Compound amino acid injection 20AA composition and preparation method thereof

Abstract

The invention provides a compound amino acid injection 20AA composition and a preparation method thereof.The preparation method comprises the steps that S1, in the whole preparation process, the whole process is away from light, and red light is turned on for protection; s2, under the nitrogen atmosphere, adding the first group of liquid medicine into water, stirring for the first time, cooling for the first time, adding the second group of liquid medicine, stirring for the second time, cooling for the second time, adding the third group of liquid medicine, stirring for the third time, fixing the volume with water, filtering, filling and sterilizing to obtain the compound amino acid injection 20AA composition. The preparation method comprises the following steps: pre-treating insoluble and light amino acid in a pulping manner, and then transferring the pre-treated amino acid into a mixing tank, so that the dissolution of the amino acid can be promoted, and meanwhile, the loss caused by material flying can be prevented; components are dissolved step by step by adopting programmed cooling, and light-sensitive amino acids are protected by adopting whole-course red light, so that the degradation of the amino acids is reduced. The safety and the quality stability of the prepared product are better than those of similar products.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a compound amino acid injection 20AA composition and a preparation method thereof. Background Art

[0002] Compound amino acid injection (20AA) is a sterile aqueous solution prepared from 20 kinds of amino acids and excipients. It is clinically used for parenteral nutrition to provide amino acids for patients with severe liver dysfunction and those who are about to develop or have developed hepatic encephalopathy.

[0003] Since amino acids are unstable in aqueous solution, they are easily oxidized to produce a series of complex substances that are harmful to the human body. The production of compound amino acid injections at home and abroad mainly adds sulfite (sodium bisulfite or sodium pyrosulfite, etc.) antioxidants, and adds activated carbon for adsorption during the production process to improve the stability of the drug, improve the clarity of the drug solution, and ensure that the indicators of the final product meet national standards.

[0004] Sulfite antioxidants have many adverse reactions, the most common toxic side effect is sulfite allergy (especially in asthma patients), the symptoms are bronchospasm, wheezing, dyspnea, malignant laryngeal edema, hypotension, shock and even death. Therefore, countries around the world have restricted their use in medicines and foods.

[0005] In the draft for comments on the consistency evaluation of injections released by CDE in 2017, it was proposed that activated carbon is not recommended for injection production. Activated carbon can adsorb impurities in the drug solution, but it also has the risk of introducing impurities. Due to the diverse sources of raw materials and production processes of activated carbon, it may contain different elemental impurities. Some elemental impurities are toxic, including neurotoxicity and nephrotoxicity. For example, long-term exposure to lead can lead to intellectual retardation in children. Therefore, activated carbon should be carefully selected during use.

[0006] In the prior art, in order to reduce the amount of antioxidants in the recurrent amino acid injection, most of them are to avoid oxidation of amino acids by controlling the oxygen content in the production process. For example, the patent with publication number CN109381423A discloses a method for preparing a pharmaceutical composition containing 19 kinds of amino acids, which comprises 19 kinds of amino acids, a pH regulator and water, and controls the content of dissolved oxygen in the injection and the headspace oxygen in the injection storage container, so that it can be stably present under the condition of not containing pyrosulfite or sulfite antioxidants, thereby completely solving the harm of pyrosulfite or sulfite antioxidants to the human body. However, it does not solve the problem of light-sensitive amino acids, especially aromatic amino acids such as tryptophan and histidine, which contain double bonds in their molecular structures and are easily degraded by ultraviolet light.

[0007] Aromatic amino acids such as tryptophan and histidine can absorb ultraviolet rays at 250-320nm, especially acetyltyrosine and tryptophan, which have strong absorption near 280nm, which is the main cause of amino acid photodegradation. The energy absorbed by acetyltyrosine will also be quickly transferred to tryptophan, promoting the latter's photodegradation chemical reaction. Under ultraviolet irradiation, tryptophan undergoes photooxidation reaction, which is also the main cause of yellowing of the liquid medicine.

[0008] Therefore, it is worthwhile to explore the pharmaceutical composition of twenty kinds of amino acids and its preparation process without adding sulfite antioxidants, edetic acid and its salts, metal ion chelating agents, pH regulators, etc. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide a compound amino acid injection and a preparation method thereof. The composition is composed of 20 kinds of amino acids, and the prescription does not contain sulfite antioxidants, edetic acid and its salts, metal ion chelating agents, and pH regulators, and contains other acid radicals such as hydrochloride, acetate, sulfate, citrate, maleate, and fumarate. The preparation method includes pre-treating the insoluble and light amino acids by pulping, and then transferring them to a batching tank, which can promote their dissolution while preventing the material from flying and causing losses; the preparation process uses red light protection throughout, and each component is dissolved in steps by program cooling, protecting light-sensitive amino acids and reducing their degradation. The acid salt-protected ornithine and lysine significantly reduce the impurity content of ornithine and lysine degradation, and ornithine and lysine are more stable; the safety and quality stability of the product thus obtained are better than similar products.

