Cardioplegic and organ preservation solutions and methods of making same

Abstract

The application provides a cardioplegic solution and organ preservation solution and a preparation method thereof, and belongs to the technical field of medicines. The cardioplegic solution and organ preservation solution are packaged by using a double-chamber bag, and the bag comprises an A chamber and a B chamber. Contents are stored separately, and the contents are mixed before use. After mixing, 1000ml of the solution contains the following components: 0.8766g of sodium chloride, 0.6710g of potassium chloride, 0.1842g of potassium alpha-ketoglutarate, 0.8132g of magnesium chloride hexahydrate, 3.7733g of L-histidine hydrochloride monohydrate, 27.9289g of histidine, 0.4085g of tryptophan, 5.4651g of mannitol, 0.0022g of calcium chloride dihydrate, the pH is 7.02-7.20, and the osmotic pressure is 310±20mOsm / kg. The alpha-ketoglutarate is not in the same chamber with the L-histidine hydrochloride monohydrate and the histidine. The product improves the stability, safety and use convenience of the product, and effectively reduces the production and circulation costs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a cardiac arrest fluid and organ preservation fluid and their preparation methods. Background Technology

[0002] HTK solution (histidine-tryptophan-ketoglutarate solution) is a low-sodium isotonic solution with histidine as the main buffer system. It is mainly used for myocardial protection, cardiac arrest, and preservation of ex vivo organs during cardiac surgery, and is a widely used organ protection product in clinical practice. The classic HTK solution currently used clinically is Custodiol®, developed by Dr. Franz Köhler Chemie GmbH in Germany. Its standard formula contains the following per 1000 mL solution: sodium chloride 0.8766 g, potassium chloride 0.6710 g, potassium hydrogen 2-ketoglutarate 0.1842 g, magnesium chloride hexahydrate 0.8132 g, L-histidine hydrochloride monohydrate 3.7733 g, histidine 27.9289 g, tryptophan 0.4085 g, mannitol 5.4651 g, and calcium chloride dihydrate 0.0022 g.

[0003] Although this product has been used clinically for a long time, there is a risk of interactions between its components, and it is prone to degradation and impurity generation under sterilization and storage conditions. A series of long-standing problems still exist in the production, quality control, storage, and transportation processes. 1. Potassium α-ketoglutarate in the formula has poor stability and is prone to degradation by reacting with high concentrations of histidine during high-temperature sterilization and long-term storage, resulting in a decrease in the content of the active ingredient. To ensure that the product's content meets the requirements within its shelf life, the production process requires excessive feeding, which not only increases production costs but also violates conventional quality control principles.

[0004] 2. According to the process disclosed in patent CN103238586B, a 25% excess of potassium α-ketoglutarate is required to ensure the content is up to standard. The domestically produced cardioprotective cardioplegic solution (trade name: Hertkin) marketed based on this patented technology has the same formulation, storage conditions, and expiration date as the imported Custodiol®, yet it still fails to solve the aforementioned problem.

[0005] 3. The product needs to be stored in the dark and refrigerated at 2℃~8℃ and transported in a cold chain throughout the process. The storage and distribution conditions are harsh, which significantly increases the supply chain cost and the difficulty of ensuring supply.

[0006] 4. The product has a short shelf life of only 12 months. Even under the specified refrigeration conditions, the content of potassium α-ketoglutarate continues to decrease (about 18%), accompanied by an increase in impurities, posing potential risks to quality stability and safety of use.

[0007] 5. Tryptophan and histidine coexist directly in the formula. During high-temperature sterilization and long-term storage, they are prone to interact and generate impurities, which further affect the purity and quality stability of the product.

[0008] Currently, the HTK solution used clinically in China is mainly imported, which is expensive and has limited accessibility. Domestically produced similar products still use traditional single-compartment packaging and formulations, failing to fundamentally improve the stability of components during sterilization and storage. Therefore, without altering the classic formulation and clinical function of HTK solution, developing a new type of cardiac arrest solution and organ preservation solution with higher stability, room temperature storage and transportation capabilities, lower impurity levels, and more controllable quality is of great significance for meeting clinical needs, reducing medical costs, and improving product supply security. Summary of the Invention

[0009] The purpose of this invention is to propose a cardiac arrest solution and organ preservation solution and their preparation method, overcoming the shortcomings of existing HTK solutions such as poor stability due to component compatibility, the need for refrigeration, short shelf life, and many impurities. It fundamentally solves the instability problem of potassium α-ketoglutarate during sterilization and storage, as well as the problem of impurities generated by the contact between tryptophan and histidine, and provides a cardiac arrest solution and organ preservation solution with high stability, room temperature storage and transportation, and better safety, as well as their preparation method.

[0010] The technical solution of this invention is implemented as follows: This invention provides a cardiac arrest solution and organ preservation solution, packaged in a double-chamber bag. The double-chamber bag is divided into chamber A and chamber B by a loose weld, and the contents of each chamber are stored separately. Before use, the contents of the two chambers are mixed. Each 1000ml of the mixed solution contains: 0.8766g sodium chloride, 0.6710g potassium chloride, 0.1842g potassium α-ketoglutarate, 0.8132g magnesium chloride hexahydrate, 3.7733g L-histidine hydrochloride monohydrate, 27.9289g histidine, 0.4085g tryptophan, 5.4651g mannitol, and 0.0022g calcium chloride dihydrate. The pH of the mixed solution is 7.02-7.20, and the osmotic pressure is 310±20mOsm / kg. Potassium α-ketoglutarate is not in the same chamber as L-histidine hydrochloride monohydrate or histidine.

[0011] As a further improvement of the present invention, the tryptophan is not stored in the same chamber as L-histidine hydrochloride monohydrate and histidine, but in the same chamber as potassium α-ketoglutarate.

[0012] Unstable potassium α-ketoglutarate and tryptophan were physically isolated from high concentrations of L-histidine hydrochloride monohydrate and histidine, and placed in different chambers.

[0013] As a further improvement of the present invention, the sodium chloride, potassium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate, and mannitol can be placed individually in chamber A or chamber B, or they can be placed separately in two chambers.

[0014] As a further improvement of the present invention, the A chamber contains potassium α-ketoglutarate and tryptophan, and the B chamber contains L-histidine hydrochloride monohydrate, histidine, and mannitol.

[0015] As a further improvement of the present invention, the volume ratio of chamber A to chamber B is 1:9 to 1:5, preferably 1:9.

[0016] As a further improvement of the present invention, the pH of component A is 5.0 to 7.0, and the pH of component B is 7.10 to 7.20.

[0017] The present invention identifies the following optimization scheme: This optimized solution is based on the following component contents per 1000 ml of the mixed solution: sodium chloride 0.8766 g, potassium chloride 0.6710 g, potassium hydrogen α-ketoglutarate 0.1842 g, magnesium chloride hexahydrate 0.8132 g, L-histidine hydrochloride monohydrate 3.7733 g, histidine 27.9289 g, tryptophan 0.4085 g, mannitol 5.4651 g, and calcium chloride dihydrate 0.0022 g.

