A Tivirmatimab composition, its preparation method and application

Abstract

The present invention relates to the field of medicine, and provides a tecovirimat composition, a preparation method thereof and an application. The composition contains an active ingredient tecovirimat, a complexing agent cyclodextrin, a solubilizer meglumine and a basic additive, and the weight ratio is 1:(15-30):(0.05-0.2):(0.1-0.2). Among them, the combined use of the three auxiliary materials of the complexing agent, the solubilizer and the basic additive can greatly improve the solubility of the active ingredient tecovirimat. Compared with the prior art, the dosage of each auxiliary material in the composition of the present invention can meet the specified limits in relevant regulations, and the risks of allergy, hemolysis and irritation are relatively low, and the safety of the preparation is better. The preparation method of the composition provided by the present invention is simple and easy to implement, the quality of the preparation finished product is more stable, and the application is more convenient. The composition can be used for treating or preventing virus infections and related diseases caused by orthopoxviruses.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a tevirima composition and a preparation method and application thereof. Background Art

[0002] Tecovirimat (English name: Tecovirimat; R&D code: ST-246; formerly known as Tecovir; Formula 1), chemical name N -[(3a R ,4 R ,4a R ,5a S ,6 S ,6a S )-3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-vinylcyclopropyl[ f ]Isoindole-2(1 H )-yl]-4-(trifluoromethyl)benzamide monohydrate is an effective broad-spectrum inhibitor of orthopoxvirus infections such as smallpox, monkeypox, and cowpox. The compound targets the conserved protein VP37 of orthopoxviruses, preventing them from forming virus-specific encapsulation complexes, thereby inhibiting the production of infectious enveloped virus particles and blocking the cell-to-cell and long-distance transmission of the virus. However, the solubility of tivirimab in water is about 3.35 μg / mL, which is a nearly insoluble or insoluble compound. This property severely limits the clinical application of the drug.

[0003]

[0004] Formula 1: Structural formula of Tevirimab

[0005] The formulation patent CN103281898 discloses a formulation of Tepirima injection, which uses hydroxypropyl-β-cyclodextrin as an excipient to increase the solubility of Tepirima (up to 21.23 mg / mL). Tepirima injection developed based on this patent (marketed trade name: TPOXX ® ; hereinafter referred to as binary prescription, binary combination, TPOXX ® The dosage of hydroxypropyl-β-cyclodextrin in TPOXX injection is as high as 40%, which far exceeds the regulatory limit according to its clinical use plan. Studies have shown that high doses of hydroxypropyl-β-cyclodextrin have the risk of hemolysis and kidney-related toxic side effects, so TPOXX ® The injection is contraindicated for patients with severe renal impairment. It is worth mentioning that TPOXX has been approved for monkeypox indications, but among monkeypox patients, children with renal dysplasia and patients with severe renal impairment account for a large proportion, which greatly limits the use of TPOXX. ® Clinical application of TPOXX. ®The injection is a solution dosage form and needs to be stored and transported at 2-8°C.

[0006] Patent CN107625967 provides another injection formulation of Tivirima (hereinafter referred to as the ternary composition), which uses cyclodextrin and meglumine as excipients, in which the amount of meglumine (calculated according to the dosing regimen of similar injections on the market) far exceeds the 75 mg / day limit stipulated in the US Excipient Database (IID), and due to insufficient safety data on excessive use of meglumine excipients, it cannot be directly used for commercial development. In addition, the patented formulation needs to be prepared under high temperature (60°C), which has high requirements for preparation conditions.

[0007] Therefore, developing a new prescription with higher safety, better stability and suitable for commercial development is an urgent problem that needs to be solved. Summary of the invention