[0010] The technical solution of the present invention is achieved as follows: The present invention provides a method for preparing a compound amino acid injection 20AA composition, the preparation method comprising:

[0011] Step S1: During the entire preparation process, avoid light and turn on red light for protection;

[0012] Step S2: adding 20 kinds of amino acids into water in three groups at 30° C. to 75° C. under a nitrogen atmosphere to obtain a compound amino acid injection 20AA composition;

[0013] The first group of solutions includes leucine, isoleucine, valine, aspartic acid, phenylalanine, threonine, and methionine;

[0014] The second group of solutions included lysine acetate, arginine, histidine, alanine, glycine, proline, serine, and glutamic acid;

[0015] The third group of solutions includes asparagine, N-acetyltyrosine, ornithine hydrochloride, tryptophan and N-acetylcysteine.

[0016] On the basis of the above technical solution, preferably, the salt in the ornithine hydrochloride is selected from at least one of hydrochloride, sulfate, oxalate, acetate, malate, citrate, maleate, fumarate and tartrate.

[0017] On the basis of the above technical solution, preferably, the salt in the lysine acetate is selected from at least one of hydrochloride, sulfate, oxalate, acetate, malate, citrate, maleate, fumarate and tartrate.

[0018] On the basis of the above technical solution, preferably, in step S1, the wavelength of the red light is 620-750 nm.

[0019] On the basis of the above technical solution, preferably, for each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is:

[0020] Isoleucine 7.92-9.68g, leucine 12.24-14.96g, lysine acetate 9.54-11.66g, methionine 1.08-1.32g, phenylalanine 1.44-1.76g, threonine 4.14-5.06g, tryptophan 1.35-1.65g, valine 9.54-11.66g, alanine 7.47-9.13g, arginine 7.92-9.68g, histidine 4. 23~5.17g, proline 6.39~7.81g, serine 3.33~4.07g, glycine 5.67~6.93g, aspartic acid 2.25~2.75g, glutamic acid 5.13~6.27g, asparagine 0.495~0.605g, N-acetyltyrosine 0.774~0.946g, ornithine hydrochloride 1.494~1.826g, N-acetylcysteine ​​0.72~0.88g.

[0021] On the basis of the above technical solution, preferably, for each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is:

[0022] Isoleucine (C6H 13 NO2) 8.8 g, leucine (C6H 13 NO2) 13.6 g, Lysine acetate (C6H 14 N2O2·C2H4O2) 10.6 g, methionine (C5H 11 NO2S) 1.2g, phenylalanine (C9H 11 NO2) 1.6g, threonine (C4H9NO3) 4.6g, tryptophan (C 11 H 12 N2O2) 1.5 g, valine (C5H 11NO2) 10.6g, alanine (C3H7NO2) 8.3g, arginine (C6H 14 N4O2) 8.8g, histidine (C6H9N3O2) 4.7g, proline (C5H9NO2) 7.1g, serine (C3H7NO3) 3.7g, glycine (C2H6NO2) 6.3g, aspartic acid (C4H7NO4) 2.5g, glutamic acid (C5H9NO4) 5.7g, asparagine (C4H8N2O2H2O) 0.55g, N-acetyltyrosine (C 11 H 13 NO4) 0.86 g, ornithine hydrochloride (C5H 12 N2O2 HCL) 1.66g, N-acetylcysteine ​​(C5H9NO3S) 0.8g.

[0023] On the basis of the above technical scheme, preferably, in the step S2, the pretreated first group of drug liquid is added to water, stirred for 1, cooled for 1, the second group of drug liquid is added, stirred for 2, cooled for 2, the third group of drug liquid is added, stirred for 3, fixed to volume with water, filtered, filled, and sterilized to obtain the compound amino acid injection 20AA composition.

[0024] On the basis of the above technical solution, preferably, the pretreatment method is pulping and premixing, and the pulping and premixing time is 5 minutes to 4 hours.

[0025] On the basis of the above technical solution, preferably, the temperature of the stirring 1 is 60-75° C., and the time of the stirring 1 is 10 min-1 h.

[0026] On the basis of the above technical solution, preferably, the temperature of the stirring 2 is 50-60° C., and the time of the stirring 2 is 10 min-1 h.

[0027] On the basis of the above technical solution, preferably, the temperature of the stirring 3 is 30-45° C., and the time of the stirring 3 is 10 min-1 h.

[0028] According to another aspect of the present application, a compound amino acid injection 20AA composition prepared by the preparation method described above is provided, wherein the headspace residual oxygen content in the compound amino acid injection 20AA composition is below 2.0%.

[0029] On the basis of the above technical solution, preferably, the dissolved oxygen content in the compound amino acid injection 20AA composition is below 2 mg / L.

[0030] On the basis of the above technical solution, preferably, the pH value of the compound amino acid injection 20AA composition is 5.5-6.5.

[0031] On the basis of the above technical solution, preferably, the compound amino acid injection 20AA composition is packaged in a glass infusion bottle or a soft bag, and the specifications are selected from 50ml, 100ml, 250ml, 500ml or 1000ml.

