TWI781262B - A stable cardioplegic solution for cardiac surgery - Google Patents

Abstract

The present invention is a cardioplegic solution that demonstrates better stability in pH, particulate matter formation and osmolality but at the same time demonstrates superior ability to preserve heart functions than currently available cardioplegic solutions. The cardioplegic solution comprises potassium (K⁺ ), magnesium (Mg²⁺ ), sodium (Na⁺ ), chloride (Cl^- ), gluconate, acetate, sulfate (SO₄ ^2- ), THAM and mannitol dissolved in water.

Description

Stabilized cardioplegia solution for heart surgery

The present invention relates to the field of cardioplegia fluid, in particular to a cardioplegia fluid with stable pH value, formation of particulate matter and osmotic pressure while maintaining heart function.

Cardioplegic fluids are used to induce cardiac paralysis while maintaining the viability of the heart, often for purposes such as heart surgery. For example, a cardioplegia solution may temporarily interrupt myocardial activity by means of modified depolarization, reduce energy expenditure in the organ, promote anaerobic glycolysis during cardioplegia, and prevent the accumulation of calcium ions in cardiomyocytes Cause myocardial damage, scavenge hydrogen ions, and protect compounds with high-energy phosphate bonds. Cardioplegia solution, as a key drug in cardiac surgery, should be stable enough during storage and use, easy to prepare and use, be able to quickly induce cardioplegia and last until the end of cardiac surgery, maintain important heart functions, and be reasonably priced.

Currently commonly used cardioplegia solutions include Custodiol ^® HTK, Plegisol ^® , and del Nido solution. Custodiol ^® HTK is characterized by its low concentration of sodium ions and calcium ions, which are 15mEq/L and 0.015mEq/L respectively, and achieves the effect of cardioplegia through hyperpolarization. In addition, the cardioplegia solution also contains tryptophan and ketoglutarate ions, and uses histidine as a buffer solution. The cardioplegia effect of Custodiol ^® HTK can last up to 4 hours, and the cardioplegia solution needs to be stored at 2 to 8 degrees Celsius, and the shelf life is one year. However, the clinical use of this drug is limited by its relatively high price. The selling price of 1,000mL of Custodiol ^® HTK in the United States is expected to exceed US$1,000, which is four times the price of Plegisol ^® .

Another affordable option is Plegisol ^® , a cardioplegic solution that achieves depolarization with a relatively high potassium ion concentration of 16mEq/L. The cardioplegia solution can be stored at room temperature, and its shelf life is 2 years, but Plegisol ^® is limited by its cardioplegia effect of only about 20 to 30 minutes, and open heart surgery may sometimes take up to 2 hours of action (need multiple administrations of cardioplegia). In addition, Plegisol has also been limited by supply shortages caused by manufacturing delays. ^1,2

The third option is Denido Cardioplegic Solution, Denido Cardioplegic Solution is 16.3 mL of 20% mannitol, 4 mL of 50% magnesium sulfate, 13 mL of 8.4% sodium bicarbonate, and 13 mL of 2 mEq/mL chloride Potassium, add 1 liter of Plasma-Lyte A solution. Each liter of Plasma-Lyte A solution contains 140mEq sodium ion (Na ⁺ ), 5mEq potassium ion (K ⁺ ), 3mEq magnesium ion (Mg ²⁺ ), 98mEq chloride ion (Cl ^- ), 27mEq acetate ion, and 23mEq gluconate ion ion. Alternatively, 13 mL of 1% lidocaine and 20% fully oxygenated patient blood can be added to the solution. Table 1 shows the crystalloid composition of Denido cardioplegia solution before adding lidocaine and oxygenated patient's blood. The cardioplegia solution has the advantage of high potassium content and does not contain calcium ions, which can prevent calcium ions from entering myocardial cells. In addition, a single dose of Denidol cardioplegia is sufficient to induce cardioplegia for a sufficient duration of action, which can reduce the possible cardiac damage caused by multiple administrations of cardioplegia and simplify the surgical procedure. Denido cardioplegic solution is not patented, but Denido's patent application for U.S. Patent No. 5,407,793 claims a different cardioplegic solution comprising histidine, at least one energy-supplying substance (glucose or fructose) ), sodium ions, potassium ions, adenosine, regular insulin, lidocaine and calcium ions. However, neither Denido cardioplegic solution nor the cardioplegic solution of the present invention contains adenosine, histidine or insulin.