[0008] One object of the present invention is to provide a composition of tevirima and its key proportions that can be used for the treatment of orthopoxvirus infection. In order to solve the problems existing in the existing tevirima injection, the present invention explores a new prescription. Cyclodextrin is used in the solubilization prescription of various poorly soluble drugs, but there are safety risks such as nephrotoxicity and hemolysis when used in high doses, so the amount of cyclodextrin needs to be controlled within an appropriate range. In addition, tevirima is a weakly acidic compound, and alkaline conditions can promote the dissolution of the compound, but the tevirima molecule contains an amide structure, and excessive use of alkali can easily lead to the degradation of the compound, increase the impurity content, and affect the stability of the preparation. The present invention investigated a variety of excipients and their dosages, and found a composition that can greatly improve the solubility of tevirima, and unexpectedly found that the inclusion agent cyclodextrin, the solubilizer meglumine and the alkaline additive (referred to as a quaternary prescription and a quaternary composition in the present invention) are used simultaneously, so that the active ingredient tevirima can be completely dissolved under the condition that the dosage of each excipient meets the requirements of relevant regulations, and the stability is good. However, if one or two of the inclusion agent cyclodextrin, the solubilizer meglumine and the alkaline additive are used as excipients, there are problems such as the inability to completely dissolve tevirima or the risk of excipient safety. Therefore, the combination of the three excipients, the inclusion agent cyclodextrin, the solubilizer meglumine and the alkaline additive, is the key to ensuring the complete dissolution of the active ingredient tevirima and the safety of the preparation.

[0009] The dosage of each auxiliary material of the tevirima quaternary composition provided by the present invention meets the regulatory requirements, and the safety of the auxiliary materials is fully guaranteed; while improving the solubility of the active ingredient tevirima, the stability of the preparation is enhanced; the prescription has low risks of allergy, hemolysis and irritation, the preparation is highly safe, and is suitable for commercial development.

[0010] Another object of the present invention is to provide a method for preparing the tevirima composition.

[0011] Another object of the present invention is to provide the use of the tivirima composition.

[0012] To achieve the above-mentioned purpose, on the one hand, the present invention provides a tevirima composition, which contains at least tevirima, a clathrate, a solubilizer and an alkaline additive, and the weight ratio thereof is 1: (15-30): (0.05-0.2): (0.1-0.2);

[0013] The inclusion agent is selected from a combination of one or more of β-cyclodextrin, methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin, 2,3-dihydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin sodium;

[0014] The solubilizing agent is selected from a combination of one or more of meglumine, hydroxypropyl methylcellulose and copovidone;

[0015] The alkaline additive is selected from one or more combinations of organic bases or inorganic bases.

[0016] According to some specific embodiments of the present invention, the weight ratio of tivirimab, inclusion agent, solubilizer and alkaline additive is 1: (15-20): (0.05-0.15): (0.1-0.15).

[0017] According to some specific embodiments of the present invention, the weight ratio of tivirima, inclusion agent, solubilizer and alkaline additive is 1:15:0.1:0.1.

[0018] According to some specific embodiments of the present invention, the inorganic base is selected from hydroxides, carbonates or bicarbonates of alkali metals or alkaline earth metals; and the organic base is selected from triethanolamine or arginine.

[0019] According to some specific embodiments of the present invention, the inorganic base is selected from sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate.

[0020] According to some specific embodiments of the present invention,

[0021] The inclusion agent is a combination of one or more of 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin and 2,3-dihydroxypropyl-β-cyclodextrin; preferably a combination of one or two of 2-hydroxypropyl-β-cyclodextrin and 3-hydroxypropyl-β-cyclodextrin; further preferably 2-hydroxypropyl-β-cyclodextrin;

[0022] The solubilizer is a combination of one or more of meglumine, hydroxypropyl methylcellulose and copovidone; preferably meglumine;

[0023] The alkaline additive is a combination of one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, triethanolamine, and arginine; preferably sodium hydroxide or potassium hydroxide; further preferably sodium hydroxide.

[0024] According to some specific embodiments of the present invention, the inclusion agent is selected from a combination of one or more of 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin and 2,3-dihydroxypropyl-β-cyclodextrin;

[0025] The solubilizing agent is meglumine;

[0026] The alkaline additive is sodium hydroxide.

[0027] According to some specific embodiments of the present invention, the inclusion agent is selected from one or a combination of 2-hydroxypropyl-β-cyclodextrin and 3-hydroxypropyl-β-cyclodextrin.

[0028] According to some specific embodiments of the present invention, the inclusion agent is 2-hydroxypropyl-β-cyclodextrin.

[0029] According to some specific embodiments of the present invention, the tevirima composition is tevirima powder injection or tevirima injection.

[0030] According to some specific embodiments of the present invention, when the tevirima composition is an injection, the concentration of tevirima is 5-50 mg / mL; preferably 10-20 mg / mL;

[0031] According to some specific embodiments of the present invention, when the tivirima composition is an injection, the pH value of the injection is 8-12.

[0032] According to some specific embodiments of the present invention, when the tivirima composition is an injection, the pH value of the injection is 9-11.