[0032] As an optional implementation mode, the present invention is implemented by the following technical solutions:

[0033] The pharmaceutical composition containing twenty kinds of amino acids is a compound amino acid injection (20AA) of different concentrations prepared by twenty kinds of amino acids and / or their salts in the following weight proportions. The formula of the compound amino acid injection (20AA) is as follows: for each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is:

[0034] Isoleucine (C6H 13 NO2) 8.8 g, leucine (C6H 13 NO2) 13.6 g, Lysine acetate (C6H 14 N2O2·C2H4O2) 10.6 g, methionine (C5H 11 NO2S) 1.2g, phenylalanine (C9H 11 NO2) 1.6g, threonine (C4H9NO3) 4.6g, tryptophan (C 11 H 12 N2O2) 1.5 g, valine (C5H 11 NO2) 10.6g, alanine (C3H7NO2) 8.3g, arginine (C6H 14 N4O2) 8.8g, histidine (C6H9N3O2) 4.7g, proline (C5H9NO2) 7.1g, serine (C3H7NO3) 3.7g, glycine (C2H6NO2) 6.3g, aspartic acid (C4H7NO4) 2.5g, glutamic acid (C5H9NO4) 5.7g, asparagine (C4H8N2O2H2O) 0.55g, N-acetyltyrosine (C 11 H 13 NO4) 0.86 g, ornithine hydrochloride (C5H 12 N2O2 HCL) 1.66g, N-acetylcysteine ​​(C5H9NO3S) 0.8g.

[0035] The composition is prepared by using the formula of compound amino acid injection (20AA), and the preparation method of the composition does not use activated carbon to adsorb and decolorize the liquid medicine. The steps are as follows:

[0036] (1) Avoid light throughout the process and turn on red light protection;

[0037] (2) Add the prescribed amount of leucine, isoleucine, valine, aspartic acid, phenylalanine, threonine and methionine to the dispensing apparatus, and add an equal weight of water for injection, and stir evenly to prepare the first group of drug solutions;

[0038] (3) Continuously fill the liquid preparation tank with nitrogen, add an appropriate amount of water for injection, and start stirring. Control the water temperature at 60-75°C, add the first group of drug solutions, and stir to dissolve;

[0039] (4) Cooling to 50-60° C., adding the prescribed amount of lysine acetate, arginine, histidine, alanine, glycine, proline, serine and glutamic acid, and stirring to dissolve;

[0040] (5) Cool down to 30-45°C, add the prescribed amount of asparagine, acetyltyrosine, ornithine hydrochloride, tryptophan and acetylcysteine, stir to dissolve, and add water for injection to make up to volume;

[0041] (6) The liquid medicine is filtered through 0.45 μm and 0.22 μm microporous filter cartridges, filled, evacuated, nitrogen-filled, plugged, capped, and sealed. After that, it is sterilized at 121°C to 123°C for 8 to 15 minutes and inspected by light.

[0042] In the pharmaceutical field, the production of light-proof products has strict requirements on lighting equipment. Red light tubes are selected as lighting equipment because they can effectively avoid ultraviolet radiation, thereby protecting products from the effects of light. According to the definition of the Chinese Pharmacopoeia, light protection mainly refers to avoiding direct sunlight and ultraviolet radiation and fluorescent lamps. Therefore, choosing red light tubes with longer wavelengths and no ultraviolet components meets the light protection requirements.

[0043] The present invention has found in the study of a pharmaceutical composition containing twenty kinds of amino acids that tryptophan in the twenty kinds of amino acids is prone to oxidative decomposition reaction with oxygen in the solution under light conditions. At this time, the color of the liquid medicine will deepen, the content will be significantly reduced, and it may even deteriorate and become unusable. However, as long as red light protection is used throughout the process and the dissolved oxygen is controlled at a certain amount, these photo-oxidative decomposition reactions will not occur. Therefore, the present invention prepares the compound amino acid injection 20AA composition in a light-proof manner and turns on red light for protection.

[0044] In addition, when preparing the composition of the present invention, the insoluble and light amino acids in the composition are premixed by pulping and then transferred to the batching tank. They can be quickly dissolved at a relatively low temperature, thereby reducing the damage to the amino acids caused by long-term high temperature and preventing the loss caused by the flying of materials.

[0045] The compound amino acid injection 20AA composition of the present invention has the following beneficial effects compared with the prior art:

[0046] The preparation method provided by the present invention comprises pre-treating the insoluble and light amino acid by pulping, and then transferring it to a batching tank, which can promote its dissolution and prevent the material from flying and causing loss. The components are dissolved in steps by programmed cooling, and the light-sensitive amino acid is protected by red light throughout the process to reduce the degradation of the amino acid; the safety and quality stability of the product prepared by the present invention are better than similar products.

[0047] Because ornithine, lysine and other amino acids in the prescription are alkaline amino acids, if acid salts are not added, the pH of the liquid will be alkaline, and the isoelectric points of most amino acids in the liquid are around 6. When the pH value of the liquid is lower or higher than the isoelectric point, the structure of the amino acid will become unstable and easily denatured or decomposed. This is because excessive positive charges will affect the interaction between molecules and thus destroy the stable structure of the amino acid. Therefore, ornithine and lysine are protected by acid salts, and the pH of the solution is close to the isoelectric point of the amino acid, making the product more stable. Among them, the content of impurities in ornithine and lysine degradation is significantly reduced when ornithine and lysine are protected by acid salts, and ornithine and lysine are more stable. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The stability effect of the pharmaceutical composition containing twenty kinds of amino acids of the present invention is further described below through relevant tests:

[0050] Example 1

[0051] In Example 1, the formula of the compound amino acid injection 20AA composition is as follows: for each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is:

[0052] Isoleucine 8.8g, leucine 13.6g, lysine acetate 10.6g, methionine 1.2g, phenylalanine 1.6g, threonine 4.6g, tryptophan 1.5g, valine 10.6g, alanine 8.3g, arginine 8.8g, histidine 4.7g, proline 7.1g, serine 3.7g, glycine 6.3g, aspartic acid 2.5g, glutamic acid 5.7g, asparagine 0.55g, N-acetyltyrosine 0.86g, ornithine hydrochloride 1.66g, N-acetylcysteine ​​0.8g, and the rest is water for injection.