[0018] 1. Compartmentalization principle: Potassium α-ketoglutarate must be compartmentalized with histidine; tryptophan should also be compartmentalized with histidine, but can be placed in the same compartment A as potassium α-ketoglutarate.

[0019] 1.1 Potassium α-ketoglutarate must be compartmentalized with histidine. Potassium α-ketoglutarate is the most unstable component in the formulation. Experimental studies have revealed that its poor stability is primarily due to the Maillard reaction (the reaction between the carbonyl group in the potassium α-ketoglutarate molecule and the amino group of histidine) that easily occurs upon contact with high concentrations of histidine during high-temperature sterilization and long-term storage. This reaction leads to a significant decrease in the potassium α-ketoglutarate content and the generation of numerous impurities. Specific grouping test results are shown in Table 1 below. Table 1

[0020] The data above show that when potassium α-ketoglutarate exists alone or in combination with L-histidine hydrochloride monohydrate, tryptophan, mannitol, or electrolytes (sodium, potassium, magnesium, and calcium salts), its content does not change significantly after high-temperature sterilization. However, when combined with histidine, the content decreases significantly by approximately 8.32% after sterilization (experimental group 2). This indicates that high concentrations of histidine are the core factor leading to the degradation of potassium α-ketoglutarate after high-temperature sterilization. The Maillard reaction (the reaction between the carbonyl group in the potassium α-ketoglutarate molecule and the amino group in histidine) is the main cause of degradation. Therefore, potassium α-ketoglutarate must be stored separately from histidine.

[0021] In addition, a sterilization comparison test between single-chamber packaging (potassium α-ketoglutarate and histidine in the same chamber, test group 2) and double-chamber packaging (potassium α-ketoglutarate and histidine in separate chambers, test group 1) showed that the content of potassium α-ketoglutarate in single-chamber packaging decreased by 8.32%, while the content in double-chamber packaging remained almost unchanged.

[0022] In addition, the results of the long-term stability study are shown in Table 2. Storage conditions: 5±3℃ for 12 months.

[0023] Table 2

[0024] The content of potassium α-ketoglutarate in the dual-compartment packaged product remained stable over 12 months (102.86% at 0 months, 101.01% at 6 months, and 101.35% at 12 months). In contrast, the content of potassium α-ketoglutarate in the traditional single-compartment packaged product (imported product) decreased by an average of 16.26% after 12 months of storage at 5±3℃, with a minimum decrease of 9.93% and a maximum decrease of 20.96%. This further verifies the necessity of storing potassium α-ketoglutarate and histidine separately, and that separate storage can significantly improve the stability of potassium α-ketoglutarate.

[0025] 1.2 Tryptophan should also be compartmentalized with histidine, but it can be placed in cavity A together with potassium α-ketoglutarate. Tryptophan, a typical unstable amino acid among commonly used amino acids, is prone to oxidative degradation and the generation of harmful impurities, affecting product safety and efficacy. Based on the premise that compartment A contains potassium α-ketoglutarate and compartment B contains histidine and L-histidine hydrochloride monohydrate, to clarify a reasonable compartmentalization scheme for tryptophan and to investigate the effects of electrolytes (sodium chloride, potassium chloride, magnesium chloride hexahydrate, and calcium chloride dihydrate) and mannitol on the stability of each component and the generation of impurities, we conducted targeted experiments: different components were combined in specific combinations, sterilized by moist heat at 121℃ for 15 minutes, and the generation of impurities was detected. The core experimental groupings and results are shown in Table 3 below. Table 3

[0026] Based on the above experimental data andFigure 1-6 It is clear that when sodium, potassium, calcium, magnesium, mannitol, potassium α-ketoglutarate, histidine (containing L-histidine hydrochloride monohydrate), and tryptophan are combined individually or in combination with tryptophan and potassium α-ketoglutarate, no obvious impurities are produced after 121°C moist heat sterilization. However, when histidine (containing L-histidine hydrochloride monohydrate) is combined with tryptophan, sodium, potassium, calcium, magnesium, and mannitol, obvious impurities are produced after sterilization. These impurities are completely consistent with those produced after sterilization with traditional single-compartment HTK solutions, and the impurity content of traditional single-compartment packaged HTK solutions (containing all components) is significantly increased after sterilization. Therefore, it can be concluded that contact between tryptophan and histidine (containing L-histidine hydrochloride monohydrate) will produce harmful impurities, and the two must be stored in separate compartments. That is, tryptophan should not be stored in the same compartment as histidine (containing L-histidine hydrochloride monohydrate), which is the core basis for the separate storage of tryptophan.

[0027] Based on the experimental data above, it can be seen that after sterilization of potassium α-ketoglutarate and tryptophan, the content changed from 98.95% to 99.33% without significant fluctuation. Furthermore, no impurities were generated after sterilization of the two compounds, indicating that tryptophan and potassium α-ketoglutarate do not have adverse interactions and can be stored in the same compartment. In summary, tryptophan should be stored separately from histidine (including L-histidine hydrochloride monohydrate), while it can be stored together with potassium α-ketoglutarate in compartment A. This compartmentalization scheme was verified through comparative experiments of single-compartment and double-compartment packaging. In single-compartment packaging, the content of potassium α-ketoglutarate decreased from 95.23% to 87.37%, a decrease of 10%. However, in double-compartment packaging (potassium α-ketoglutarate and tryptophan in compartment A, and histidine and L-histidine hydrochloride monohydrate in compartment B), the content of potassium α-ketoglutarate remained stable. This fully demonstrates that the compartmentalization principle is scientific and feasible, and can effectively ensure product quality stability.

[0028] In addition, sodium, potassium, calcium, magnesium, and mannitol have no adverse effects on the stability and impurity generation of potassium α-ketoglutarate. Mannitol is used in larger quantities and should be placed in chamber B (large volume chamber). Sodium, potassium, calcium, and magnesium can be placed in chamber A or chamber B according to actual production needs, which ensures the implementation of the above-mentioned compartmentalization principle.

[0029] 2. Volume ratio requirement of chamber A to chamber B: To prevent histidine precipitation in chamber B when used at low temperature (4-8℃), the volume ratio of chamber A to chamber B should be controlled between 1:9 and 3:7, preferably 1:9.

[0030] After clarifying that potassium α-ketoglutarate needs to be stored separately from histidine, in order to further reduce the various possible organic reactions, we also stored histidine hydrochloride and mannitol separately from potassium α-ketoglutarate. That is, histidine, histidine hydrochloride and mannitol were placed in the same compartment. Considering their usage and solubility, they should be placed in a large compartment.

[0031] Based on the fact that L-histidine has a solubility of 41.9 g / L at 25°C, the prescribed amount of histidine in 1.5L of this product is 41.85 g. Solubility at room temperature is not a major issue, and it can be completely dissolved even if the large and small chambers are divided in a 1:2 ratio. However, considering that this product is a cardiac arrest solution, the temperature needs to be lowered to 4-8°C before use. Under these conditions, its solubility may decrease sharply. Therefore, it is necessary to determine the volume ratio of the large and small chambers of this product under the operating temperature to ensure that histidine crystals will not precipitate before use.