[0033] According to some specific embodiments of the present invention, when the tevirima composition is a powder injection, after reconstitution at a tevirima concentration of 5-50 mg / mL, the pH value of the obtained solution is 8-12.

[0034] According to some specific embodiments of the present invention, when the tevirima composition is a powder injection, it can be reconstituted at a tevirima concentration of 10-20 mg / mL during use.

[0035] According to some specific embodiments of the present invention, when the tivirima composition is a powder injection, the pH value of the solution obtained after reconstitution is 9-11.

[0036] According to some specific embodiments of the present invention, when the tevirima composition is a powder injection, after reconstitution at a tevirima concentration of 10-20 mg / mL, the pH value of the obtained solution is 9-11.

[0037] According to some specific embodiments of the present invention, when the tivirima composition is a powder injection, its reconstitution solvent is one or more of water for injection, 0.9% sodium chloride and 5% glucose solution.

[0038] On the other hand, the present invention also provides a method for preparing a tivirima composition, which comprises the following steps:

[0039] (1) Take an appropriate amount of water for injection, add the inclusion agent, solubilizer and alkaline additive, and stir to dissolve to obtain a first solution;

[0040] (2) adding tevirimab to the first solution, adding the remaining water for injection, and stirring until the solid is dissolved to obtain tevirimab injection; or

[0041] Optionally, the method further comprises step (3): freeze-drying or spray-drying the injection solution obtained in step (2) to obtain a tevirimab powder injection.

[0042] According to some specific embodiments of the present invention, step (1) comprises taking 60%-80% of the total amount of water for injection, adding a complexing agent, a solubilizing agent and an alkaline additive, stirring and dissolving them to obtain a first solution.

[0043] According to some specific embodiments of the present invention, step (1) is to control the temperature of the water for injection to be no higher than 60° C., and to add the inclusion agent, solubilizer and alkaline additive.

[0044] According to some specific embodiments of the present invention, step (1) is to control the temperature of the water for injection to not less than 15° C. and add the inclusion agent, solubilizer and alkaline additive.

[0045] According to some specific embodiments of the present invention, step (1) is to control the temperature of the water for injection at 15°C-60°C, and add the inclusion agent, solubilizer and alkaline additive.

[0046] According to some specific embodiments of the present invention, step (2) comprises adding tevirima to the first solution and adding the remaining water for injection, and sterilizing to obtain tevirima injection.

[0047] According to some specific embodiments of the present invention, the sterilization in step (2) is filtration sterilization or autoclave sterilization. The filtration sterilization membrane range is 0.22 μm-0.45 μm; the autoclave sterilization parameters are 115°C-121°C, 15 min-30 min.

[0048] According to some specific embodiments of the present invention, step (2) is to control the temperature of the first solution to no higher than 60° C. and add tivirimab.

[0049] According to some specific embodiments of the present invention, step (2) is to control the temperature of the first solution to not less than 15°C and add tivirima.

[0050] According to some specific embodiments of the present invention, step (2) is to control the temperature of the first solution at 15°C-60°C and add tivirima.

[0051] The obtained Tevirima injection can be directly used in combination; and the obtained Tevirima powder injection can be reconstituted with injection water, 0.9% sodium chloride, or 5% glucose solution and then used in combination.

[0052] In another aspect, the present invention also provides the use of the tivirima composition in the preparation of a drug for treating or preventing viral infection.

[0053] According to some specific embodiments of the present invention, the virus is an orthopoxvirus.

[0054] According to some specific embodiments of the present invention, the virus is cowpox virus, monkeypox virus or vaccinia virus.

[0055] It can be understood that, under the premise of no contradiction, the specific embodiments of the present invention can be combined arbitrarily.

[0056] The term “optional” as used in the present invention means that the condition or situation may occur or exist, or may not occur or exist. For example, the term “optionally further includes step (3)” as used in the present invention means that step (3) may exist or may not exist. When step (3) exists, a tevirima powder injection is obtained, and when step (3) does not exist, a tevirima injection solution is obtained.

[0057] In summary, the present invention provides a tevirima composition and a preparation method and application thereof. The tevirima composition of the present invention has the following advantages:

[0058] 1) Compared with the existing technology, the present invention develops a quaternary formulation of Tevirima and determines its key ratio. While improving the solubility of Tevirima, the dosage of each excipient can meet the requirements of relevant regulations and is suitable for commercial development.

