Letermovir injection and preparation method thereof

Abstract

The invention belongs to the field of medicines, and provides a letemovir injection and a preparation method thereof, the letemovir injection is a high-concentration letemovir injection for intravenous drip, and the letemovir injection comprises letemovir, an alkaline reagent, a pH regulator, a buffer agent, a non-aqueous solvent for injection and water for injection. According to the injection, the solubility of letemovir is greatly improved, hydroxypropyl-beta-cyclodextrin is abandoned as a solubilizer in a prescription, so that accumulated toxicity of the kidney is avoided, and the injection prescription does not contain other known solubilizers and surfactants for pharmaceutical preparations, so that the medication safety is remarkably improved; the injection can tolerate thermocompression sterilization, no visible foreign matter exists in the sterilized solution and after compatibility, the adverse reaction risk caused by capillary obstruction is avoided or reduced, and the clinical medication safety is remarkably improved; the preparation process is simple, production amplification is easy to realize, and the method has great application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and particularly relates to a letermovir injection and a preparation method thereof. BACKGROUND

[0002] Letermovir is a highly active drug for responding to HCMV infection, and its chemical name is (4S)-2-(8-fluoro-3-(2-methoxy-5-(trifluoromethyl)phenyl)-2-(4-(3-methoxyphenyl)piperazin-1-yl)-3,4-dihydroquinazolin-4-yl)acetic acid, its molecular formula is C 29 H 28 F4N4O4, and its molecular weight is 572.55, and its structural formula is as follows: .

[0003] Letermovir is a new non-nucleoside anti-cytomegalovirus drug, which was first developed by Aicuris Company in Germany. The tablet and injection were first approved for marketing by the US FDA in 2017, and the trade name is Prevymis ® , which became the first new anti-CMV drug approved for marketing by the FDA in 15 years. In China, letermovir tablets were approved for marketing by NMPA in January 2022, and the trade name is Prevymis, which is used for preventing cytomegalovirus infection and cytomegalovirus disease in adult recipients with positive cytomegalovirus serum after receiving allogeneic hematopoietic stem cell transplantation. + ] [R

[0004] The letermovir compound patent and the preparation patent have expired in 2024, and the pharmaceutical companies or research units at home and abroad have carried out relevant research on the letermovir compound and the preparation.

[0005] The patent CN119015222A a letermovir injection and a preparation method thereof directly mixes letermovir, hydroxypropyl betacyclodextrin and sodium hydroxide in the form of dry powder, then adds injection water with a temperature of 40-70 DEG C to stir and dissolve, filters, fills, sterilizes, and obtains the letermovir injection. The patent CN110433166A a pharmaceutical preparation containing an antiviral active dihydroquinoline derivative contains at least one solubilizing agent selected from cyclodextrin, lysine and arginine to obtain a pharmaceutical composition for intravenous administration. The prescription contains cyclodextrin, which has the risk of kidney accumulation safety.

[0006] The original research product Prevymis ® (Prevymis ®The hydroxypropyl betacyclodextrin may accumulate in the kidney when the patients with moderate or severe renal impairment (CrCl < 50 mL / min) are administered with the product, which may cause safety problems and limit the use of the product in clinical patients, in particular, the CMV prevention demand of high-risk groups (such as kidney solid organ transplantation) is greatly reduced or not covered; secondly, the recommended daily dose of hydroxypropyl betacyclodextrin in the original letimovir injection is 3600 mg, which has exceeded the FDA non-active substance dosage range, which may increase the risk of local toxicity, allergy, hemolysis and organ damage, etc. Moreover, the pharmaceutical composition containing letimovir and solubilizers such as cyclodextrin, especially hydroxypropyl betacyclodextrin (HP-β-CD) has a tendency to cause particle problems when mixed with compatible solutions such as water for injection, which requires additional post-treatment before the intended use, for example, the pharmaceutical composition needs to be filtered before use. Therefore, the development of a safer and higher concentration letimovir injection becomes a technical problem to be solved in the art. SUMMARY

[0007] The purpose of the present application is to overcome the defects of the prior art, provide a high-concentration letimovir injection for intravenous infusion and a preparation method thereof, the injection liquid comprising letimovir, an alkaline reagent, a pH adjusting agent, a non-aqueous solvent for injection, a buffer, and water for injection. The solubility of letimovir is greatly improved, and hydroxypropyl betacyclodextrin is abandoned as a solubilizer in the prescription, thereby avoiding kidney accumulation toxicity. The injection liquid prescription does not contain other known solubilizers and surfactants for pharmaceutical preparations, significantly improving the safety of drug use. The injection can withstand heat sterilization, and there is no visible foreign matter in the solution after sterilization and after compounding, avoiding or reducing the risk of adverse reactions caused by capillary obstruction, significantly improving the safety of clinical drug use. The preparation process is simple, easy to realize production scaling-up, and has great application value.