[0032] To address this issue, we prepared chambers of varying sizes with different volume ratios, formulated the product according to the prescribed composition, filled and sterilized them, and observed the precipitation of histidine by placing them at 4–8°C for 8 hours. The results are shown in Table 4 below.

[0033] Table 4

[0034] The data in the table above shows that as the volume of the large chamber decreases, the solubility of histidine gradually decreases. According to the formulation ratio of HTK solution, when the ratio of the large chamber to the small chamber exceeds 2:8, histidine will precipitate under low temperature conditions. Considering the actual situation of the mold in the production line, this product selects a large chamber to small chamber volume ratio of 1:9 (experimental group 15).

[0035] 2. Component allocation: In a preferred embodiment, chamber A contains: potassium α-ketoglutarate, tryptophan, sodium chloride, potassium chloride, magnesium chloride hexahydrate, and calcium chloride dihydrate; chamber B contains: L-histidine hydrochloride monohydrate, histidine, and mannitol.

[0036] To determine the optimal distribution of each component in chambers A and B, different component distribution combinations were designed based on the core principle of compartmentalization of potassium α-ketoglutarate, tryptophan, and histidine. After moist heat sterilization at 121℃ for 15 minutes, the changes in potassium α-ketoglutarate content and the morphological stability under low temperature (2–8℃) conditions were detected. The core test results are shown in Table 5 below: Table 5

[0037] The results show that the content of potassium α-ketoglutarate in single-chamber packaged products decreased by about 5% during preparation and by about 8% after sterilization, for a total decrease of about 13% (test group 16). In double-chamber packaged products, the content remained almost unchanged (test groups 17–20).

[0038] Simultaneously, the volume ratios of components A and B were examined at 2:8 and 1:9. Component B with a volume ratio of 2:8 showed crystal precipitation during storage at 2–8°C, while the product with a volume ratio of 1:9 showed no change in properties during storage.

[0039] The final composition of compartment A was determined to include sodium chloride, potassium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate, tryptophan, and potassium hydrogen α-ketoglutarate; the composition of compartment B was determined to include L-histidine hydrochloride monohydrate, histidine, and mannitol. The volume ratio of components A to B was 1:9 (experimental group 19).

[0040] 3. pH control: Based on the volume ratio and formula of the above-mentioned experimental group 19, pH control was performed. The pH of the A chamber solution was adjusted to 5.0-7.0, and the pH of the B chamber solution was adjusted to 7.10-7.20 to ensure that the pH after mixing is 7.02-7.20, without affecting the stability of each component.

[0041] The pH range of the HTK solution is 7.02–7.20. To ensure that the pH of the dual-chamber bag design meets the requirements after mixing in chambers A and B, we designed an experiment to investigate the pH of chambers A and B. The results are shown in Tables 6 and 7 below.

[0042] Table 6. pH values ​​of chambers A and B before and after sterilization.

[0043] Table 7. Mixed pH values ​​of chambers A and B before and after sterilization.

[0044] The results show that the pH of component A (5.0–7.0) and component B (7.02–7.20) had no significant effect on the potassium α-ketoglutarate content before and after sterilization. The pH of component A (5.0–7.0) had little impact on the pH of the mixture, while the pH of component B (7.10–7.20) ensured that the pH of the mixture was between 7.02 and 7.20. Therefore, the pH of component A was ultimately determined to be 5.0–7.0, and the pH of component B to be 7.10–7.20.

[0045] This invention further protects a method for the above-mentioned cardioplegic solution and organ preservation solution, comprising the following steps: (1) Solution preparation: Prepare the A component solution corresponding to chamber A and the B component solution corresponding to chamber B respectively; (2) Filtration: After coarse filtration of component A and component B through a 0.45μm filter element, they are then finely filtered through a 0.22μm filter element; (3) Packaging: The filtered A component liquid and B component liquid are filled into the A chamber and B chamber of the double chamber bag respectively. Nitrogen is continuously filled during the filling process. After filling, the double chamber bag is sealed and then a high-barrier outer packaging film is added to the sealed double chamber bag for vacuum packaging. (4) Sterilization: Sterilize the vacuum-packed product to obtain cardiac arrest fluid and organ preservation fluid.

[0046] As a further improvement of the present invention, in step (1), when preparing component A solution, potassium hydroxide is used to adjust the pH of component A solution to 5.0 to 7.0; when preparing component B solution, under continuous nitrogen purging conditions, potassium hydroxide is used to adjust the pH of component B solution to 7.10 to 7.20.

[0047] As a further improvement of the present invention, in step (4), the sterilization conditions are moist heat sterilization at 121°C for 8 to 15 minutes.

[0048] The present invention has the following beneficial effects: 1. Product stability has been significantly improved. This invention employs a compartmentalized storage design to physically block the contact between potassium α-ketoglutarate and histidine, preventing degradation reactions between the two during sterilization and storage. Test results show that the potassium α-ketoglutarate content of traditional single-compartment products decreases by approximately 10% after sterilization at 121℃; however, the content of the product of this invention does not decrease significantly after sterilization, and remains above 98% of its initial value after 12 months of storage at room temperature. This eliminates the need for excessive feed, resulting in more stable and controllable quality.

[0049] 2. Storage and transportation are more convenient. The product can withstand 121℃ excessive sterilization process and can be stored and transported at room temperature for a long time without the need for refrigeration at 2℃~8℃ and protection from light, as well as a complete cold chain. This greatly reduces logistics costs and supply chain management difficulty, and improves product accessibility.

[0050] 3. Impurity levels have been significantly reduced. The compartmentalized design avoids direct contact between tryptophan and histidine, effectively inhibiting their interaction and the generation of impurities. Compared with traditional single-compartment products, the total amount of impurities after sterilization is significantly reduced, and no new impurities are generated or exceed the impurity limit even after long-term storage, resulting in significantly improved safety.

[0051] 4. Product shelf life is significantly extended. Thanks to its excellent long-term stability, the shelf life of the product of this invention can be extended from 12 months for existing imported products to 24 months, significantly reducing inventory and expiration date management pressure and reducing resource waste.

[0052] 5. Maintain equivalent therapeutic effect Animal experiments (ex vivo heart Langendorff perfusion model) show that the product of this invention is superior to or equivalent to traditional HTK solution and control cardioplegic solution in inducing cardiac arrest, reducing ischemia-reperfusion injury, and protecting myocardial function. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 The image shows the HPLC results of product group 8 after sterilization.

[0055] Figure 2 The image shows the HPLC results of product 9 in the experimental group after sterilization.

[0056] Figure 3 The image shows the HPLC results of product 10 in the experimental group after sterilization.

[0057] Figure 4 The image shows the HPLC results of product 11 in test group after sterilization.

[0058] Figure 5 The image shows the HPLC results of product 12 in the experimental group after sterilization.