[0059] 2) Compared with the patent CN107625967, the preparation method of the quaternary preparation of Tevirima of the present invention is simple and has a low impurity content, which ensures the repeatability and quality stability of the product.

[0060] 3) The quaternary composition provided by the present invention has lower risks of allergy, hemolysis and irritation. Compared with the TPOXX that has been marketed ® The injection has better safety and can cover a wider population (such as patients with severe renal impairment).

[0061] 4) The quaternary composition provided by the present invention and TPOXX ® Compared with the injection, the solubility of Tevirima is significantly improved, and it can tolerate higher temperatures and has better stability. It can be transported, stored and used at room temperature, greatly improving the convenience of clinical application.

[0062] The present invention provides a novel tevirima composition and a preparation method thereof, which is simple in preparation method, higher in safety and better in stability, and is suitable for commercial development. The composition can be used to treat or prevent viral infections and related diseases caused by orthopoxviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 These are the results of a comparative study of in vitro hemolysis between the quaternary composition and the binary composition of Tivirima in Example 13. DETAILED DESCRIPTION

[0064] The implementation process of the present invention and the beneficial effects produced are described in detail below through specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention.

[0065] Example 1: Proportion of the Tevirima Composition

[0066] Preparation method: As shown in Table 1, weigh the corresponding weight of hydroxypropyl-β-cyclodextrin, meglumine and sodium hydroxide in each prescription, dissolve them in a certain volume of water for injection at room temperature, stir and mix evenly, then weigh 1 g of tivirima and add it to the solution, add water for injection to 100 mL, stir at room temperature, and observe the dissolution after 24 hours.

[0067] Table 1 Dissolution of different ratios of tivirima compositions

[0068]

[0069] Experimental results: According to the relevant regulations of the US FDA and the EU on pharmaceutical excipients, the maximum dosage of each excipient is as follows: 200 mg / kg / day for hydroxypropyl-β-cyclodextrin, 75 mg / day for meglumine, and 78 mg / day for sodium hydroxide. Combined with the dosage regimen of similar preparations that have been marketed (see Table 6), for people of normal weight, under the conditions of the above dosage limits, it was found that only when the three excipients are used at the same time, can tevirima be completely dissolved; using any two of the three excipients will not make tevirima completely dissolved. Therefore, the ratio and combination of the three excipients of inclusion agent cyclodextrin, solubilizer meglumine, and alkaline additive sodium hydroxide is the key to ensure the complete dissolution of the active ingredient tevirima and the safety of the preparation.

[0070] Example 2: Solubility of the Tevirima Quaternary Composition

[0071] Preparation method: As shown in Table 2, weigh the corresponding weight of hydroxypropyl-β-cyclodextrin, meglumine and sodium hydroxide in each prescription, dissolve them in a certain volume of water for injection at room temperature, stir and mix evenly, then weigh 2-5 g of tivirimab and add it to the solution, adjust the pH and add water for injection to 100 mL, continue stirring, and observe the dissolution after 24 hours.

[0072] Table 2 Solubility of the quaternary composition of Tevirima

[0073]

[0074] Experimental results: All the above prescriptions can completely dissolve Tevirima, and the prescription provided by the present invention can increase the solubility of Tevirima to 50 mg / mL, which is helpful for the industrial production of Tevirima preparations. During the experiment, it was also observed that as the amount of Tevirima increased, the time required for the prescription to dissolve became longer; when the content of Tevirima was limited, the more the amount of each excipient in the formula was used, the shorter the time required for the prescription to completely dissolve.

[0075] Example 3: Comparison of preparation conditions of quaternary and ternary compositions of Tevirima

[0076] The quaternary composition of Tivirima was prepared according to the preparation method in Example 2, and the ternary composition was prepared according to the preparation method in patent CN107625967. The preparation volume was 100 mL. Each prescription was stirred at a specific temperature. The dissolution state of each composition was recorded after 24 hours. The results are shown in Table 3.

[0077] Table 3 Comparison of dissolution states of each formulation of the quaternary combination of Tevirima and the ternary combination

[0078]

[0079] Note: The ratio of the ternary composition is quoted from the preferred formula of patent CN107625967. Ternary composition 1 is tevirima: meglumine: hydroxypropyl-β-cyclodextrin = 2 g: 4 g: 12 g, and ternary composition 2 is tevirima: meglumine: hydroxypropyl-β-cyclodextrin = 5 g: 10 g: 30 g.