[0008] The present application provides a letimovir injection liquid, which comprises a pH adjusting agent and water for injection, and further comprises letimovir, an alkaline reagent, a non-aqueous solvent for injection, and a buffer.

[0009] The content of the letimovir is 1-100 mg / mL, the molar ratio of the letimovir to the alkaline reagent is 1:(0.5-10), preferably 1:(1-5); the mass ratio of the letimovir to the buffer is 1:(0.05-10), preferably 1:(0.3-6.0); the volume concentration of the non-aqueous solvent for injection in the system is 1-60%, preferably 5%-30%, and the rest is water for injection.

[0010] The non-aqueous solvent for injection is selected from one or more of anhydrous ethanol, polyethylene glycol (PEG), propylene glycol, glycerol, isopropyl alcohol, polyethylene glycol tetrahydrofuran, wherein the polyethylene glycol is selected from one or more of PEG 300, PEG 400. The use of non-aqueous solvent for injection not only has a certain solubilization effect, but also increases the stability of the sample.

[0011] The basic reagent comprises one or more of inorganic base, inorganic salt, organic base and basic amino acid, wherein the inorganic base is selected from sodium hydroxide; the inorganic salt is selected from one or more of sodium carbonate, potassium carbonate; the organic base is selected from one or more of choline hydroxide, meglumine, ethylenediamine, triethanolamine, tromethamine; the basic amino acid is selected from one or more of arginine, lysine, histidine. The addition of the basic reagent greatly increases the solubility of the poorly soluble drug letmovir.

[0012] The buffer is selected from one or more of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), phosphate buffered saline (PBS), tris-hydroxymethyl aminomethane (Tris). The phosphate buffered saline is composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, and the molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is (1-35):1, preferably 20:1. The use of the buffer in the prescription greatly improves the stability of clinical compounding.

[0013] The pH regulator comprises one or more of basic pH regulator and acidic pH regulator, wherein the basic pH regulator is selected from one or more of sodium hydroxide, sodium carbonate, potassium carbonate, preferably sodium carbonate; the acidic pH regulator is selected from one or more of citric acid, acetic acid, hydrochloric acid, phosphoric acid, preferably citric acid.

[0014] The injection prepared by containing the basic reagent, the non-aqueous solvent for injection and the buffer can withstand heat pressure sterilization, and after sterilization, it is a clear solution, no visible foreign matter, no insoluble particles are qualified, the preparation and compounding stability are good, there is no visible transparent foreign particle or precipitate, the risk of adverse reactions caused by capillary obstruction is avoided or reduced, and the safety of the preparation is significantly improved.

[0015] Further, the present application provides a preparation method of the letmovir injection, which specifically comprises the following steps: (1) mixing the basic reagent with 20%-80% of the total volume of water for injection to make the solution pH≥8, to obtain a basic solution; weighing the prescription amount of letmovir and adding it to the basic solution, heating and stirring until completely dissolved to obtain a clear solution, denoted as solution 1; (2) adding the non-aqueous solvent for injection into solution 1 and stirring to obtain a clear solution under room temperature conditions; then adding the buffer and stirring to dissolve, denoted as solution 2; (3) Adjust the pH of solution 2 to 7.0-8.5 with a pH regulator, and then make up the volume or weight, and filter through a 0.22 μm filter membrane to obtain solution 3; (4) The solution 3 is divided into ampoules or vials, sealed or plugged, and then autoclaved at 121℃ for 15 min to obtain letemovir injection.

[0016] Further, the step (1) can also be to weigh the prescribed amount of letemovir and alkaline reagent, add 20-80% of the total volume of water for injection, and heat and stir to obtain a solution, which is solution 1.

[0017] The heating temperature in the step (1) is 25-80℃, and the subsequent materials can be added directly without waiting for the solution to cool down after the previous step.