[0059] Figure 6 The image shows the HPLC results of product 13 in test group after sterilization. Detailed Implementation

[0060] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] Example 1: Preparation of Cardiac Arrest Fluid and Organ Preservation Fluid In this embodiment, a dual-chamber cardiac arrest fluid and organ preservation solution with a total volume of 2000L was prepared. The volume ratio of chambers A to B was 1:9 (chamber A 200L, chamber B 1800L). The content of each component in each chamber was precisely calculated based on the formula ratio per 1000mL after mixing. The specific preparation steps are as follows: 1. Solution preparation: (1) A chamber fluid (small chamber, 200L) Weigh the following raw materials according to the specified proportions: 1753.2g sodium chloride, 1342g potassium chloride, 1626.4g magnesium chloride hexahydrate, 4.4g calcium chloride dihydrate, 817g tryptophan, and 368.4g potassium α-ketoglutarate. Add 160L of purified water to a mixing tank and heat to 40℃. Add the above electrolytes sequentially, stirring until completely dissolved. Then add tryptophan and potassium α-ketoglutarate, continuing to stir until dissolved. Add purified water to bring the volume to 200L. Stir and cool to 25℃, then adjust the pH to 6.0 with 5mol / L potassium hydroxide solution to obtain chamber A solution.

[0062] (2) Chamber B liquid (large chamber, 1800L) Weigh the raw materials according to the following proportions: mannitol 10930.2g, L-histidine hydrochloride monohydrate 7546.6g, and histidine 55857.8g. Add 1440L of purified water to another preparation vessel and heat to 40℃. Add mannitol, L-histidine hydrochloride monohydrate, and histidine sequentially, stirring under nitrogen purging until completely dissolved. Add purified water to bring the volume to 1800L. Stir and cool to 25℃. Under nitrogen purging, adjust the pH to 7.15 using 5mol / L potassium hydroxide solution to obtain chamber B solution.

[0063] 2. Filtration and Filling: The solutions in chambers A and B are filtered through 0.45μm and 0.22μm polyethersulfone filters, respectively. Under nitrogen protection, the solutions in chamber A (200ml) and chamber B (1800ml) are filled into non-PVC double-chamber bags (total volume 2000ml), heat-sealed, and vacuum-packed with a high-barrier film.

[0064] 3. Sterilization: Sterilize at 121℃ with moist heat for 12 minutes, then cool to obtain the finished product.

[0065] Examples 2-6 and Comparative Example 1 Prepare the solution by changing the distribution positions of sodium chloride, potassium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate, and mannitol in chambers A and B, or by adjusting the chamber volume ratio, following a method similar to Example 1.

[0066] Example 2: Preparation of Cardiac Arrest Fluid and Organ Preservation Fluid In this embodiment, a dual-chamber cardiac arrest fluid and organ preservation fluid with a total volume of 2000L was prepared. The volume ratio of chambers A to B was 2:8 (chamber A 400L, chamber B 1600L). The core principle was to store potassium α-ketoglutarate, tryptophan and histidine, and L-histidine hydrochloride monohydrate separately in different chambers. The electrolyte distribution was adjusted, and the preparation was carried out according to the following method: 1. Solution preparation: (1) A chamber solution (400L): Sodium chloride 1753.2g, potassium chloride 1342g, magnesium chloride hexahydrate 1626.4g, calcium chloride dihydrate 4.4g, tryptophan 817g, potassium α-ketoglutarate 368.4g. Add 320L of purified water to the preparation tank, heat to 40℃, add the above electrolytes in sequence, and stir until completely dissolved; then add tryptophan and potassium α-ketoglutarate, continue stirring to dissolve, and add purified water to make up to 400L. Stir and cool to 25℃, and adjust the pH to 5.5 with 5mol / L potassium hydroxide solution.

[0067] (2) Chamber B solution (1600L): Mannitol 10930.2g, L-histidine hydrochloride monohydrate 7546.6g, histidine 55857.8g. Add 1280L of purified water to another preparation tank, heat to 40℃, add the above raw materials in sequence, and stir under nitrogen protection until completely dissolved. Add purified water to make up to 1600L. Stir and cool to 25℃, and adjust the pH to 7.12 with 5mol / L potassium hydroxide solution under nitrogen protection.

[0068] 2. Filtration and filling: The solutions in chambers A and B are precisely filtered through 0.45μm and 0.22μm polyethersulfone filter elements, respectively. Under nitrogen protection throughout the process, the filtered solutions in chambers A and B are filled into the corresponding chambers of the double-chamber bag, heat-sealed, and vacuum-packed with a high-barrier outer packaging film to control the residual oxygen content of the solution to ≤3mg / L and the headspace residual oxygen content to ≤2%.

[0069] 3. Sterilization: Place the sealed double-chamber bag in a sterilization cabinet and sterilize it at 121℃ for 12 minutes. After sterilization, cool it to room temperature to obtain the finished product.

[0070] Example 3: Preparation of Cardiac Arrest Fluid and Organ Preservation Fluid In this embodiment, a dual-chamber cardiac arrest fluid and organ preservation solution with a total volume of 2000L was prepared. The volume ratio of chambers A to B was 1:9 (chamber A 200L, chamber B 1800L). The core principle was to store potassium α-ketoglutarate, tryptophan and histidine, and L-histidine hydrochloride monohydrate separately in chambers. All electrolytes were placed in chamber B, and the solution was prepared as follows: 1. Solution preparation: (1) A chamber solution (200L): Weigh 817g of tryptophan and 368.4g of potassium hydrogen α-ketoglutarate. Add 160L of purified water to the preparation tank, heat to 40℃, add the above raw materials in sequence, stir until completely dissolved, and add purified water to make up to 200L. Stir and cool to 25℃, and adjust the pH to 6.5 with 5mol / L potassium hydroxide solution.

[0071] (2) Chamber B solution (1800L): Weigh 10930.2g mannitol, 7546.6g L-histidine hydrochloride monohydrate, 55857.8g histidine, 1753.2g sodium chloride, 1342g potassium chloride, 1626.4g magnesium chloride hexahydrate, and 4.4g calcium chloride dihydrate. Add 1440L of purified water to another preparation tank, heat to 40℃, add electrolyte first and stir to dissolve, then add the remaining raw materials, and stir under nitrogen protection until completely dissolved. Add purified water to make up to 1800L. Stir and cool to 25℃, and adjust the pH to 7.18 with 5mol / L potassium hydroxide solution under nitrogen protection.

[0072] 2. Filtration and filling: The solutions in chambers A and B are precisely filtered through 0.45μm and 0.22μm polyethersulfone filter elements, respectively. Under nitrogen protection throughout the process, the filtered solutions in chambers A and B are filled into the corresponding chambers of the double-chamber bag, heat-sealed, and vacuum-packed with a high-barrier outer packaging film to control the residual oxygen content of the solution to ≤3mg / L and the headspace residual oxygen content to ≤2%.

[0073] 3. Sterilization: Place the sealed double-chamber bag in a sterilization cabinet and sterilize it at 121℃ for 12 minutes. After sterilization, cool it to room temperature to obtain the finished product.