[0080] Experimental results: Each formulation of the quaternary combination of Tevirimab can be completely dissolved at a preparation temperature of 25°C-60°C, while the ternary combination prepared according to the patented formulation of CN107625967 can only be completely dissolved when the preparation temperature is raised to 60°C.

[0081] Example 4: Impurity determination of the quaternary composition solution of Tivirima

[0082] The solution prepared in Example 2 was placed at room temperature for 12 h, and the appearance, pH and content of related substances were measured. The results are shown in Table 4.

[0083] Table 4 Solution stability of quaternary composition

[0084]

[0085] Experimental results: After being placed for 12 hours, the appearance and pH of each formulation of the quaternary combination of Tevirima did not change, and the impurity content was at a low level, indicating that the quaternary combination solution had good stability.

[0086] Example 5: Study on the reconstitution and compatibility stability of the quaternary composition of Tevirima

[0087] The quaternary composite solution was prepared according to the method in Example 2, and the powder injection was obtained after being packaged (10 mL / bottle) and freeze-dried. It was reconstituted with 20 mL of water for injection, and 40 mL of 5% glucose or 0.9% sodium chloride was added to obtain a compatible solution. The compatible solution was placed at room temperature for 6 hours, and the pH and the content of related substances were measured. The results are shown in Table 5.

[0088] Table 5 Quaternary composition freeze-dried powder * The compatibility stability

[0089]

[0090] Note: * The freeze-drying process here is: (1) pre-freezing and heat preservation: -40℃ pre-freezing and heat preservation; (2) vacuuming; (3) when the vacuum degree is lower than 0.15mbar, start heating: increase the temperature from -20℃ to 35℃.

[0091] Experimental results: After the freeze-dried powder of the quaternary combination of Tivirima was reconstituted and placed at room temperature for 6 hours, there was no obvious change in each test item, indicating that the freeze-drying process of each prescription of the quaternary combination was stable and the compatibility stability was good.

[0092] Example 6: Comparison of excipient dosages of quaternary composition prescription 10 and existing patented prescriptions at clinically intended doses

[0093] According to TPOXX ® Injection instructions, calculate prescription 10, TPOXX respectively ® The dosage of each excipient for injection, CN103281898 patented prescription, and ternary combination in people with different weights (see Tables 6 to 8).

[0094] Table 6 Comparison of dosage of hydroxypropyl-β-cyclodextrin excipients in different prescriptions

[0095]

[0096] Note: The EU's "Annex to the Guidelines for Excipients in Labelling and Package Instructions for Medicinal Products for Human Use" stipulates that the limit of hydroxypropyl-β-cyclodextrin in injections is 200 mg / kg / day.

[0097] aPrescription from TPOXX ® Injection instructions.

[0098] b is from patent CN103281898, which is the formula with the lowest dosage of hydroxypropyl-β-cyclodextrin among all the embodiments in the patent.

[0099] Table 7 Comparison of dosage of excipients of meglumine in different prescriptions

[0100]

[0101] Note: The U.S. Excipient Database (IID) shows that the excipient limit for meglumine in injections (including lyophilized powder) is 75 mg / day.

[0102] a The formula of the ternary composition is extracted from patent CN107625967.

[0103] Table 8 Amount of NaOH auxiliary material used in quaternary composition prescription 10

[0104]

[0105] Note: The U.S. Excipient Database (IID) shows that the limit of NaOH in injections is 78 mg / day.

[0106] Research results: The dosage of each excipient in the four-component combination prescription 10 of Tepcoviridad meets the corresponding excipient limit requirements at the clinical dosage. ®The dosage of the excipient hydroxypropyl-β-cyclodextrin in the injection prescription and patent CN103281898 prescription far exceeds the limit specified in the EU "Annex to the Guide to Excipients in Labelling and Package Instructions for Medicinal Products for Human Use"; while the dosage of the excipient meglumine in the ternary composition from patent CN107625967 far exceeds the dosage specified in the US Excipient Database (IID). The above results show that the excipient safety of the quaternary composition of Tevirima is better and suitable for commercial development.