[0018] Preferably, the letemovir injection prepared by the present application has a content of 1-100 mg / mL, preferably 10-100 mg / mL, a pH of 7.0-8.5, and a transmittance of >90%.

[0019] The obtained letemovir injection is a new type of non-nucleoside anti-cytomegalovirus drug, which is suitable for preventing cytomegalovirus infection and cytomegalovirus disease in adult recipients of allogeneic hematopoietic stem cell transplantation (HSCT) who are seropositive for cytomegalovirus (CMV) [R + ].

[0020] In specific applications, the letemovir injection provided by the present application can be combined with a compatible solution for drip infusion. The compatible solution is selected from one of sterile water for injection, 0.9% sodium chloride injection, 5% glucose injection, 5% glucose and 0.9% sodium chloride injection, and sodium lactate Ringer's injection, and as one of the preferred embodiments, 5% glucose injection or 0.9% sodium chloride injection is preferred.

[0021] Compared with the prior art, the present application has the following beneficial effects: (1) Greatly improve the solubility The present application uses letemovir and alkaline reagent to form a salt, which improves the solubility of letemovir. On this basis, a small amount of non-aqueous solvent for injection is added, which can significantly improve the stability of letemovir. The letemovir injection prepared by the present application has a concentration of up to 100 mg / mL, which significantly improves the solubility of the drug compared with the commercially available injection (20 mg / mL).

[0022] (2) Significantly improve safety The addition of a large amount of hydroxypropyl betacyclodextrin of the original research has exceeded the FDA non-active material usage range, and there is a safety of kidney accumulation; according to the growth standard of children under 7 years old of the People's Republic of China health industry standard: the body weight value of 2-year-old children P50 is 11.9 kg, the ADI of HP-β-CD injection administration route is 200 mg / kg / day, so the maximum daily acceptable exposure is 2.38 g, and the value in letmovir injection is 3.6 g, which is much higher than the maximum daily acceptable exposure, and a safer letmovir injection is needed to meet the clinical needs, such as children's medication.

[0023] The application discards the use of cyclodextrin, adopts innovative solubilization technology, the prescription does not contain other known solubilizers and surfactants for pharmaceutical preparations, the irritability is reduced, the kidney accumulation toxicity is avoided, the use risk of patients with moderate or severe renal function impairment is significantly reduced, and the application is particularly suitable for preventing CMV disease patients of adult kidney transplant recipients (donor cytomegalovirus [CMV] positive / recipient CMV negative [D+ / R-]) in high risk, and has better safety.

[0024] The injection prepared by the application can withstand heat pressure sterilization, and after sterilization, it is a clear solution, no visible foreign matter, no insoluble particles qualified, good stability, no visible transparent foreign particle or precipitate, avoids or reduces the risk of adverse reactions caused by capillary obstruction, and significantly improves the safety of the preparation.

[0025] (3) improve compliance The letmovir injection prepared by the application can be directly diluted by 0.9% sodium chloride injection or 5% glucose injection and other compatible solutions when used clinically, the concentration after dilution is 1-2 mg / mL, the stability after compounding is good, it can be directly intravenous infusion, no additional post-treatment is needed before predetermined use, and the dosing is more accurate, which improves the clinical use compliance.

[0026] (4) low cost, easy to produce The auxiliary materials in the prescription are simple and cheap, the process is simple, only stirring, mixing, dissolving, filtering and other processes are needed, no special equipment is needed, process control is easy, industrialization is easy, the preparation difficulty of letmovir injection is effectively reduced, the occurrence of bubbles, wall sticking, opalescence and precipitation in the preparation process is avoided, the operation is simple, and ordinary injection production line can be realized, and the industrial feasibility is high.

[0027] Therefore, the preparation method provided by the application has simple process, low production cost, is easy to realize, and has great application value. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The appearance morphology diagram of the letmovir injection prepared in Example 1 is shown. Figure 2 The content determination high performance liquid chromatogram of the letimovir injection obtained in Experimental Example 1 is shown. Figure 3 The related substance determination high performance liquid chromatogram of the letimovir injection obtained in Experimental Example 1 is shown. DETAILED DESCRIPTION

[0029] The following examples can be used to illustrate the present application, but are not used to limit the scope of the present application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art, or according to the product instruction is used.