[0074] Example 4: Preparation of Cardiac Arrest Fluid and Organ Preservation Fluid In this embodiment, a dual-chamber cardiac arrest fluid and organ preservation solution with a total volume of 2000L was prepared. The volume ratio of chambers A to B was 3:7 (chamber A 600L, chamber B 1400L). The core principle was to store potassium α-ketoglutarate, tryptophan and histidine, and L-histidine hydrochloride monohydrate separately in chambers A and B, and mannitol was placed in chambers A and B respectively, prepared according to the following method: 1. Solution preparation: (1) A chamber solution (600L): Weigh 1753.2g sodium chloride, 1342g potassium chloride, 1626.4g magnesium chloride hexahydrate, 4.4g calcium chloride dihydrate, 817g tryptophan, 368.4g potassium α-ketoglutarate, and 3279.06g mannitol. Add 480L of purified water to the preparation tank, heat to 40℃, first add the electrolyte and mannitol and stir to dissolve, then add tryptophan and potassium α-ketoglutarate, continue stirring to dissolve, and add purified water to make up to 600L. Stir and cool to 25℃, and adjust the pH to 5.0 with 5mol / L potassium hydroxide solution.

[0075] (2) Chamber B solution (1400L): Weigh 7651.14g mannitol, 7546.6g L-histidine hydrochloride monohydrate, and 55857.8g histidine. Add 1120L of purified water to another preparation tank, heat to 40℃, add the above raw materials in sequence, and stir under nitrogen protection until completely dissolved. Add purified water to make up to 1400L. Stir and cool to 25℃, and adjust the pH to 7.10 with 5mol / L potassium hydroxide solution under nitrogen protection.

[0076] 2. Filtration and filling: The solutions in chambers A and B are precisely filtered through 0.45μm and 0.22μm polyethersulfone filter elements, respectively. Under nitrogen protection throughout the process, the filtered solutions in chambers A and B are filled into the corresponding chambers of the double-chamber bag, heat-sealed, and vacuum-packed with a high-barrier outer packaging film to control the residual oxygen content of the solution to ≤3mg / L and the headspace residual oxygen content to ≤2%.

[0077] 3. Sterilization: Place the sealed double-chamber bag in a sterilization cabinet and sterilize it at 121℃ for 12 minutes. After sterilization, cool it to room temperature to obtain the finished product.

[0078] Example 5: Preparation of Cardiac Arrest Fluid and Organ Preservation Fluid In this embodiment, a dual-chamber cardiac arrest fluid and organ preservation solution with a total volume of 2000L was prepared. The volume ratio of chambers A to B was 1:9 (chamber A 200L, chamber B 1800L). The core principle was to store potassium α-ketoglutarate, tryptophan and histidine, and L-histidine hydrochloride monohydrate separately in chambers A and B, respectively. A portion of the electrolytes and mannitol were placed in chamber A, and the remainder in chamber B. The preparation was carried out as follows: 1. Solution preparation: (1) A chamber solution (200L): Weigh 1753.2g sodium chloride, 1342g potassium chloride, 817g tryptophan, 368.4g potassium α-ketoglutarate, and 1093.02g mannitol. Add 160L of purified water to the preparation tank, heat to 40℃, first add the electrolyte and mannitol and stir to dissolve, then add tryptophan and potassium α-ketoglutarate, stir slowly until completely dissolved, and add purified water to make up to 200L. Stir and cool to 25℃, and adjust the pH to 7.0 with 5mol / L potassium hydroxide solution.

[0079] (2) Chamber B solution (1800L): Weigh 9837.18g mannitol, 7546.6g L-histidine hydrochloride monohydrate, 55857.8g histidine, 1626.4g magnesium chloride hexahydrate, and 4.4g calcium chloride dihydrate. Add 1440L of purified water to another preparation tank, heat to 40℃, add the above raw materials in sequence, and stir under nitrogen protection until completely dissolved. Add purified water to make up to 1800L. Stir and cool to 25℃, and adjust the pH to 7.20 with 5mol / L potassium hydroxide solution under nitrogen protection.

[0080] 2. Filtration and filling: The solutions in chambers A and B are precisely filtered through 0.45μm and 0.22μm polyethersulfone filter elements, respectively. Under nitrogen protection throughout the process, the filtered solutions in chambers A and B are filled into the corresponding chambers of the double-chamber bag, heat-sealed to ensure no air bubbles remain, and vacuum-packed with a high-barrier outer packaging film to control the residual oxygen content of the solution to ≤3mg / L and the headspace residual oxygen content to ≤2%.

[0081] 3. Sterilization: Place the sealed double-chamber bag in a sterilization cabinet and sterilize it at 121℃ for 12 minutes. After sterilization, cool it to room temperature to obtain the finished product.

[0082] Example 6: Preparation of Cardiac Arrest Fluid and Organ Preservation Fluid This embodiment prepares a dual-chamber cardiac arrest fluid and organ preservation solution with a total volume of 2000L. The volume ratio of chambers A to B is 2:8 (chamber A 400L, chamber B 1600L). The core principle is to store potassium α-ketoglutarate, tryptophan and histidine, and L-histidine hydrochloride monohydrate separately in chambers. Magnesium and calcium salts are placed in chamber A, and sodium and potassium salts are placed in chamber B. The preparation is carried out as follows: 1. Solution preparation: (1) A chamber solution (400L): Weigh 1626.4g magnesium chloride hexahydrate, 4.4g calcium chloride dihydrate, 817g tryptophan, and 368.4g potassium α-ketoglutarate. Add 320L of purified water to the preparation tank, heat to 40℃, first add the electrolyte and stir to dissolve, then add tryptophan and potassium α-ketoglutarate, stir until completely dissolved, and add purified water to make up to 400L. Stir and cool to 25℃, and adjust the pH to 6.0 with 5mol / L potassium hydroxide solution.

[0083] (2) Chamber B solution (1600L): Weigh 10930.2g mannitol, 7546.6g L-histidine hydrochloride monohydrate, 55857.8g histidine, 1753.2g sodium chloride, and 1342g potassium chloride. Add 1280L of purified water to another preparation tank, heat to 40℃, first add sodium and potassium salts and stir to dissolve, then add the remaining raw materials, and stir under nitrogen protection throughout the process until completely dissolved. Add purified water to make up to 1600L. Stir and cool to 25℃, and adjust the pH to 7.15 with 5mol / L potassium hydroxide solution under nitrogen protection.

[0084] 2. Filtration and filling: The solutions in chambers A and B are respectively filtered through 0.45μm and two-stage 0.22μm polyethersulfone filters. Under nitrogen protection throughout the process, the filtered solutions in chambers A and B are filled into the corresponding chambers of the double-chamber bag, heat-sealed, and vacuum-packed with a high-barrier outer packaging film to control the residual oxygen content of the solution to ≤3mg / L and the headspace residual oxygen content to ≤2%.

[0085] 3. Sterilization: Place the sealed double-chamber bag in a sterilization cabinet and sterilize it at 121℃ for 12 minutes. After sterilization, cool it to room temperature to obtain the finished product.

[0086] Example 7: Preparation of Cardiac Arrest Fluid and Organ Preservation Fluid The only difference from Example 1 is that the raw material of the fluid in cavity A is different.