[0053] According to the formula of the above-mentioned compound amino acid injection 20AA composition, test group 1, i.e., light-protection group (full red light protection), was prepared. The specific preparation method includes the following steps:

[0054] (1) Avoid light throughout the process and turn on red light with a wavelength of 620nm for protection;

[0055] (2) Add the prescribed amount of leucine, isoleucine, valine, aspartic acid, phenylalanine, threonine and methionine to the dispensing apparatus, and add an equal weight of water for injection, stir and premix for 5 minutes to prepare the first group of drug solutions;

[0056] (3) Continuously fill the liquid preparation tank with nitrogen, add an appropriate amount of water for injection, and start stirring. Control the water temperature at 60°C, add the first group of drug solutions, and stir for 1 hour to dissolve;

[0057] (4) Cooling to 50°C, adding the prescribed amount of lysine acetate, arginine, histidine, alanine, glycine, proline, serine and glutamic acid, and stirring for 1 hour to dissolve;

[0058] (5) Cool to 30°C, add the prescribed amount of asparagine, acetyltyrosine, ornithine hydrochloride, tryptophan and acetylcysteine, stir for 1 h to dissolve, and add water for injection to make up to volume;

[0059] (6) The liquid medicine is filtered through 0.45 μm and 0.22 μm microporous filter cartridges, filled, evacuated, nitrogen-filled, plugged, capped, and sealed. The mixture is sterilized at 121°C for 12 minutes and inspected by light.

[0060] Comparative Example 1

[0061] Comparative Example 1 is Experimental Group 2, which is a non-light-shielded group (irradiated with D50 fluorescent lamp). Except for step (1) which is different from the preparation of Experimental Group 1, the remaining preparation steps are consistent with the preparation method of Experimental Group 1. The specific changes in conditions are shown in Table 1.

[0062] Table 1 Two groups of 20AA samples with different preparation conditions

[0063] Full red light protection D50 fluorescent lamp irradiation Dissolved oxygen <2.0mg / L Headspace oxygen <2.0% Experimental Group 1 yes no yes yes Experimental Group 2 no yes yes yes

[0064] Test group 2 in Table 1 is the non-light-shielded group. The sterilized test groups 1 and 2 were taken to examine key quality indicators such as pH value, transmittance, related substances and content of the samples. The results are shown in Table 2.

[0065] Table 2: Test results of samples from test groups 1 and 2.

[0066]

[0067] The test results of test groups 1 and 2 are shown in Table 2. The results show that the content of kynurenine can reflect the degree of photooxidation of the sample. The lower the content of kynurenine, the smaller the degree of photooxidation of the sample and the better the stability of the sample. It can be seen that the treatment scheme of full red light protection is effective and can significantly reduce the degradation of light-sensitive amino acids in the sample.

[0068] Example 2

[0069] In Example 2, the formula of the compound amino acid injection 20AA composition is as follows: for each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is:

[0070] Isoleucine 7.92g, leucine 12.24g, lysine acetate 9.54g, methionine 1.08g, phenylalanine 1.44g, threonine 4.14g, tryptophan 1.35g, valine 9.54g, alanine 7.47g, arginine 7.92g, histidine 4.23g, proline 6.39g, serine 3.33g, glycine 5.67g, aspartic acid 2.25g, glutamic acid 5.13g, asparagine 0.495g, N-acetyltyrosine 0.774g, ornithine hydrochloride 1.494g, N-acetylcysteine ​​0.72g, and the rest is water for injection.

[0071] According to the formula of the above-mentioned compound amino acid injection 20AA composition, test group 3 was prepared. The specific preparation method includes the following steps:

[0072] (1) Avoid light throughout the process and turn on red light with a wavelength of 750nm for protection;

[0073] (2) Add the prescribed amount of leucine, isoleucine, valine, aspartic acid, phenylalanine, threonine and methionine to the dispensing apparatus, and add an equal weight of water for injection, stir and premix for 4 hours to prepare the first group of drug solutions;

[0074] (3) Continuously fill the liquid preparation tank with nitrogen, add an appropriate amount of water for injection, and start stirring. Control the water temperature at 75°C, add the first group of drug solutions, and stir for 10 minutes to dissolve;

[0075] (4) Cooling to 60°C, adding the prescribed amount of lysine acetate, arginine, histidine, alanine, glycine, proline, serine and glutamic acid, and stirring for 10 min to dissolve;

[0076] (5) Cool to 45°C, add the prescribed amount of asparagine, acetyltyrosine, ornithine hydrochloride, tryptophan and acetylcysteine, stir for 10 min to dissolve, and add water for injection to make up to volume;

[0077] (6) The liquid medicine is filtered through 0.45 μm and 0.22 μm microporous filter cartridges, filled, evacuated, nitrogen-filled, plugged, capped, and sealed. The mixture is sterilized at 121°C for 12 minutes and inspected by light.