[0030] Example 1 A letimovir injection, the prescription is as follows: The preparation method comprises the following steps: (1) The prescription amount of arginine and letimovir is weighed, 80 mL of water for injection is added, and the mixture is heated to dissolve in a 60 °C water bath, and stirred until completely dissolved, and is recorded as solution 1; (2) The prescription amount of PEG400 is added to solution 1, and stirred to dissolve under room temperature conditions; the prescription amount of disodium hydrogen phosphate and sodium dihydrogen phosphate is added, and stirred to dissolve, and is recorded as solution 2; (3) The pH value of solution 2 is adjusted to 8.0 with 1 mol / L acetic acid solution, and the volume is fixed to 100 mL, and the solution is filtered with a 0.22 μm pore size filter membrane to obtain solution 3; (4) Solution 3 is divided into vials, 5 mL per vial, and sterilized at 121 °C for 15 min, to obtain the letimovir injection.

[0031] As shown in Figure 1 , the letimovir injection obtained in Example 1 is clear and transparent, and the pH value is 8.0.

[0032] Example 2 A letimovir injection, the prescription is as follows: The preparation method comprises the following steps: (1) The prescription amount of meglumine and letimovir is weighed, 700 mL of water for injection is added, and the mixture is heated to dissolve in a 50 °C water bath, and stirred until completely dissolved, and is recorded as solution 1; (2) The prescription amount of PEG300 is added to solution 1, and stirred to dissolve under room temperature conditions; the prescription amount of disodium hydrogen phosphate and sodium dihydrogen phosphate is added, and stirred to dissolve, and is recorded as solution 2; The subsequent steps are the same as those in Example 1.

[0033] The letermovir injection obtained in Example 2 has a specification of 5 mL: 240 mg, and is clear and transparent in appearance, with a pH of 8.1.

[0034] Example 3 A letermovir injection, with the following prescription: The preparation method comprises the following steps: (1) The prescribed amount of letermovir and sodium carbonate is weighed, 700 mL of water for injection is added, and heating is performed in a water bath at 50°C until complete dissolution, and this is recorded as solution 1; (2) The prescribed amount of propylene glycol is measured and added to solution 1, and stirring is performed under room temperature conditions until complete dissolution; the prescribed amount of Tris is added, and stirring is performed until complete dissolution, and this is recorded as solution 2; (3) The pH value of solution 2 is adjusted to 7.5 using a 1 mol / L acetic acid solution, a fixed volume of 1000 mL is obtained, and filtration is performed using a 0.22 μm pore size filter membrane to obtain a solution, which is recorded as solution 3; (4) Solution 3 is divided into vials, 5 mL per vial, and sterilization is performed at 121°C for 15 min, thereby obtaining a letermovir injection.

[0035] The letermovir injection obtained in Example 3 has a specification of 5 mL: 240 mg, and is clear and transparent in appearance, with a pH of 7.6.

[0036] Example 4 A letermovir injection, with the prescription shown in Table 1: Table 1 Prescription composition of buffer Preparation method: According to the above prescription composition, the type (sodium carbonate is selected) and amount of the basic reagent are fixed, a 0.5M citric acid solution is used to adjust the pH, and 100 mL of an injection is prepared according to the process steps of Example 3, and is sequentially named F0-F6.

[0037] Through investigation of the type (phosphate buffer, HEPES and Tris buffer) and amount of the buffer, the experimental results are as follows: (1) The above preparations F0-F6 are sterilized at 121°C for 15 min, and after sterilization, they are all clear solutions, and no precipitate or precipitate is observed; however, the pH of preparation F0 changes greatly before and after sterilization (for example, 7.8→6.0), and the pH of preparations F1-F6 does not change significantly before and after sterilization.

[0038] (2) Preparations F0-F6 are subjected to three cycles of freeze-thaw cycles, and the test results show that no precipitate or precipitated crystals are observed.

[0039] (3) Formulations F0-F6 were respectively mixed with 5% glucose injection, and after mixing, the mixtures were placed at room temperature for 4 hours. No precipitation was observed in the solution of F0, and no precipitation was observed in the other mixtures. After being placed for 24 hours, no precipitation was observed in the mixtures of F1-F6, which indicated that the mixed stability of the formulation after adding the buffer was good.

[0040] Therefore, the pH value of the formulation before and after sterilization is more stable after adding the buffer in the prescription, and the mixed stability of the formulation is significantly improved.

