preparation process.

[0081] Investigation on the water solubility stability of Example 7

[0082] Comparison of the water solubility stability of the samples at 0 hour: The products at 0 hour prepared in Examples 1-5 and Comparative Examples 1-4 were diluted 50 times with purified water and emulsified, and then placed at high temperature for 10 days and low temperature (4°C) for 10 days respectively to investigate the stability of the samples. The results are as follows:

[0083] Table 3 Investigation on the stability of the products at 0 hour diluted 50 times

[0084]

[0085] Comparison of the water solubility stability of the samples after being placed at high temperature for 10 days: After the samples of the above examples and comparative examples were placed at high temperature for 10 days, they were diluted 50 times with purified water and emulsified, and then placed at high temperature for 10 days and low temperature (4°C) for 10 days respectively to investigate the stability of the samples. The results are as follows:

[0086] Table 4 Investigation on the stability of the samples after being placed at high temperature for 10 days diluted 50 times

[0087]

[0088]

[0089] From the above two sets of data, it can be seen that for the preparations of Examples 1-5 and Comparative Examples 1-4, the samples diluted 50 times at 0 o'clock had almost the same stability after being placed under high and low temperature conditions. However, for the samples after being placed at high temperature for 10 days, after being diluted by the same multiple, obvious stability problems occurred for the products of Examples 4 and 5 and Comparative Examples 1-3 after being placed at low temperature for 10 days. While the stability of Examples 1-3 and Comparative Example 4 under the same conditions was good. The reason for the appearance of a small amount of precipitation after the water-soluble samples were placed at low temperature for 10 days was that after the oil-based solvent in the excipients was oxidized, the solubility of the active pharmaceutical ingredient decreased, and the raw material precipitated after being placed at low temperature, which might affect the absorption of the product in the body.

[0090] Investigation on the stability during the use after opening the sample of Example 8

[0091] Compared with Comparative Example 4, in Example 1, α-tocopherol and glutathione were used synergistically as antioxidants, and there was no need to fill nitrogen during the preparation process; in Comparative Example 4, only α-tocopherol was used as an antioxidant, and nitrogen protection was required during the preparation and filling processes. The samples of Example 1 and Comparative Example 4 were placed separately after being opened and detected on the 10th, 30th, 70th, and 80th days. The results are as follows:

[0092] Table 5 Detection of the oxidation degree of the samples

[0093]

[0094]

[0095] From the above data, it can be seen that for the detection results of Example 1, as the opening time prolonged, the impurity peaks at RRT 4.93, 5.14, 5.33, and 5.52 gradually increased, but the final peak area ratio was all below 0.2%. While for the product of Comparative Example 4, the peak area ratio of the impurity peaks increased rapidly during the process from 10 days to 80 days of opening. At the 70th day of opening, the peak ratio had approached or even exceeded the limit of 0.2%, and at the 80th day of opening, the growth trend was even more rapid. This shows that although the product of Comparative Example 4 was protected by filling nitrogen during both preparation and filling, during the process of opening and using, the nitrogen content decreased, and the oil-based excipients were gradually oxidized, thus affecting the stability and palatability of the product. In Example 1, α-tocopherol and glutathione were used synergistically as antioxidants, and even without filling nitrogen, it showed good antioxidant effects.

[0096] Palatability evaluation of Example 9

[0097] The palatability tests were carried out on Example 1 and Comparative Example 4. Twenty beagle dogs and twenty Chinese rural cats were selected, and the reactions of the animals were observed after administration. The results are as follows in the table:

[0098] Table 6 Investigation on the palatability of dogs

[0099] Dog Behavior Example 1 (N = 20) Control 4 (N = 20) Dog licks the oral cavity normally 15 10 Dog has more saliva secretion 4 9 Dog shows retching and foaming 1 1 Acceptability / Palatability 75％ 50％

[0100] Table 7 Palatability Investigation of Cats

[0101] Cat Behavior Example 1 (N = 20) Control 4 (N = 20) Cat licks the oral cavity normally 12 7 Cat has more saliva secretion 7 12 Cat shows head shaking and resistance 1 1 Acceptability / Palatability 60％ 35％

[0102] The test results show that after adding glutathione to the formulation of Example 1, 75% of the dogs and 60% of the cats can accept it in the palatability results. In the palatability evaluation of Comparative Example 4, 50% of the dogs and 35% of the cats can accept it. It shows that the palatability is significantly increased after adding glutathione.

[0103] Pharmacokinetics of Example 10

[0104] Twenty-four adult healthy beagle dogs weighing 10 - 30 kg were selected and designed with a two-treatment, two-period, crossover design, and there was an 8-day washout period between the two treatments. The dogs were randomly divided into two treatment groups: the fasted dogs were administered the products of Example 1 and Comparative Example 4 according to their body weight, and 2 ml of blood samples were taken at 0, 0.5, 1, 1.5, 2, 4, 6, 12, 24, 36, 48 h after administration for detection. The results are as follows:

[0105] Table 8 Pharmacokinetic Test Results

[0106] Test Items Example 1 Control 4 <![CDATA[T 1 / 2 (h)]]> 9.21 9.35 <![CDATA[T max (h)]]> 1.22±0.40 1.12±0.35 <![CDATA[C max (ng / ml)]]> 770.2±170.3 783.2±163.4 AUC (h.ng / ml) 3621±1260 3512±1256 F(％) 35 33

[0107] The above results show that the pharmacokinetic behaviors of Example 1 and Comparative Example 4 in vivo are consistent.

Claims

1. A cyclosporine self - microemulsifying formulation, characterized in that, The preparation consists of the following components: cyclosporine, α-tocopherol, amino acids, solvent, surfactant, co-surfactant; the mass percentages of the components of the preparation are: cyclosporine 10% - 15%, α-tocopherol 0.05% - 6%, amino acids 0.05% - 2%, solvent 30% - 35%, surfactant 30% - 40%, co-surfactant 15% - 25%; the dosage ratio of α-tocopherol to amino acids in the cyclosporine preparation is 0.8:1 - 5:1; the amino acid is glutathione; the solvent is glyceryl mono-linoleate; the surfactant is polyoxyethylene hydrogenated castor oil; the co-surfactant is propylene glycol and ethanol.

2. The preparation method of a cyclosporine self-microemulsion preparation as claimed in claim 1, the steps are as follows: (1) Mix the co-surfactant, α-tocopherol and amino acids in the prescribed amounts evenly to obtain a solution for standby; (2) Add the cyclosporine in the prescribed amount to the solution in step (1), start stirring at room temperature to completely dissolve the cyclosporine, and obtain a solution for standby; (3) Add the solvent and surfactant in the prescribed amounts to the solution obtained in step (2), mix evenly to obtain the cyclosporine preparation; (4) Directly fill the cyclosporine preparation to obtain the finished product.

3. The application of a cyclosporine self-microemulsion preparation as claimed in claim 1 in the preparation of a drug for preventing and treating atopic dermatitis or allergic dermatitis in dogs and cats.

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