¹ https://www.beaumont.org/treatments/heart-surgery-frequently-asked-questions ² https://www.ashp.org/drug-shortages/current-shortages/Drug-Shortage-Detail.aspx？id=121

¹ https://www.beaumont.org/treatments/heart-surgery-frequently-asked-questions ² https://www.ashp.org/drug-shortages/current-shortages/Drug-Shortage-Detail.aspx? id=121

Although Denido cardioplegia has many advantages, it must be stored at 2 to 8 degrees Celsius, and the storage period is only 45 days. In addition, heat sterilization of the cardioplegic solution produces sediment or particulate matter, examples of which are shown in relation to the particulate matter in Figures 4A and 4B. Since the cost of filtration sterilization is higher than that of heat sterilization, the commercial production of cardioplegic solution usually uses heat sterilization as the main sterilization process. However, the particulate matter produced after heat sterilization makes Denido cardioplegic solution unusable. Due to the short shelf life of Denido Cardioplegic Solution and the generation of particulate matter in the solution due to heat sterilization, there is currently no commercial ready-made Denido Cardioplegic Solution. The existing practice is to prepare Denido Cardioplegic Solution in the hospital before open heart surgery Cardioplegic solution, or outsource the preparation of Denido Cardioplegic solution to a sterile pharmaceutical compounding company. But preparing the solution on the spot adds many risks of human error to the surgery, another drawback of Denido's cardioplegia solution. Therefore, it is necessary to develop another cardioplegic solution that is at least as effective as Denido cardioplegic solution in causing cardioplegia and protecting heart function, but has a longer shelf life and does not produce particulate matter after heat sterilization, so that it can be produced It is a commercialized cardioplegia solution, which substantially reduces the need for on-the-spot preparation before use.

Compared with the existing cardioplegia solution, the present invention is a cardioplegia solution that is more stable in terms of pH value, particulate matter formation and osmotic pressure, and has better ability to maintain heart function. The cardioplegia solution contains potassium ions (K ⁺ ), magnesium ions (Mg ²⁺ ), sodium ions (Na ⁺ ), chloride ions (Cl ^- ), gluconate ions, acetate ions, sulfate ions ( SO ₄ ^2- ), trishydroxymethylaminomethane (THAM) and mannitol. The present invention also discloses a method for preparing the cardioplegic solution of the present invention, a use of the cardioplegic solution of the present invention for preparing a composition for inducing temporary cardiac paralysis, and a method of administering the cardioplegic solution of the present invention.

Figure 1 compares the pH values of the four cardioplegic solutions studied in this experiment. The Y axis represents the pH value, and the X axis represents the number of days of the experiment. The four kinds of cardioplegia solutions in this study were stored at 25°C. They were the WAW cardioplegia solution of the present invention containing 10mmol/L THAM as shown in Table 1, and the WAW heart of the present invention containing 20mmol/L THAM as shown in Table 1. Anesthetic solution, heat sterilized Denido cardioplegic solution, and filter sterilized Denido cardioplegic solution.

Figure 2 compares the pH values of the four cardioplegic solutions studied in this experiment, and the Y axis represents the pH value, and the X-axis represents the number of experimental days. The four kinds of cardioplegia solutions in this study were stored at 16°C. They were respectively the WAW cardioplegia solution of the present invention containing 10mmol/L THAM as shown in Table 1, and the WAW heart of the present invention containing 20mmol/L THAM as shown in Table 1. Anesthetic solution, heat sterilized Denido cardioplegic solution, and filter sterilized Denido cardioplegic solution.