[0041] Example 5: An injection of letermovir was prepared according to the prescription shown in Table 2. Table 2: Prescription composition of the injection of letermovir Preparation method: Formulations F7-F12 were respectively prepared according to the above prescription and the preparation method in Example 3. The corresponding alkaline reagent was weighed in a clean container, and then dissolved in water with a target volume of about 70%. The prescription amount of letermovir was added to make the final API concentration 48 mg / mL, and then stirred and dissolved. Subsequently, the injection non-aqueous solvent (F12 batch without injection non-aqueous solvent) was added to achieve the target concentration. After the buffer was completely dissolved, the pH of the solution was adjusted to the appropriate range using a pH adjuster, and finally the injection water (appropriate amount) was used to add the formulation F7-F12 to the target volume.

[0042] The stability of the injection was investigated by fixing the type and amount of the added buffer, changing different injection non-aqueous solvents and alkaline reagents, and the experimental results are as follows: (1) After the injection was sterilized at 121°C for 15 minutes, all the formulations were clear without precipitation or turbidity, and no precipitate or precipitate was observed. The pH value after sterilization did not change significantly compared with that before sterilization.

[0043] (2) The freeze-thaw cycle test and low-temperature cycle test were respectively performed on F7-F12, and the results showed that the formulations of F7-F11 remained stable after the above conditions, no crystallization was observed, and the color, pH value, clarity, content and related substances of the solution did not change significantly. The sample of F12 batch was observed to precipitate after two rounds of freeze-thaw cycle, and the stability was poorer than that of other batches. Subsequent mixed stability was not investigated. The present application found that the addition of the injection non-aqueous solvent increased the stability of the formulation.

[0044] (3) The pH values of the preparations F7-F11 are maintained between 7.5-8.0 after being mixed with 5% glucose injection to the clinical concentration, and all of them are clear solutions within 24 hours without precipitation or turbidity increase. Unexpectedly, the preparations F7-F11 are all clear solutions without precipitation or turbidity increase within 72 hours, and have good stability.

[0045] The above results show that the injection prepared by adding a buffer to the prescription, selecting different non-aqueous solvents for injection, and arbitrarily combining different alkaline reagents has good quality and good stability. In the present application, the addition of a buffer to the prescription can not only maintain the pH value of the preparation stable before and after sterilization, but also significantly improve the compatibility stability of the injection and the convenience of clinical use.

[0046] Comparative Example 1 The preparation method comprises the following steps: (1) Weigh the prescription amount of letromovir, sodium chloride and sodium hydroxide, add an appropriate amount of water, and place in a 50°C water bath for heating; (2) To the solution obtained in (1), add hydroxypropyl betacyclodextrin and sodium chloride in sequence, dissolve, and then adjust the pH value to 7.5 with 1 mol / L hydrochloric acid; (3) The volume of the solution obtained in (2) is made up to 240 mL, and filtered through a 0.22 μm pore size filter membrane; (4) The solution obtained in (3) is divided into vials, sterilized at 121°C for 15 min, and letromovir injection is obtained.

[0047] Comparative Example 2 The existing patent CN115515565A contains a pharmaceutical composition of 2-[(4S)-8-fluoro-2-[4-(3-methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-4H-quinazolin-4-yl]acetate and sodium ion, and the prescription contains polyalkoxy compound (poloxamer, PVP PF12), excipient (phenylalanine), sodium ion and buffer. According to the patent example 4 (lyophilization after 7 days and reconstitution in Ringer's lactate solution), the preparation method comprises four steps of preliminary drying, preparation of solution and solubility analysis, lyophilization and reconstitution in Ringer's lactate.

[0048] The inventors prepared two letromovir free base samples (20 mg / mL) according to the example 4 of the patent CN115515565A, and prepared the composition, detected the content and related substances, and investigated the compatibility stability. The patent has the following obvious defects: (1) the composition of the present application needs to be prepared into a freeze-dried powder injection by a freeze-drying process, and is not resistant to high-temperature sterilization, and is turbid after sterilization, and the sterile level is difficult to guarantee.

[0049] After the non-aqueous solvent for injection is creatively added in the preparation, the present application can tolerate hot pressure sterilization (121℃ 15min), and the guarantee degree of sterile level is high.