[0087] Specifically as follows: A-chamber fluid (small chamber, 200L) Weigh the following raw materials according to the specified proportions: sodium chloride 1753.2g, potassium chloride 1342g, magnesium chloride hexahydrate 1626.4g, calcium chloride dihydrate 4.4g, tryptophan 817g, potassium α-ketoglutarate 368.4g, and amiloride 2.66g. Add 160L of purified water to a preparation vessel and heat to 40℃. Add the above electrolytes sequentially, stirring until completely dissolved. Then add tryptophan and potassium α-ketoglutarate, continuing to stir until dissolved. Add purified water to bring the volume to 200L. Stir and cool to 25℃, then adjust the pH to 6.0 with 5mol / L potassium hydroxide solution to obtain cavity A solution.

[0088] Comparative Example 1: Preparation of Single-Compartment Packaging HTK Liquid Comparative Example 1 prepared a single-compartment packaged conventional HTK solution with a total volume of 2000L. The formulation was consistent with the formulation of this invention after mixing, and no compartment storage was performed. It was prepared according to the following method: 1. Solution Preparation: Weigh out 1753.2g sodium chloride, 1342g potassium chloride, 1626.4g magnesium chloride hexahydrate, 4.4g calcium chloride dihydrate, 817g tryptophan, 368.4g potassium hydrogen α-ketoglutarate, 10930.2g mannitol, 7546.6g L-histidine hydrochloride monohydrate, and 55857.8g histidine. Add 1600L of purified water to a preparation tank, heat to 40℃, and add all the above raw materials sequentially, stirring under nitrogen throughout the process until completely dissolved. Add purified water to bring the volume to 2000L. Stir and cool to 25℃, then adjust the pH to 7.15 with 5mol / L potassium hydroxide solution.

[0089] 2. Filtration and filling: The above solution is precisely filtered through 0.45μm and 0.22μm polyethersulfone filter cartridges; it is filled into single-chamber infusion bags under nitrogen protection throughout the process, heat-sealed, and vacuum-packed with a high-barrier outer packaging film to control the residual oxygen content of the solution ≤3mg / L and the headspace residual oxygen content ≤2%.

[0090] 3. Sterilization: Place the packaged single-chamber bag in a sterilizer and sterilize it at 121℃ for 12 minutes. After sterilization, cool it to room temperature to obtain the finished product.

[0091] Performance testing: The dual-chamber products of Examples 1-7 of this invention and the conventional single-chamber packaging product of Comparative Example 1 were subjected to simultaneous accelerated stability and long-term stability studies. The immediate change in potassium α-ketoglutarate content after sterilization was also measured. The study conditions and results are as follows: 1. Immediate changes in content after sterilization: After the dual-chamber products of the present invention were sterilized at 121°C, the content of potassium α-ketoglutarate did not decrease significantly and remained above 99% of the initial content; after the single-chamber packaged products of Comparative Example 1 were sterilized under the same conditions, the content of potassium α-ketoglutarate decreased directly by about 10%, accompanied by the generation of trace impurities.

[0092] 2. Accelerated stability test: The samples were placed at a constant temperature of 40℃ for 6 months. The potassium α-ketoglutarate content of the products in each embodiment of the present invention remained above 98% of the initial content. No increase was observed in tryptophan and histidine-related impurities. The contents of various electrolytes and mannitol did not change significantly. The solution was clear and no crystals precipitated. Under the same conditions, the potassium α-ketoglutarate content of the single-chamber product of Comparative Example 1 decreased by more than 25% compared with the initial value, the impurity content increased significantly, and the solution became slightly turbid.

[0093] 3. Long-term stability test: The samples were placed at room temperature (25°C) for 12 months. The potassium α-ketoglutarate content of the products in each embodiment of the present invention remained above 98% of the initial content. The content of all components met the quality standard requirements. There were no new impurities or excessive content. No crystallization occurred when placed at low temperature (4-8°C). In contrast, the potassium α-ketoglutarate content of the single-chamber product of Comparative Example 1 continued to decrease. After 12 months, it was only about 65% of the initial content. The content of related degradation impurities and tryptophan-histidine reaction impurities far exceeded the quality control limit. Significant crystallization occurred when placed at low temperature.

[0094] Experimental conclusion: As long as the core principle of separate storage of potassium α-ketoglutarate, tryptophan, and histidine in this invention is followed, the prepared dual-chamber HTK solution can significantly improve the sterilization stability and storage stability of potassium α-ketoglutarate and effectively inhibit the generation of impurities. Among them, the component allocation and volume ratio design of Example 1 (all electrolytes, potassium α-ketoglutarate, and tryptophan are placed in chamber A, and histidine, L-histidine hydrochloride monohydrate, and mannitol are placed in chamber B, with an A:B volume ratio of 1:9) is the optimal scheme. Under this scheme, the product not only achieves long-term stability of potassium α-ketoglutarate content, but also takes into account low-temperature solubility, with no crystal precipitation throughout the process, and has the best overall stability.

[0095] Test Example 1: Evaluation of Functional Recovery During Isolated Heart Pause Induction and Reperfusion I. Experimental Objective The purpose of this invention is to verify the ability of the cardiac arrest solution and organ preservation solution (HTK solution) to induce rapid and reversible cardiac arrest in isolated mouse hearts, to evaluate the recovery effect of cardiac function and coronary perfusion after reperfusion, and to clarify its cardiac arrest induction efficacy and myocardial protective effect.

[0096] II. Experimental Materials Experimental animals: 32 healthy, clean-grade adult mice, half male and half female, weighing 21.9–22.3 g, were randomly divided into the Sham group (sham operation group), Example 1 HTK group, Example 7 HTK group, and Comparative Example 1 HTK group, with 8 mice in each group; Experimental reagents: HTK solution prepared in Example 1 of this invention (pre-cooled to 4°C), HTK solution prepared in Example 7 (pre-cooled to 4°C), HTK solution prepared in Comparative Example 1 (pre-cooled to 4°C), KH perfusion fluid (pH 7.35-7.45, osmotic pressure 300±5mOsm / L), heparin sodium solution, and pentobarbital sodium solution; Experimental instruments: Langendorff isolated heart perfusion system, biological signal acquisition and analysis system, electrocardiogram monitoring module, coronary blood flow monitor, low-temperature operating table, etc.

[0097] III. Experimental Methods Animal pretreatment and heart removal: After intraperitoneal injection of heparin sodium for anticoagulation and pentobarbital sodium for anesthesia, the heart was completely removed by thoracotomy, placed in 4°C pre-cooled KH solution to drain residual blood, and connective tissue was trimmed. Perfusion system connection: Fix the aortic cannula to the Langendorff perfusion frame, perform retrograde perfusion at a constant pressure (80 mmHg) of 37℃ KH fluid, continuously introduce a 95% O2-5% CO2 mixed gas, and balance the perfusion for 15-20 minutes until cardiac function is stable. Record baseline cardiac function indicators (LVDP, ±dp / dt, HR, CF). Group treatment: The Sham group was continuously perfused with 37°C KH solution; other groups were switched to cardiac arrest solution and organ preservation solution prepared at 4°C according to Example 1, Example 7 or Comparative Example 1, and perfused at a perfusion pressure of 40-50 mmHg for 8 minutes to induce cardiac arrest, and then re-perfused with 37°C KH solution for 60 minutes. Indicator detection: Record the pause time (from the start of perfusion to the complete cessation of electromechanical activity) and the spontaneous rhythm recovery time (from the start of reperfusion to the recovery of spontaneous rhythm) in the HTK group; at 10, 30 and 60 minutes after reperfusion, the recovery rates of LVDP, ±dp / dt, HR and CF were measured in both groups (recovery rate = reperfusion index value / baseline index value × 100%).