Figure 3 is a comparison of the pH values of the four cardioplegic solutions studied in this experiment. The Y-axis represents the pH value, and the X-axis represents the number of days of the experiment. The four kinds of cardioplegia solutions in this study were stored at 4°C. They were the WAW cardioplegia solution of the present invention containing 10mmol/L THAM as shown in Table 1, and the WAW heart of the present invention containing 20mmol/L THAM as shown in Table 1. Anesthetic solution, heat sterilized Denido cardioplegic solution, and filter sterilized Denido cardioplegic solution.

Figures 4A and 4B compare the particulate matter concentrations of the four cardioplegic fluids studied in this experiment, respectively illustrating the concentration of particulate matter >=10 μm and the concentration of particulate matter >=25 μm, and the Y axis represents the number of particulate matter per mL , and the X-axis represents the number of experimental days. The four kinds of cardioplegia solutions in this study are stored at room temperature, and they are the WAW cardioplegia solution of the present invention containing 10mmol/L THAM as shown in Table 1, and the WAW heart of the present invention containing 20mmol/L THAM as shown in Table 1. Anesthetic solution, heat sterilized Denido cardioplegic solution, and filter sterilized Denido cardioplegic solution.

Figure 5 is a comparison of the osmotic pressures of the four cardioplegic solutions studied in this experiment. The Y-axis represents the osmotic pressure in mOsm/kg, and the X-axis represents the number of days of the experiment. The four kinds of cardioplegia solutions in this study are stored at room temperature, and they are the WAW cardioplegia solution of the present invention containing 10mmol/L THAM as shown in Table 1, and the WAW heart of the present invention containing 20mmol/L THAM as shown in Table 1. Anesthetic solution, heat sterilized Denido cardioplegic solution, and filter sterilized Denido cardioplegic solution.

Figures 6A and 6B show the expression levels of Bcl-2 in rat hearts after administration of the three cardioplegic solutions studied in this experiment. The three kinds of cardioplegia solutions in this study are respectively the components as shown in Table 1, containing 20mmol/L THAM's WAW Cardioplegic Solution (Denoted W), Denido Cardioplegic Solution (Denoted D), and HTK Cardioplegic Solution (Denoted H) of the present invention.

Figure 7 shows the expression ratio of Bax/Bcl-2 in rat hearts after administration of the three cardioplegic solutions studied in this experiment. The three cardioplegia solutions used in this study are the WAW cardioplegia solution of the present invention (labeled as WAW), Denido cardioplegia solution (labeled as DND), and HTK cardioplegia solution as shown in Table 1. liquid (labeled as HTK).

8A and 8B show rat hearts and survival rates of heart cells. More specifically, Figure 8A compares the number of rats in the six groups used in the experiment to achieve the necessary n=6 per group, and the number of rats in the final data collection. The Y-axis represents the number of rats, and the X-axis represents the experiment Six solutions were administered. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as P), HTK cardioplegia solution (marked as H), Denido cardioplegia solution (marked as D), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (marked as W) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (marked as K) of the positive control group, and normal saline (marked as S) of the negative control group. Figure 8B compares the ratio of myocardial cell death due to local ischemia after the rat heart was given the five solutions studied in this experiment. solution. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as P), HTK cardioplegia solution (marked as H), Denido cardioplegia solution (marked as D), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (marked as W) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (marked as K) of the positive control group, and normal saline (marked as S) of the negative control group.