[0078] According to the formula of the above-mentioned compound amino acid injection 20AA composition, the pulping premixed group (test group 3) and the non-premixed comparison test (test group 4) samples were prepared according to Table 3 below. The samples in test group 4 were prepared according to the preparation conditions of Table 3, and the remaining preparation steps were consistent with the steps of test group 3.

[0079] Table 3 Two groups of 20AA samples with different preparation conditions.

[0080] Direct feeding without premixing Pre-mixing Time required for dissolution of the first group Dissolved oxygen <2.0mg / L Headspace oxygen <2.0% Experimental Group 3 no yes 10min yes yes Experimental Group 4 yes no 20min yes yes

[0081] When dissolving the first group of drug solutions of test group 3 and test group 4, the samples were stirred for 5 min, 10 min, 15 min and 20 min respectively, and the first group of drug solutions were taken to detect the content of the most insoluble amino acid in the drug solutions. The results are shown in Table 4. The sterilized test groups 3 and 4 were also taken to detect key quality indicators such as pH value, transmittance, related substances and content of the samples. The results are shown in Table 5.

[0082] The results of the content test at 5min, 10min, 15min and 20min for the first group of drug solutions in test groups 3 and 4 are shown in Table 4.

[0083] Table 4: The test results of the first group of drug solution content at different stirring times.

[0084]

[0085] After sterilization of test groups 3 and 4, the test results of the samples are shown in Table 5.

[0086] Table 5: Test results of samples after sterilization in test groups 3 and 4.

[0087] Experimental groups Controls in samples Dissolution time pH Light transmittance Methionine sulfoxide (mg / L) Experimental Group 3 Pre-mixing 10min 6.2 99.8％ 2.5 Experimental Group 4 Direct feeding without premixing 20min 6.2 99.6％ 8.2

[0088] Analyzing the results of Tables 4 and 5, after pulping and premixing, the amino acids dissolved faster and were subjected to high temperature for a shorter time at the same thermometer stirring speed. The content of methionine sulfoxide, an impurity caused by methionine degradation, in the sterilized sample was lower, so the content and stability of the amino acids were better. This treatment scheme is effective, and pulping and premixing can significantly reduce the damage of high temperature to amino acids.

[0089] Example 3 is a comparative test sample of ornithine with ornithine acid salt added and without ornithine acid salt added in the prescription.

[0090] In Example 3, the formula of the compound amino acid injection 20AA composition is as follows: for each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is:

[0091] Isoleucine 9.68g, leucine 14.96g, lysine acetate 11.66g, methionine 1.32g, phenylalanine 1.76g, threonine 5.06g, tryptophan 1.65g, valine 11.66g, alanine 9.13g, arginine 9.68g, histidine 5.17g, proline 7.81g, serine 4.07g, glycine 6.93g, aspartic acid 2.75g, glutamic acid 6.27g, asparagine 0.605g, N-acetyltyrosine 0.946g, ornithine hydrochloride 1.826g, N-acetylcysteine ​​0.88g, and the rest is water for injection.

[0092] According to the formula of the above-mentioned compound amino acid injection 20AA composition, test groups 5 to 14 were prepared, wherein the preparation of ornithine hydrochloride salt was prepared according to the conditions in Table 6. The specific preparation method includes the following steps:

[0093] (1) Avoid light throughout the process and turn on red light with a wavelength of 650nm for protection;

[0094] (2) Add the prescribed amount of leucine, isoleucine, valine, aspartic acid, phenylalanine, threonine and methionine to the dispensing apparatus, and add an equal weight of water for injection, stir and premix for 2 hours to prepare the first group of drug solutions;

[0095] (3) Continuously fill the liquid preparation tank with nitrogen, add an appropriate amount of water for injection, and start stirring. Control the water temperature at 65°C, add the first group of drug solutions, and stir for 30 minutes to dissolve;

[0096] (4) Cooling to 55°C, adding the prescribed amount of lysine acetate, arginine, histidine, alanine, glycine, proline, serine and glutamic acid, and stirring for 30 minutes to dissolve;

[0097] (5) Cool to 40°C, add the prescribed amount of asparagine, acetyltyrosine, ornithine hydrochloride, tryptophan and acetylcysteine, stir for 30 min to dissolve, and add water for injection to make up to volume;

[0098] (6) The liquid medicine is filtered through 0.45 μm and 0.22 μm microporous filter cartridges, filled, evacuated, nitrogen-filled, plugged, capped, and sealed. The mixture is sterilized at 121°C for 12 minutes and inspected by light.

[0099] Table 6 20AA samples of different acid salts of ornithine.

[0100]

[0101] The sterilized test groups 5 to 14 were selected to examine key quality indicators such as pH value, transmittance, related substances and content of the samples. The results are shown in Table 7.

[0102] Table 7: Test results of samples from test groups 5 to 14.

[0103]

[0104]

[0105] The test results of test groups 5 to 14 are shown in Table 7. Analysis of the results in Table 7 shows that ornithine dimer, the main degradation impurity of ornithine, can reflect the degree of ornithine degradation. The higher the content of ornithine dimer, the more ornithine is degraded. As can be seen from Table 7, the ornithine without acid salt protection has the highest ornithine dimer content after sterilization, and the ornithine content decreases significantly, while the ornithine with acid salt protection has a significantly reduced content of ornithine degradation impurities, and the ornithine is more stable. In addition, the protection effect of adding organic acid salts is better than that of inorganic acid salts.