[0050] (2) the last step of the preparation method is to be dissolved in Ringer's lactate solution to obtain a final concentration of 20mg / mL, and the related substances are detected according to the detection method of related substances in Experimental Example 1, and the result is that the total impurities (%) are 0.96, which is obviously higher than those in each embodiment of the present application.

[0051] (3) the compatible solution such as 5% glucose injection is compatible to the clinical use concentration of the original research product, and is placed at room temperature, and the compatible stability within 24h is investigated, and the related substances increase significantly at 6h, and the precipitation phenomenon appears at 12h, and the stability is poor.

[0052] Comparative Example 3: a letmovir injection, the prescription is as follows: The preparation method comprises the following steps: (1) the prescription amount of sodium carbonate is weighed, added into 70mL of water for injection, and dissolved completely to obtain an alkaline solution, which is recorded as solution 1; (2) the prescription amount of letmovir is weighed, added into solution 1, and placed in a 50℃ water bath for heating until completely dissolved, which is recorded as solution 2; (3) 0.5mol / L citric acid or 1mol / L sodium carbonate solution is used to adjust the pH value of solution 2 to 7.5, a fixed volume is added to 100mL, and then filtered through a filter membrane with a pore size of 0.22μm to obtain a solution, which is recorded as solution 3; (4) solution 3 is divided into a westlin bottle, and sterilized by hot pressure at 121℃ for 15min to obtain a letmovir injection.

[0053] Comparative Example 4: a letmovir injection, the prescription is as follows: The preparation method comprises the following steps: (1) the prescription amount of sodium hydroxide is weighed, added into 400mL of water for injection, and stirred until completely dissolved to obtain solution 1; (2) the prescription amount of letmovir is weighed, added into solution 1, and placed in a 50℃ water bath for heating and stirring until completely dissolved to obtain solution 2; (3) the prescription amount of glycerol is weighed, added into solution 2, and placed in a 50℃ water bath for heating and stirring until dissolved to obtain a clear solution, which is recorded as solution 3; (4) Adjust the pH value of solution 3 to 7.8 with 1 mol / L acetic acid solution, and dilute to 500 mL. Filter through a 0.22 μm pore size filter membrane to obtain solution 4; (5) Divide solution 4 into 5 mL per vial, and sterilize at 121°C for 15 min to obtain letimovir injection.

[0054] Experimental Example 1 Determination of content and related substances The determination of content and related substances is carried out according to the following method.

[0055] 1. Determination method of content Chromatographic conditions: octadecylsilane-bonded silica gel is used as the filler (Agilent Eclipse XDB-C18 250 mm x 4.6 mm, 5 μm is recommended); 10 mM phosphate buffer (take 1.36 g of potassium dihydrogen phosphate, add 1000 mL of water, shake well; adjust the pH value to 2.5 with phosphoric acid) is used as the mobile phase A, and water-acetonitrile (10:90) is used as the mobile phase B; the detection wavelength is 234 nm; the injection volume is 10 μl.

[0056] Reference substance solution: take about 25 mg of the reference substance, dilute to the mark in a 25 mL volumetric flask with the solvent, shake well; accurately take 1 mL, dilute to the mark in a 20 mL volumetric flask with the solvent, shake well.

[0057] Test sample solution: accurately take 5 mL of the product in a 100 mL volumetric flask, dilute to the mark with the solvent, shake well, accurately take 1 mL, dilute to the mark in a 10 mL volumetric flask with the solvent, shake well.

[0058] Solvent: 10% acetonitrile 2. Test method of related substances Chromatographic conditions: octadecylsilane-bonded silica gel is used as the filler (Sunniest C18 specification: 250 x 4.6 mm, 5 μm); 10 mM phosphate buffer (take 1.36 g of potassium dihydrogen phosphate, add 1000 mL of water, shake well; adjust the pH value to 2.5 with phosphoric acid) is used as the mobile phase A, and water-acetonitrile (10:90) is used as the mobile phase B; the detection wavelength is 234 nm; the injection volume is 20 μl.

[0059] Test sample solution: accurately take 1 mL of the product, dilute to the mark in a 20 mL volumetric flask with the solvent, shake well.

[0060] Table 3 Gradient elution table for test method of related substances of letimovir injection The content, related substance detection method according to the above was used to detect examples 1-3, preparations F7-11 and comparative examples 1-2, and the results are shown in table 4; and the prepared letmovir injection was subjected to visible foreign matter detection according to the Chinese Pharmacopoeia.