Figure 9 is a comparison of the electrocardiogram voltages of rat hearts administered with the five solutions studied in this experiment. The Y-axis represents the voltage in mV, and the X-axis represents the time. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Figure 10 is a comparison of the left ventricle systolic pressure (LVSP) of rat hearts administered with the five solutions studied in this experiment. The Y-axis represents the left ventricular systolic pressure in mmHg, and the X-axis represents time. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Figure 11 is a comparison of the left ventricle diastolic pressure (LVDP) of rat hearts administered with the five solutions studied in this experiment. The Y-axis represents the left ventricular diastolic pressure in mmHg, and the X-axis represents time. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Figure 12 is a comparison of the heart rate (heart rate, HR) of the rat heart administered with the five solutions studied in this experiment. The Y-axis represents the heart rate in bpm, and the X-axis represents the time. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Fig. 13 is a comparison of the coronary blood flow (coronary flow, CF) in the heart of rats administered with the five solutions studied in this experiment. The Y-axis represents the coronary blood flow in mL/min, and the X-axis represents time. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Figure 14 is a comparison of the expression of lactate dehydrogenase (lactate dehydrogenase, LDH) after the rat heart was given five kinds of solutions studied in this experiment. The Y axis represents the expression of lactate dehydrogenase in U/L, and the X axis represents time. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Table 1 lists the ion composition and concentration of Denido Cardioplegic Solution and an example of the cardioplegic solution of the present disclosure (labeled as WAW Cardioplegic Solution) dissolved in water.

Table 2 lists the experimental data used in drawing Figure 9, comparing the changes in the voltage (in mV) of the electrocardiogram over time after the rat heart was given the five solutions of this experimental study. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Table 3 lists the experimental data used in drawing Figure 10, comparing the changes in left ventricular systolic pressure (unit: mmHg) over time after the five solutions in this experimental study were administered to the rat heart. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Table 4 lists the experimental data used in drawing Figure 11, comparing the changes in left ventricular diastolic pressure (unit: mmHg) over time after rat hearts were administered with the five solutions studied in this experiment. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Table 5 lists the experimental data used in drawing Figure 12, comparing the changes in heart rate (unit: bpm) over time after the five solutions in this experimental study were administered to the heart of rats. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention in THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Table 6 lists the experimental data used in drawing Figure 13, comparing the changes in coronary blood flow (in mL/min) over time after rat hearts were administered with the five solutions studied in this experiment. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

Table 7 lists the experimental data used in drawing Figure 14, comparing the changes in the expression of lactate dehydrogenase (unit: U/L) over time after rat hearts were administered with the five solutions of this experimental study. The five kinds of solutions in this study are respectively ^Plegisol® cardioplegia solution (marked as Plegisol), HTK cardioplegia solution (marked as HTK), Denido cardioplegia solution (marked as DND), and the ingredients are as shown in Table 1, containing 20mmol/L WAW cardioplegia solution (labeled as WAW) of the present invention of THAM, KH buffer solution (Krebs-Henseleit buffer) (labeled as KH) of the positive control group, and normal saline (labeled as Saline) of the negative control group.

In the following content used in the specification and patent application, unless otherwise specified in the text, the singular forms "a" and "the" include plural references. Thus, for example, reference to "an ingredient" includes a mixture of ingredients, reference to "an active agent" includes more than one active agent, and the like.

In this disclosure, the word "about" is used as a modifier of an amount to mean including + or -5% of the modified amount.

In this disclosure, the term "effective amount" or "therapeutically effective amount" of a drug or pharmaceutical active ingredient is used to mean a non-toxic amount of the drug or active agent that is sufficient to provide the desired therapeutic effect. This amount that is "effective" will vary from individual to individual, depending on the individual's age and general condition, the particular active agent, and the like. An appropriate "effective" amount in any particular instance can be determined by one of ordinary skill in the art using routine experimentation.