[0106] Example 4 is a comparative test sample of lysine prepared with and without acid salt in the prescription.

[0107] In Example 4, the formula of the compound amino acid injection 20AA composition is as follows: for each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is:

[0108] Isoleucine 8.51g, leucine 13.53g, lysine acetate 10.52g, methionine 1.16g, phenylalanine 1.56g, threonine 4.5g, tryptophan 1.55g, valine 10.56g, alanine 8.52g, arginine 8.68g, histidine 4.86g, proline 7.56g, serine 3.68g, glycine 6.28g, aspartic acid 2.45g, glutamic acid 5.56g, asparagine 0.512g, N-acetyltyrosine 0.828g, ornithine hydrochloride 1.58g, N-acetylcysteine ​​0.82g, and the rest is water for injection.

[0109] According to the formula of the above-mentioned compound amino acid injection 20AA composition, test groups 15 to 24 were prepared, wherein the preparation of lysine acetate salt was prepared according to the conditions in Table 8. The specific preparation method includes the following steps:

[0110] (1) Avoid light throughout the process and turn on red light with a wavelength of 700nm for protection;

[0111] (2) Add the prescribed amount of leucine, isoleucine, valine, aspartic acid, phenylalanine, threonine and methionine to the dispensing apparatus, and add an equal weight of water for injection, stir and premix for 3 hours to prepare the first group of drug solutions;

[0112] (3) Continuously fill the liquid preparation tank with nitrogen, add an appropriate amount of water for injection, and start stirring. Control the water temperature at 65°C, add the first group of drug solutions, and stir for 35 minutes to dissolve;

[0113] (4) Cooling to 55°C, adding the prescribed amount of lysine acetate, arginine, histidine, alanine, glycine, proline, serine and glutamic acid, and stirring for 35 minutes to dissolve;

[0114] (5) Cool to 40°C, add the prescribed amount of asparagine, acetyltyrosine, ornithine hydrochloride, tryptophan and acetylcysteine, stir for 35 min to dissolve, and add water for injection to make up to volume;

[0115] (6) The liquid medicine is filtered through 0.45 μm and 0.22 μm microporous filter cartridges, filled, evacuated, nitrogen-filled, plugged, capped, and sealed. The mixture is sterilized at 121°C for 12 minutes and inspected by light.

[0116] Table 8 20AA samples of different acid salts of lysine

[0117]

[0118] The sterilized test groups 15 to 24 were selected to examine key quality indicators such as pH value, transmittance, related substances and content of the samples. The results are shown in Table 8.

[0119] Table 8: Test results of samples from test groups 15 to 24.

[0120] Experimental groups Types of Lysine pH Light transmittance Cyclolysine(mg / L) Lysine(%) Test group 15 Lysine 6.2 99.2 100.5 93％ Test group 16 Lysine Hydrochloride 6.2 99.6 50.6 96％ Test Group 17 Lysine sulfate 6.2 99.6 51.3 95％ Test group 18 Lysine Oxalate 6.2 99.8 32.2 98％ Test Group 19 Lysine acetate 6.2 99.9 33.6 97％ Test group 20 Lysine Malate 6.2 99.7 35.2 97％ Test group 21 Lysine Citrate 6.2 99.8 31.9 98％ Test Group 22 Lysine Maleate 6.2 99.8 32.2 99％ Test Group 23 Lysine Fumarate 6.2 99.7 34.8 98％ Test Group 24 Lysine tartrate 6.2 99.9 30.9 99％

[0121] The test results of test groups 15 to 24 are shown in Table 8. Analysis of the results in Table 8 shows that cyclolysine, the main degradation impurity of lysine, can reflect the degree of degradation of lysine. The higher the cyclolysine content, the more lysine is degraded. As can be seen from Table 8, the lysine without acid salt protection has the highest cyclolysine content after sterilization, and the lysine content decreases significantly, while the lysine with acid salt protection has a significantly reduced content of lysine degradation impurities, and lysine is more stable. In addition, the protection effect of adding organic acid salts is better than that of inorganic acid salts.

[0122] 1. Comprehensive stability study of samples

[0123] In order to examine the quality of the product of the present invention, we took products from the test group 5 to 24 after sterilization and subjected them to accelerated testing for 180 days, and tested them according to the method of the draft for comments on compound amino acid injection (20AA).

[0124] The test results show that: this product uses infrared protection throughout the process, and the dissolved oxygen is controlled at 2.0 mg / L, the headspace oxygen is controlled at 2.0%, and the samples protected by acid salts meet all the requirements. The specific test results are shown in Tables 9 and 10.

[0125] Table 9: Quality test results of an injection of a pharmaceutical composition containing twenty kinds of amino acids

[0126]

[0127] Table 10: Quality test results of an injection of a pharmaceutical composition containing twenty kinds of amino acids

[0128]

[0129]

[0130] From the test results of Tables 9 and 10, the samples without acid salt protection, after sterilization and accelerated placement for 6 months, the content of ornithine and lysine is lower than the standard requirements. For the samples with acid salt protection, the content of ornithine and lysine is almost unchanged compared with 0 days after sterilization, and the content of each amino acid meets the standard requirements. Because ornithine, lysine, etc. in the prescription are alkaline amino acids, if acid salts are not added, the pH of the liquid medicine will be alkaline, and the isoelectric point of most amino acids in the liquid medicine is about 6. When the pH value of the liquid medicine is lower than or higher than the isoelectric point, the structure of the amino acid will become unstable and easily denatured or decomposed. This is because excessive positive charge will affect the interaction force between molecules, thereby destroying the stable structure of the amino acid. Therefore, ornithine and lysine are protected by acid salts, and the pH of the solution is close to the isoelectric point of the amino acid, which makes the product more stable. It also reflects that the technical solution of the present invention is feasible and effective.