[0098] IV. Experimental Results 1. Baseline indicators: There were no statistically significant differences in baseline body weight and cardiac function indicators (LVDP, ±dp / dt, HR, CF) among the four groups of mice (P > 0.05), and the baseline data were comparable; 2. Effects of pause and re-pumping: Results are shown in Table 8.

[0099] Table 8

[0100] As shown in the table above, both the HTK group in Example 1 and the HTK group in Example 7 can rapidly induce complete cardiac arrest with little individual variation; after reperfusion, spontaneous heart rhythm is successfully restored in both groups without irreversible cardiac arrest; the average cardiac arrest time and average spontaneous heart rhythm recovery time in the HTK group in Comparative Example 1 are significantly prolonged.

[0101] 3. Cardiac function recovery: Results are shown in Table 9.

[0102] Table 9

[0103] As shown in the table above, the LVDP recovery rate, +dp / dt recovery rate, and -dp / dt recovery rate were all high in the HTK group of Example 1 and the HTK group of Example 7 after 60 minutes of reperfusion, while the recovery rate of the HTK group of Comparative Example 1 was low, and there was no statistically significant difference between the two groups and the Sham group at the same time point (P > 0.05).

[0104] 4. Coronary blood flow and heart rate recovery: Results are shown in Table 10.

[0105] Table 10

[0106] As shown in the table above, the coronary blood flow recovery rate and heart rate recovery rate were high in both the HTK group of Example 1 and the HTK group of Example 7 after 60 minutes of reperfusion, and there was no statistically significant difference compared with the Sham group at the same time point (P > 0.05), indicating stable coronary microcirculation perfusion. The effect was significantly reduced in the HTK group of Comparative Example 1.

[0107] V. Experimental Conclusions The cardiac arrest solution and organ preservation solution prepared in Examples 1 and 7 of this invention can rapidly and stably induce electromechanical arrest in isolated mouse hearts, and the arrest has good reversibility. After reperfusion, cardiac function, coronary blood flow and heart rate can be restored to baseline levels without significant myocardial damage. The cardiac arrest induction efficacy and myocardial protection effect are definite, and the effect is significantly better than that of the cardiac arrest solution and organ preservation solution prepared in Comparative Example 1.

[0108] Test Example 2: Isolated Ischemia-Reperfusion (I / R) Myocardial Protection Experiment I. Experimental Objective The clinical ischemia-reperfusion injury scenario was simulated to verify the protective effect of the cardiac arrest fluid and organ preservation fluid of the present invention on ischemia-reperfusion injury of isolated mouse hearts, and to clarify their inhibitory effects on myocardial infarction, cardiac function recovery and cardiomyocyte damage.

[0109] II. Experimental Materials Experimental animals: Forty healthy, clean-grade adult mice, half male and half female, weighing 21.9–22.5 g, were randomly divided into the Sham group (sham operation group), I / R group (ischemia-reperfusion group), Example 1 HTK+I / R group (pretreated with HTK solution prepared in Example 1), Example 7 HTK+I / R group (pretreated with HTK solution prepared in Example 7), and Comparative Example 1 HTK+I / R group (pretreated with HTK solution prepared in Comparative Example 1), with 8 mice in each group.

[0110] Experimental reagents: HTK solution (pre-cooled to 4°C) prepared in Examples 1, 7 and Comparative Example 1 of this invention, KH perfusion fluid, heparin sodium solution, sodium pentobarbital solution, 1% TTC staining solution, LDH / cTnI / SOD / MDA detection kit; Experimental instruments: Langendorff isolated heart perfusion system, biological signal acquisition and analysis system, high-speed refrigerated centrifuge, ImageJ image analysis software, constant temperature water bath, etc.

[0111] III. Experimental Methods Heart removal and balanced perfusion: Same as in test case 1, mice were anesthetized and anticoagulated, their hearts were removed, connected to the Langendorff perfusion system, and perfused at constant pressure with KH solution at 37℃ for 15-20 minutes until cardiac function was stable. Baseline cardiac function indicators were recorded. Group processing: Sham group: continuous constant pressure perfusion with 37℃ KH solution for 120 minutes, without ischemia treatment; I / R group: Perfusion was stopped after equilibration, ischemia was observed for 45 minutes, followed by reperfusion with 37°C KH solution for 60 minutes; HTK+I / R group: After balanced perfusion, pretreatment was performed by perfusion with 4℃ HTK solution for 10 minutes, followed by 45 minutes of ischemia and then reperfusion with 37℃ KH solution for 60 minutes; Indicator Testing: ① At 60 minutes after reperfusion, the cardiac function recovery rate of LVDP, ±dp / dt, and CF was measured. Coronary effusion was collected, and LDH activity and cTnI content were measured. ② After the experiment, the heart was removed, and TTC staining was used to calculate the proportion of myocardial infarction area (infarct area / total myocardial area × 100%) using ImageJ software. ③ Extract myocardial tissue, detect myocardial ATP content, SOD activity, and MDA content, and assess myocardial energy metabolism and oxidative stress levels; ④ Detect the positive rate of cardiomyocyte apoptosis and the Bcl-2 / Bax protein ratio to evaluate the degree of cardiomyocyte apoptosis.

[0112] IV. Experimental Results 1. Baseline indicators: There were no statistically significant differences in baseline body weight and cardiac function indicators (LVDP, ±dp / dt, HR, CF) among the four groups of mice (P > 0.05), ensuring the comparability of experimental results; 2. Infarct area: Results are shown in Table 11.

[0113] Table 11

[0114] Note: ** indicates P < 0.01 compared to the I / R group.

[0115] As shown in the table above, the Sham group had no obvious infarct area (infarct area ratio 0.8±0.3%); the I / R group had a significantly increased infarct area ratio (42.6±5.8%); the infarct area ratios of the HTK+I / R group in Example 1 and the HTK+I / R group in Example 7 were significantly reduced (Example 1 18.3±4.2%, Example 7 10.9±3.9**, both P<0.01 vs I / R group); the effect of Example 1 was significantly worse than that of Example 1 and Example 7.

[0116] 3. Cardiac function recovery: Results are shown in Table 12.

[0117] Table 12

[0118] Note: ** indicates P < 0.01 compared to the I / R group.

[0119] As shown in the table above, the recovery rates of LVDP, +dp / dt, and CF in the I / R group at 60 minutes of reperfusion were 45.8±6.3%, 42.5±5.9%, and 48.2±6.5%, respectively. Compared with the I / R group, the recovery rates of LVDP, +dp / dt, and CF in the HTK+I / R group of Example 1 and the HTK+I / R group of Example 7 were significantly improved at 60 minutes of reperfusion (P < 0.01 vs I / R group), while there was no statistically significant difference compared with the Sham group (P > 0.05). The effect of Comparative Example 1 was significantly worse than that of the Example 1 and Example 7 groups.