[0061] Note: The clarity detector model is YB-2, and the manufacturer is Tianda Tianfa Science and Technology Co., Ltd.

[0062] Table 4: Detection results of content, related substances, etc. Figure 2 The content determination high performance liquid chromatogram of letmovir injection obtained in example 1 is shown; Figure 3 The related substance determination high performance liquid chromatogram of letmovir injection obtained in example 1 is shown.

[0063] It can be seen that: (1) the related substance result of the injection prepared in comparative example 1 is obviously higher than that of the examples, and there are visible particles under the clarity detector, which has great risk; (2) the pH of the injection prepared in comparative example 2 increases (7.8→9.0) by about 1 unit after sterilization, while the pH values of examples 3-5 and preparation F7-11 have no obvious change before and after sterilization, so the addition of buffer in the preparation improves the stability of the preparation; (3) examples 1-3 and preparation F7-11 have no visible foreign matter and clear solution after sterilization, which meets the standard of the Chinese Pharmacopoeia 2025 edition, and the content and related substances are within the standard, so the quality of the injection is good; the related substance of the injection prepared in comparative example 1 is obviously higher than that of the examples, and there are obvious visible particles under the clarity detector, which has poor stability and great safety risk in clinical use.

[0064] Experimental example 2: compatibility stability The samples prepared in examples 1-3, F8-9 and comparative examples 1 and 2 were diluted to the clinical use concentration with 0.9% sodium chloride injection and 5% glucose injection respectively, and placed at room temperature (25℃) for 24 h to investigate the stability under clinical use conditions. The dilution stability was investigated by combining the content and related substance analysis detection methods in experimental example 1, and the results are shown in tables 5-7.

[0065] The prepared injection was diluted, and placed at room temperature (25℃) for 24 h, and the properties, pH, content and related substances were detected, and the results are shown in tables 5-7.

[0066] Table 5: pH value determination results of the diluted solution (5% glucose injection) Note: " / " means not detected, because the comparative example 1, comparative example 2 compatible solution 24h have been precipitated, not detected.

[0067] Table 6 Content determination results of the compatible solution of example 1 Table 7 Determination results of related substances of the compatible solution of example 1 The injection solutions prepared in examples 1-3, F8-9 were respectively mixed with 5% glucose injection solution, and no crystals were precipitated after being placed at room temperature (25℃) for 24h. As shown in Table 5, the pH values of the compatible solutions were all in the range of 7.5-8.0.

[0068] From the above results, it can be seen that the content and related substances of the compatible solution prepared by mixing example 1 with 5% glucose injection solution and 0.9% sodium chloride injection solution respectively and placing at room temperature (25℃) for 24h did not change significantly, indicating that the prepared letromovir injection solution has good compatibility stability.

[0069] Experimental example 3 Freeze-thaw cycle test The letromovir injection solution prepared in example 1 was subjected to freeze-thaw cycle test, which should include 3 cycles, each cycle being placed at-20 to-18℃ for 2 days, and then at 40℃ for 2 days. After each cycle, samples were taken for detection. The results are shown in Table 8.

[0070] Table 8 Freeze-thaw cycle test results The injection solution was detected for its properties using a clarity detector, and no precipitation was observed after 3 cycles. As shown in Table 8, compared with 0 days, the pH value, clarity, color, content, and related substances did not change significantly, indicating that the letromovir injection solution can withstand freeze-thaw test.

[0071] Experimental example 4 Stability investigation The letromovir injection solutions of preparations F9-F11 were subjected to stability study, including influence factor test, long-term test, and accelerated test, to investigate the changes in properties and pH value, and to determine the content of letromovir and related substances according to experimental example 1 above. The results are shown in Table 9.

[0072] Influence factor test: 60℃±2℃; long-term test: 25℃±2℃ / 60%RH±5%RH; accelerated test: 40℃±2℃ / 75%RH±5%RH.

[0073] Table 9 Stability investigation results Conclusion: From the above table, it can be seen that the preparations F9-F11 of letemovir injection have no significant changes in the conditions of influencing factor test, long-term test and accelerated test, respectively for 30 days and 6 months in terms of properties, pH, content, related substances and other indexes.

[0074] The above results show that the letemovir injection prepared by the present application has good stability during the stability investigation period. The temporary storage condition of letemovir injection is room temperature, and the temporary effective period is 2 years.