[0131] 2. Safety test of samples (allergic, vascular irritation and hemolytic):

[0132] In order to investigate the safety of the composition of the present invention and provide safety reference information for clinical research, inorganic salts were used for acid protection to prepare 20AA samples of implementation group 25 (ornithine and lysine were both hydrochloric acid salts), and the resulting products were used as the test group to conduct special safety tests related to local and systemic administration, such as irritation, hemolysis and allergy. 0.9% sodium chloride injection was used as a negative control, and 20% human albumin was used as a positive control.

[0133] Test methods and results:

[0134] 1. Vascular irritation test

[0135] 1.1 Test methods

[0136] Take 3 rabbits as above, slowly inject 10mL / kg of the test sample solution into the left ear edge through intravenous injection in the test group, and inject sodium chloride injection solution into the right ear edge at the same speed and in equal amounts. Administer the drug once a day for 5 consecutive days. During the administration period, observe the local blood vessels for thrombosis and the surrounding tissues for inflammatory reactions such as redness, swelling and blood stasis. The animals were killed 48 hours after the last administration, and the ear tissues were quickly taken (2-3cm from the injection point to the heart), fixed with 10% formalin, embedded in paraffin, routinely prepared, stained with HE, and observed the morphological changes of blood vessels and surrounding tissues under a light microscope. Attach pathological photos.

[0137] 1.2 Test results

[0138] According to naked eye observation, no obvious redness, swelling, bruises, etc. were observed during the local administration of local administration in the ear vein of each group of rabbits. No significant difference was found between the test group and the control group. The results are shown in Table 11 below.

[0139] Table 11 Symptoms of rabbit samples in the test group and the control group.

[0140]

[0141]

[0142] Note: No thrombus (-) Small thrombus 1-4 mm (+) Medium thrombus 5-14 mm (++) Large thrombus (+++)

[0143] No inflammatory changes (-) Mild inflammation 3 cm (+) Moderate inflammation 1 / 3 of the ear shell (++)

[0144] Intensity of inflammation: 1 / 2 ear shell to the entire ear (+++)

[0145] Histological observation: The test sample and the control group had no obvious irritation to the rabbit blood vessels

[0146] 2. Allergy test

[0147] 2.1 Test methods

[0148] Take 20 healthy guinea pigs weighing 250-350g and randomly divide them into 3 groups (test group, negative control group, positive control group), including 8 in the test group and 6 in each of the other groups. For 3 consecutive times, inject the test solution (0.5mL / guinea pig), sodium chloride injection and 0.5mL / guinea pig of 20% human albumin intraperitoneally every other day for sensitization. On the 12th day after the last injection, inject 1mL / guinea pig of the test solution, sodium chloride injection and 20% human albumin intravenously for stimulation. Immediately to 30 minutes after the intravenous injection, observe the reaction of each guinea pig in detail according to the symptoms described in Table 12, the time of appearance and disappearance of the symptoms, and the longest observation is 3 hours. And evaluate the degree of allergic reaction according to the evaluation criteria in Table 13. Calculate the incidence of allergic reaction. Make a comprehensive judgment based on the incidence of reaction and the symptoms of allergic reaction. Guinea pigs in the negative control group shall not have allergic reaction. Guinea pigs in the positive control group should all have allergic reaction.

[0149] 2.2 Test results

[0150] All guinea pigs in the test group and negative control group did not show allergic reactions within 30 minutes after intravenous administration of the test sample and negative control group. After intravenous administration of 20% human albumin, all 6 guinea pigs showed symptoms such as sneezing, coughing, dyspnea, gait, instability, and convulsions, and 2 guinea pigs died. The results are shown in Table 12.

[0151] Table 12 Allergic reaction symptoms

[0152]

[0153]

[0154] Table 13 Evaluation criteria for systemic sensitization

[0155] 0 — Allergic reaction negative 1-4 symptoms + Allergic reaction weak positive 5-10 symptoms ++ Positive allergic reaction 11-19 Symptoms +++ Strong positive allergic reaction 20 ++++ Allergic reaction is very positive

[0156] According to the evaluation criteria of Tables 12 and 13, the results of the allergy test are shown in Table 14: Under the above test conditions, the test compound amino acid injection and the commercially available control compound amino acid injection had consistent reactions, and no systemic allergic reaction was found in the allergy test.

[0157] Table 14 Allergy test results

[0158]

[0159] 3. Hemolysis test

[0160] 3.1 Test methods

[0161] Take 7 test tubes, add 2% red blood cell suspension and saline in the following proportions, mix well, place in a 37±0.5℃ constant temperature incubator for half an hour, then add different volumes of test sample stock solution to tubes 1 to 5 (see Table 15 for specific volume usage), add saline to tube 6 as a negative control tube, add water for injection to tube 7 as a positive control, shake well, and place in a 37±0.5℃ constant temperature incubator. Observe every 15 minutes at first, and observe every 1 hour after 1 hour, indicating that there is red blood cell agglutination.