[0107] Example 7: Comparative study on the stability of different prescription preparations

[0108] According to the method in Example 5, a lyophilized powder of the quaternary composition prescription 10 was prepared and reconstituted with 0.9% sodium chloride injection or 5% glucose injection. ® Injection formula: binary composition, each volume is 20 mL. The above different prescription preparations are diluted with 2 times the volume of 0.9% sodium chloride injection or 5% glucose injection. The diluted solution is placed at 2-8℃, and various indicators are measured. The results are shown in Table 9.

[0109] Table 9 Comparative study on the stability of different prescription preparations

[0110]

[0111] Note: The ratio of binary prescription is 1:40 of tevirimab:hydroxypropyl-β-cyclodextrin, stir until dissolved.

[0112] Experimental results: The four-component combination of tevirima, prescription 10 and the two-component combination were mixed with 0.9% sodium chloride or 5% glucose, respectively. After being placed at 2-8°C for 168 hours, there was no significant change in the test items of prescription 10, while the two-component prescription injection became turbid 48 hours after being mixed with 0.9% sodium chloride or 5% glucose. The above results show that the compatibility stability of prescription 10 is better than that of the two-component combination.

[0113] Example 8: Study on high temperature stability of different formulations

[0114] According to the method in Example 5, a quaternary composition freeze-dried powder (prescription 10) was prepared. It was placed at 60°C for 10 days or at 40°C for 30 days, reconstituted with 20 mL of water for injection, and various indicators were measured. According to Example 7 and Example 3, a binary composition and a ternary composition 1 were prepared respectively, placed at 60°C for 10 days or at 40°C for 30 days, and various indicators were measured. The results are shown in Table 10.

[0115] Table 10 High temperature stability of different formulations

[0116]

[0117] Experimental results: After preparation, the initial impurity level of ternary composition 1 was higher than that of prescription 10 and binary composition. After each prescription preparation was placed at 60°C for 10 days or at 40°C for 30 days, the impurity growth trend in binary composition and ternary composition 1 was obvious, which was much higher than the impurity level of the quaternary composition prescription 10 preparation.

[0118] Example 9: High humidity stability study of quaternary compositions

[0119] According to the method in Example 5, the quaternary composition freeze-dried powder was prepared with prescription 10, which was placed under RH 92.5% for 30 days and reconstituted with 20 mL of water for injection on day 0, day 10 and day 30, respectively. Various indicators were measured. The results are shown in Table 11.

[0120] Table 11 Stability test results of formulation 10 under high humidity conditions

[0121]

[0122] Experimental results: After the preparation of the quaternary combination of Tevirima, prescription 10, was placed under RH 92.5% for 30 days, there was no significant change in each measurement item, indicating that it has good stability under high humidity conditions.

[0123] Example 10: Study on the photostability of the quaternary composition of Tevirima

[0124] According to the method in Example 5, the quaternary composition freeze-dried powder was prepared with prescription 10, which was placed under 4500±500 Lx conditions for 30 days and reconstituted with 20 mL of water for injection on the 0th day, the 10th day, and the 30th day, respectively. Various indicators were measured. The results are shown in Table 12.

[0125] Table 12 Stability test results of formulation 10 under light conditions

[0126]

[0127] Experimental results: After the quaternary combination prescription 10 of Tevirima was placed under 4500±500 Lx conditions for 30 days, there was no obvious change in each measurement item, indicating that it has good stability under light conditions.

[0128] Example 11: Stability study of the quaternary composition of Tevirima at 40°C and RH75%

[0129] According to the method in Example 5, the quaternary composition freeze-dried powder was prepared with prescription 10, and placed at 40°C and RH75% for 90 days. It was reconstituted with 20 mL of water for injection on the 0th day, the 30th day, the 60th day, and the 90th day, and various indicators were measured. The results are shown in Table 13.

[0130] Table 13 Stability test results of formulation 10 at 40°C and RH75%

[0131]

[0132] Experimental results: After the four-component combination prescription 10 of Tevirima was placed at 40°C and RH75% for 90 days, there was no significant change in the measured items, indicating that it has good stability at 40°C and RH75%.

[0133] Example 12: Stability study of the quaternary composition of Tevirima at 25°C and RH 60%

[0134] According to the method in Example 5, the quaternary composition freeze-dried powder was prepared with prescription 10, and placed at 25°C and RH60% for 90 days. It was reconstituted with 20 mL of water for injection on the 0th day, the 30th day, the 60th day, and the 90th day, and various indicators were measured. The results are shown in Table 14.