[0075] For those skilled in the art, the specific embodiments are only exemplary descriptions of the present application, and obviously the specific implementation of the present application is not limited by the above method. Various non-essential improvements or direct application of the concept and technical solutions of the present application to other occasions without improvement are within the protection scope of the present application.

Claims

1. A letimovir injection solution comprising a pH adjusting agent, water for injection, characterized in that, The injection solution also comprises letimovir, an alkaline reagent, a non-aqueous solvent for injection, a buffer; wherein the content of letimovir is 1-100 mg / mL, the molar ratio of letimovir to alkaline reagent is 1:0.5-10, the mass ratio of letimovir to buffer is 1:0.05-10, the volume concentration of non-aqueous solvent for injection in the system is 1-60%, and the rest is water for injection; the non-aqueous solvent for injection is one or more of anhydrous ethanol, polyethylene glycol, propylene glycol, glycerol, isopropyl alcohol, and polyethylene glycol tetrahydrofuran.

2. The letimovir injection according to claim 1, characterized in that, The molar ratio of letimovir to alkaline reagent is 1:1-5; the mass ratio of letimovir to buffer is 1:0.3-6.0; the volume concentration of non-aqueous solvent for injection in the system is 5%-30%.

3. The letimovir injection of claim 1, wherein, The alkaline reagent comprises one or more of inorganic base, inorganic salt, organic base, and basic amino acid, wherein the inorganic base is selected from sodium hydroxide; the inorganic salt is selected from one or more of sodium carbonate and potassium carbonate; the organic base is selected from one or more of choline hydroxide, meglumine, ethylenediamine, triethanolamine, and tromethamine; and the basic amino acid is selected from one or more of arginine, lysine, and histidine.

4. The letimovir injection of claim 1, wherein, The polyethylene glycol is selected from one or more of PEG300 and PEG400.

5. The letimovir injection of claim 1, wherein the letimovir injection is a solution. The buffer is selected from one or more of 4-hydroxyethylpiperazine ethanesulfonic acid, phosphate buffer, and tris(hydroxymethyl)aminomethane, wherein the phosphate buffer is composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, and the molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1-35:1; The pH adjuster comprises one or more of alkaline pH adjuster and acidic pH adjuster, wherein the alkaline pH adjuster comprises one or more of sodium hydroxide, sodium carbonate, and potassium carbonate; and the acidic pH adjuster comprises one or more of citric acid, acetic acid, hydrochloric acid, and phosphoric acid.

6. The letimovir injection of claim 5, wherein, The molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate in the phosphate buffer is 20:

1.

7. The letimovir injection according to any one of claims 1 to 6, characterized in that, The content of the letimovir injection is 10-100 mg / mL, the pH is 7.0-8.5, and the transmittance is >90%.

8. The letimovir injection of claim 7, wherein, The letimovir injection can be used after being compatible with a compatible solution, wherein the compatible solution is selected from one of water for injection, 0.9% sodium chloride injection, 5% glucose injection, 5% glucose and 0.9% sodium chloride injection, and sodium lactate Ringer's injection.

9. A method for preparing letimovir injection, characterized by, The method comprises the following steps: (1) mixing an alkaline reagent with 20%-80% water for injection to prepare a total volume to make the pH of the solution ≥8 to obtain an alkaline solution; weighing a prescription amount of letimovir and adding it to the alkaline solution; heating and stirring until complete dissolution to obtain a clear solution, which is denoted as solution 1; (2) adding a non-aqueous solvent for injection to solution 1 and stirring to obtain a clear solution under room temperature conditions; then adding a buffer and stirring to dissolve it, which is denoted as solution 2; (3) adjusting the pH of solution 2 to 7.0-8.5 with a pH adjuster, and then performing constant volume or constant weight, and filtering through a 0.22 μm filter membrane to obtain a solution, which is denoted as solution 3; (4) The solution 3 is divided into ampoules or vials, sealed or plug-rolled, and autoclaved at 121℃ for 15 minutes to obtain letmovir injection.

10. The method for preparing lemetmovir injection according to claim 8, characterized in that, The prescription amount of letmovir and alkaline reagent is weighed in step (1), 20-80% of the total volume of injection water is added, and heated and stirred to obtain a solution, which is solution 1; the heating temperature in step (1) is 25-80℃, and the subsequent material can be directly added without waiting for the solution to cool down after the previous step.

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