[0162] Table 15 Compound Amino Acid Injection (20AA) Hemolysis Test Proportional Amount (mL)

[0163]

[0164]

[0165] 3.2 Test results

[0166] When tube No. 7 was kept at 37°C for 15 minutes, the solution showed hemolysis. However, the solutions in tubes No. 1 to 5 and tube No. 6 with sodium chloride injection added did not turn transparent red or show brown-red flocculent precipitation within the specified time, indicating that the compound amino acid injection did not cause hemolysis and agglutination reaction on rabbit red blood cells. The results are shown in Table 16 below.

[0167] Table 16 Hemolysis test results of compound amino acid injection (20AA)

[0168] Test tube number No. 1 pipe No. 2 pipe No. 3 pipe No. 4 pipe No. 5 pipe No. 6 pipe No. 7 pipe Keep at 37℃ for 15 minutes — — — — — — +++ Keep at 37℃ for 30 minutes — — — — — — +++ Keep at 37℃ for 45 minutes — — — — — — +++ Keep warm at 37℃ for 1 hour — — — — — — +++ Keep warm at 37℃ for 2 hours — — — — — — +++ Keep warm at 37℃ for 3 hours — — — — — — +++

[0169] Note: +++Complete hemolysis; ++Partial hemolysis; +Agglutination; -No hemolysis and no agglutination.

[0170] Through special safety tests related to local and systemic administration, such as irritation, hemolysis and allergy, the results show that the compound amino acid injection (20AA) produced by this prescription and preparation method has no obvious irritation to rabbit veins, does not cause hemolysis and agglutination reactions to rabbit red blood cells, and does not cause allergic reactions in guinea pigs, and is safe for clinical use.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a compound amino acid injection 20AA composition, characterized in that: The preparation method comprises: Step S1: During the entire preparation process, avoid light and turn on red light for protection; Step S2: adding 20 kinds of amino acids into water in three groups at 30° C. to 75° C. under a nitrogen atmosphere to obtain a compound amino acid injection 20AA composition; The first group of solutions includes leucine, isoleucine, valine, aspartic acid, phenylalanine, threonine, and methionine; The second group of solutions included lysine acetate, arginine, histidine, alanine, glycine, proline, serine, and glutamic acid; The third group of solutions includes asparagine, N-acetyltyrosine, ornithine hydrochloride, tryptophan and N-acetylcysteine.

2. The preparation method according to claim 1, characterized in that The salt in the ornithine hydrochloride is selected from at least one of hydrochloride, sulfate, oxalate, acetate, malate, citrate, maleate, fumarate and tartrate.

3. The preparation method according to claim 1, characterized in that: The salt in the lysine acetate is selected from at least one of hydrochloride, sulfate, oxalate, acetate, malate, citrate, maleate, fumarate and tartrate.

4. The preparation method according to claim 1, characterized in that: In the step S1, the wavelength of the red light is 620-750 nm.

5. The preparation method according to claim 1, characterized in that: For each preparation of 1000 mL of the compound amino acid injection 20AA composition, the amount of each raw material is as follows: Isoleucine 7.92-9.68g, leucine 12.24-14.96g, lysine acetate 9.54-11.66g, methionine 1.08-1.32g, phenylalanine 1.44-1.76g, threonine 4.14-5.06g, tryptophan 1.35-1.65g, valine 9.54-11.66g, alanine 7.47-9.13g, arginine 7.92-9.68g, histidine 4. 23~5.17g, proline 6.39~7.81g, serine 3.33~4.07g, glycine 5.67~6.93g, aspartic acid 2.25~2.75g, glutamic acid 5.13~6.27g, asparagine 0.495~0.605g, N-acetyltyrosine 0.774~0.946g, ornithine hydrochloride 1.494~1.826g, N-acetylcysteine ​​0.72~0.88g.

6. The preparation method according to claim 1, characterized in that: In the step S2, the pretreated first group of liquid medicines are added to water, stirred for 1, cooled for 1, the second group of liquid medicines are added, stirred for 2, cooled for 2, the third group of liquid medicines are added, stirred for 3, fixed to volume with water, filtered, filled, and sterilized to obtain a compound amino acid injection 20AA composition; The pretreatment method is pulping and premixing, and the pulping and premixing time is 5min to 4h; The temperature of the stirring 1 is 60-75° C., and the time of the stirring 1 is 10 min-1 h.

7. The preparation method according to claim 6, characterized in that: The temperature of the stirring 2 is 50-60° C., and the time of the stirring 2 is 10 min-1 h.

8. The preparation method according to claim 6, characterized in that: The temperature of the stirring 3 is 30-45° C., and the time of the stirring 3 is 10 min-1 h.

9. The compound amino acid injection 20AA composition prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The headspace residual oxygen content in the compound amino acid injection 20AA composition is below 2.0%; The dissolved oxygen content in the compound amino acid injection 20AA composition is below 2 mg / L.

10. The compound amino acid injection 20AA composition according to claim 9, characterized in that: The pH value of the compound amino acid injection 20AA composition is 5.5-6.5.

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