[0135] Table 14 Stability test results of formulation 10 at 25°C and RH 60%

[0136]

[0137] Experimental results: After the four-component combination prescription 10 of Tevirima was placed at 25°C and RH60% for 90 days, there was no significant change in the measured items, indicating that it has good stability at 25°C and RH60%.

[0138] Example 13: Comparative study on hemolysis risk of different prescription preparations

[0139] According to the method in Example 5, a quaternary composition and a corresponding excipient control group (excluding tevirima) were prepared with prescription 10, and a binary composition and a corresponding excipient control group (excluding tevirima) were prepared according to Example 7. The above four compositions were prepared in the same way. The corresponding amount of the prepared solution was added to the in vitro hemolysis test system of New Zealand rabbit red blood cells, and the mixture was placed in a 36.1°C incubator for 3 hours, and the hemolysis was recorded. The results are shown in Tables 15 and Figure 1 .

[0140] Table 15 Hemolysis test results of different prescription preparations

[0141]

[0142] Experimental results: Hemolysis occurred in two high-dose tubes of the binary composition and its excipient control group, while no hemolysis was observed in the quaternary composition and its excipient control group, indicating that the quaternary composition prescription 10 preparation has no hemolysis risk and its hemolysis safety is higher than that of the binary composition.

[0143] Example 14: Study on the risk of allergy to the quaternary combination of Tevirima

[0144] According to the method in Example 5, a quaternary composition preparation was prepared with prescription 10. It was prepared with 0.9% sodium chloride injection and set aside for use. An active sensitization experiment was carried out in a guinea pig model using 0.9% sodium chloride injection as a negative control and albumin as a positive control. All groups of animals were administered by intraperitoneal injection during the sensitization period and intravenous injection during the challenge period. After the last sensitization injection, the animals were challenged twice on the 14th and 21st days. During this period, cage-side observations, detailed clinical observations, weight records, post-challenge observations, allergy evaluations, etc. were carried out.

[0145] Experimental results: All animals in the positive control group died after stimulation, while the negative control group and the prescription 10 preparation group showed no abnormalities, indicating that the prescription 10 preparation of the quaternary combination of tivirima has no allergic risk.

[0146] Example 15: Study on the risk of vascular irritation in rabbits with the quaternary combination of Tevirima

[0147] According to the method in Example 5, a quaternary composition preparation was prepared with prescription 10. It was prepared with 0.9% sodium chloride solution and then used. Vascular stimulation studies were conducted in New Zealand white rabbit models, with the administration method being twice a day, intravenous drip for 60 min each time, for 7 consecutive days, and recovery for 14 days. During this period, cage-side observation, detailed clinical observation, observation of the administration site, body weight recording, and post-anatomy evaluation were performed. The experimental results are shown in Table 16.

[0148] Table 16 Results of rabbit vascular stimulation study on the four-component combination of Tevirima

[0149]

[0150] Experimental results: The 10 preparations of the quaternary combination prescription of Tevirima had no irritation risk.

[0151] Example 16: Pharmacokinetic Analysis of the Tevirima Quaternary Composition in Cynomolgus Monkeys

[0152] The quaternary composition (prescription 10) was prepared according to the method of Example 5 and prepared with 0.9% sodium chloride solution for use. The 10 mg / kg dose was intravenously infused into cynomolgus monkeys for 4 hours, and blood was collected at 14 blood sampling points before and after administration. The plasma concentration of tevirimab at each time point was determined and the main pharmacokinetic parameters were calculated. The results are shown in Table 17.

[0153] Table 17 Pharmacokinetic parameters of the quaternary composition in cynomolgus monkeys

[0154]

[0155] The experimental results showed that after intravenous infusion of the quaternary composition, the plasma concentration of tivirima reached a peak at the end of the infusion, and the corresponding C max , AUC (0-∞) They were 5048 ng / mL and 23672 h•ng / mL respectively.

[0156] Example 17: Experimental evaluation of the efficacy of Tivirima against orthopoxvirus

[0157] The gradient diluted tivirima was incubated with Vero cells and orthopoxviruses (such as cowpox virus, vaccinia virus, monkeypox virus, etc.) for 3-5 days, and the anti-orthopoxvirus activity of tivirima was detected by cell micropathogenicity (inhibition) method (EC 50 : half effective inhibitory concentration). A similar system (without virus) was used to detect the cytotoxicity of tivirimab (CC 50 The experimental results are shown in Table 18.