[0120] 4. Markers of myocardial injury: Results are shown in Table 13.

[0121] Table 13

[0122] Note: ** indicates P < 0.01 compared to the I / R group.

[0123] As shown in the table above, the LDH activity and cTnI content in the coronary effluent and myocardial tissue of the HTK+I / R group in Example 1 and the HTK+I / R group in Example 7 were significantly lower than those in the I / R group (P < 0.01 vs I / R group), while the myocardial ATP content was significantly higher (P < 0.01 vs I / R group). The effect of Comparative Example 1 was significantly worse than that of the groups in Example 1 and Example 7.

[0124] 5. Oxidative stress and apoptosis: Results are shown in Table 14.

[0125] Table 14

[0126] Note: ** indicates P < 0.01 compared to the I / R group.

[0127] As shown in the table above, the SOD activity in the myocardial tissue of the HTK+I / R group in Example 1 and the HTK+I / R group in Example 7 was significantly increased, and the MDA content was significantly decreased (P < 0.01 vs I / R group); the positive rate of myocardial cell apoptosis decreased, and the Bcl-2 / Bax ratio was significantly increased (P < 0.01 vs I / R group). The effect of Comparative Example 1 was significantly worse than that of the groups in Example 1 and Example 7.

[0128] 6. Myocardial edema and recovery of spontaneous heart rhythm: Results are shown in Table 15.

[0129] Table 15

[0130] Note: ** indicates P < 0.01 compared to the I / R group.

[0131] As shown in the table above, the myocardial water content in the HTK+I / R group of Example 1 and the HTK+I / R group of Example 7 decreased to 77.8±1.8%, which was not statistically different from the Sham group (P>0.05); the spontaneous heart rhythm recovery time was significantly shortened, significantly shorter than that of the I / R group (128.6±18.5 seconds, P<0.01 vs I / R group). The effect of Comparative Example 1 was significantly worse than that of the Example 1 and Example 7 groups.

[0132] Therefore, the HTK solution pretreatment in Example 1 of this invention can significantly reduce ischemia-reperfusion injury in isolated mouse hearts, decrease myocardial infarction area, and effectively promote the recovery of cardiac function and spontaneous rhythm; it can reduce the release of myocardial injury markers, improve myocardial energy metabolism, inhibit oxidative stress and cardiomyocyte apoptosis, and reduce myocardial edema, showing significant multi-target protective effects on ischemic-reperfused myocardium. The HTK solution pretreatment in Example 7 is superior to that in Example 1 because the addition of amiloride simultaneously inhibits NHE1 and NBC, resulting in significant myocardial protection; the dual-channel blockade can more thoroughly prevent Na+ oxidative stress.+ Inflow and secondary Ca 2+ Overload reduces ischemic re-injury and has a synergistic effect. Therefore, the formulation of Example 7 has good application prospects. In Comparative Example 1, a single-chamber design was used. After high-temperature sterilization, the effective concentrations of potassium α-ketoglutarate and tryptophan in the solution decreased significantly, and their effects were significantly reduced. Therefore, this also highlights the advantages of the dual-chamber design of the present invention and the separate treatment of specific potassium α-ketoglutarate and tryptophan.

[0133] The HTK solution prepared by this invention can rapidly and reversibly induce cardiac arrest and has a significant cardioprotective effect on ischemia-reperfusion injury of isolated hearts. It can effectively maintain cardiac function, reduce myocardial necrosis and apoptosis, and improve myocardial energy metabolism. Its protective effect is related to mechanisms such as maintaining the acid-base balance of the myocardial environment, scavenging oxygen free radicals, inhibiting calcium overload and cell apoptosis. The cardioprotective efficacy of the product is definite and meets the effectiveness requirements for clinical application and registration application.

[0134] 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 within the protection scope of the present invention.

Claims

1. A cardiac arrest fluid and organ preservation fluid, characterized in that, The solution is packaged in a dual-chamber bag, which is divided into chamber A and chamber B by a loose weld. The contents of each chamber are stored separately, and the contents of the two chambers are mixed before use. Each 1000ml of the mixed solution contains: 0.8766g sodium chloride, 0.6710g potassium chloride, 0.1842g potassium α-ketoglutarate, 0.8132g magnesium chloride hexahydrate, 3.7733g L-histidine hydrochloride monohydrate, 27.9289g histidine, 0.4085g tryptophan, 5.4651g mannitol, and 0.0022g calcium chloride dihydrate. The pH of the mixed solution is 7.02-7.20, and the osmotic pressure is 310±20mOsm / kg. Notably, potassium α-ketoglutarate is not in the same chamber as L-histidine hydrochloride monohydrate or histidine.

2. The cardiac arrest fluid and organ preservation fluid according to claim 1, characterized in that, The tryptophan is not stored in the same chamber as L-histidine hydrochloride monohydrate or histidine, but in the same chamber as potassium α-ketoglutarate.

3. The cardiac arrest fluid and organ preservation fluid according to claim 1 or 2, characterized in that, The sodium chloride, potassium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate, and mannitol can be placed individually in chamber A or chamber B, or they can be placed separately in two chambers.

4. The cardiac arrest fluid and organ preservation fluid according to claim 3, characterized in that, Chamber A contains potassium α-ketoglutarate and tryptophan, and chamber B contains L-histidine hydrochloride monohydrate, histidine, and mannitol.

5. The cardiac arrest fluid and organ preservation fluid according to claim 4, characterized in that, The volume ratio of chamber A to chamber B is 1:9 to 1:5, preferably 1:

9.

6. The cardiac arrest fluid and organ preservation fluid according to claim 5, characterized in that, The pH of component A is 5.0–7.0, and the pH of component B is 7.10–7.

20.

7. A method for preparing the cardioplegic solution and organ preservation solution as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Solution preparation: Prepare the A component solution corresponding to chamber A and the B component solution corresponding to chamber B respectively; (2) Filtration: After coarse filtration of component A and component B through a 0.45μm filter element, they are then finely filtered through a 0.22μm filter element; (3) Packaging: The filtered A component liquid and B component liquid are filled into the A chamber and B chamber of the double chamber bag respectively. Nitrogen is continuously filled during the filling process. After filling, the double chamber bag is sealed and then a high-barrier outer packaging film is added to the sealed double chamber bag for vacuum packaging. (4) Sterilization: Sterilize the vacuum-packed product to obtain cardiac arrest fluid and organ preservation fluid.

8. The preparation method according to claim 7, characterized in that, In step (1), when preparing component A solution, potassium hydroxide is used to adjust the pH of component A solution to 5.0-7.0; when preparing component B solution, potassium hydroxide is used to adjust the pH of component B solution to 7.10-7.20 under continuous nitrogen purging conditions.

9. The preparation method according to claim 7, characterized in that, In step (4), the sterilization conditions are moist heat sterilization at 121°C for 8 to 15 minutes.

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