[0158] Table 18 Anti-OPV Activity and Cytotoxicity of Tevirimab

[0159]

[0160] Experimental results: Tevirimab has good antiviral activity against orthopoxviruses (vaccinia virus, cowpox virus, monkeypox virus), and no cytotoxicity was detected in the system.

[0161] In summary, the present invention provides a quaternary composition of tivirima and its preparation method and application, the formula includes active ingredient tivirima, inclusion agent cyclodextrin, solubilizer meglumine and alkaline additive. The weight ratio of each component is tivirima: inclusion agent cyclodextrin: solubilizer meglumine: alkaline additive = 1: (15~30): (0.05~0.2): (0.1~0.2). It is worth mentioning that the ratio and combination of the three excipients are the key to ensure the complete dissolution of the active ingredient tivirima and the safety of the preparation. Compared with the existing technology, the preferred quaternary composition of the present invention is simple to prepare. While improving the solubility of tivirima, the amount of each excipient can meet the requirements of relevant regulations. Combined with the results of allergy, hemolysis and irritation experiments, the quaternary composition has good preparation safety. The results of multiple stability experiments such as influencing factors (high temperature, high humidity, light), re-dissolution / compatibility, acceleration, and long-term show that the quaternary composition has good stability and can be stored, transported and used at room temperature, which is similar to TPOXX. ®Compared with injection, it is more convenient to use. The results of the efficacy experiment show that tevirima has a broad spectrum of anti-orthopoxvirus activity and is non-cytotoxic. Therefore, the present invention provides a new type of tevirima quaternary composition with higher safety, better stability and easy large-scale preparation, which is suitable for commercial development. The composition can be used to treat or prevent viral infections and related diseases caused by orthopoxviruses.

Claims

1. A tevirima composition, the composition comprising at least tevirima, a inclusion agent, a solubilizer and an alkaline additive, wherein the weight ratio is 1: (15-30): (0.05-0.2): (0.1-0.2); The inclusion agent is selected from a combination of one or more of β-cyclodextrin, methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin, 2,3-dihydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin sodium; The solubilizing agent is selected from a combination of one or more of meglumine, hydroxypropyl methylcellulose and copovidone; The alkaline additive is selected from one or more combinations of organic bases or inorganic bases.

2. The tivirima composition according to claim 1, wherein The inclusion agent is a combination of one or more of 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin and 2,3-dihydroxypropyl-β-cyclodextrin; The solubilizer is a combination of one or more of meglumine, hydroxypropyl methylcellulose and copovidone; The alkaline additive is a combination of one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, triethanolamine, and arginine.

3. The tivirima composition according to claim 1 or 2, wherein: The weight ratio of tivirimab, inclusion agent, solubilizer and alkaline additive is 1: (15-20): (0.05-0.15): (0.1-0.15).

4. The tivirima composition according to claim 1, which is in the form of a powder injection or an injection solution.

5. The tivirima composition according to claim 4, when the tivirima composition is an injection, the concentration of tivirima is 5-50 mg / mL.

6. The tivirima composition according to claim 4, when the tivirima composition is an injection, the pH value of the injection is 8-12; when the tivirima composition is a powder injection, after reconstitution at a tivirima concentration of 5-50 mg / mL, the pH value of the obtained solution is 8-12.

7. The tivirima composition according to claim 4 or 6, when the tivirima composition is a powder injection, its reconstitution solvent or compatibility solvent is one or more of water for injection, 0.9% sodium chloride and 5% glucose solution.

8. The method for preparing the tivirima composition according to any one of claims 1 to 7, wherein: The following steps are involved: (1) Take an appropriate amount of water for injection, add the inclusion agent, solubilizer and alkaline additive, and stir to dissolve to obtain a first solution; (2) adding tevirimab to the first solution, adding the remaining water for injection, and stirring until the solid is dissolved to obtain tevirimab injection; or Optionally, the method further comprises step (3): freeze-drying or spray-drying the injection solution obtained in step (2) to obtain a tevirimab powder injection.

9. The preparation method according to claim 8, wherein: Step (2) includes adding tevirimab to the first solution and adding the remaining water for injection, and obtaining tevirimab injection after sterilization.

10. Use of the tivirima composition according to any one of claims 1 to 7 in the preparation of a medicament for treating or preventing viral infection.

11. The use according to claim 10, wherein: The virus is cowpox virus, monkeypox virus or vaccinia virus.

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