The cardioplegia solution disclosed in this disclosure improves the shortcomings of the Denido cardioplegia solution mentioned in the prior art, and the ability to maintain heart function is substantially better than that of the Denido cardioplegia solution. More specifically, applicants have unexpectedly discovered that substituting the sodium hydroxide of the Denido cardioplegic solution with an appropriate concentration of trishydroxymethylaminomethane (THAM), also known as Tris, can prevent the cardioplegic solution of the present disclosure from forming particles due to heat sterilization Substances, as described below in relation to the embodiments shown in Figures 4A and 4B. In addition, the cardioplegia solution disclosed in this disclosure still maintains a stable pH value and osmotic pressure after being stored for 365 days, so that the pH value maintains the ion concentration in the critical range for causing cardioplegia, as shown in Figure 1-3, and the osmotic pressure is maintained at The harmless range to the human body is shown in Figure 5. In addition, the ingredients that can be added before use are limited to fully oxygenated patient blood and/or lidocaine, which can be selectively added according to the needs of cardiac surgeons; , the present invention substantially reduces the possibility of human error in the process of preparing the cardioplegia solution. Importantly, the Examples and related studies described below in relation to Figures 6-14 show that the cardioplegic fluids of the present disclosure are surprisingly superior to other cardioplegic fluids including Denido Cardioplegic Fluid in their ability to preserve cardiac function. For example, according to the expression level of BCL-2 and the ratio of BAX/BCL-2 discussed in the following examples related to FIG. 6 and FIG. 7 , the cardioplegia solution of the present disclosure can reduce cell apoptosis. In addition, as shown in the following embodiments related to FIGS. 8-14 , From the experimental results of electrocardiogram voltage, left ventricular systolic pressure, left ventricular diastolic pressure, heart rate, coronary blood flow, and lactate dehydrogenase expression, it can be seen that compared with Denido cardioplegic solution, the cardioplegic solution disclosed in this disclosure has greater maintenance The effect of mouse heart function is better.

In addition, the applicant of the present disclosure is the first to discover that replacing sodium bicarbonate with THAM at an appropriate concentration can not only reduce the instability of particulate matter formed after heat sterilization of cardioplegic solution, but also substantially improve the effect of maintaining heart function as described above. as above. More specifically, as far as the applicant is aware, there is no prior art showing that substituting THAM for sodium bicarbonate in the Denido cardioplegia solution can make the cardioplegia solution have the above-mentioned properties. For example, Volgushev's US Patent Application No. 15/325,501 indicated that THAM is not an essential component of cardioplegia solution and can be replaced by sodium bicarbonate or other pharmaceutically acceptable bases. ('501 Patent Application, Section 0023). This statement is inconsistent with the following examples in relation to Figures 1-3, which show that sodium bicarbonate causes problems with the instability of Denido cardioplegic solution, making it unavailable for commercialization, whereas THAM does not. the problem. Furthermore, the '501 patent application only discusses maintaining the pH of a solution within a specific range, and more specifically, the patent application only discusses maintaining a pH of a solution at a pH below 8, which target pH range is substantially lower. The cardioplegia solution disclosed herein does not include overall stability such as preventing the solution from forming precipitates during storage. ('501 patent application, section 0022 and claims 1, 2, 3 and 4). Furthermore, the cardioplegic solution described in the '501 patent application is a cardioplegic solution substantially different in composition and preparation from the present invention. For example, the cardioplegia solution described in the '501 patent application was prepared from a different compound without the sodium ions or gluconate ions included in the present invention. ('501 Patent Application, Sections 0047-0081 and Claims 1-4). The cardioplegia solution described in the '501 patent application has an osmotic pressure greater than 400 mOsm/kg, substantially greater than the osmotic pressure of the present invention, and a pH value lower than 8 is also lower than the pH value of the present invention. ('501 Patent Application, Sections 0022, 0025, and 0026).

Since the applicant is the first to discover that THAM is used at an appropriate concentration instead of sodium bicarbonate to improve Denido cardioplegic solution, it can eliminate the problem of particulate matter in the solution due to heat sterilization, and maintain the cardioplegic solution at a stable pH value of at least 365, in order to commercialize the cardioplegia solution, the present invention should conform to the Phair doctrine, and according to this principle, the present invention should be patentable. More specifically, Ex parte Phair, 1 USPQ 133,134 (Bd.App.1929) "The Phair Principle" ruled that: "An invention consists in discovering the cause of a defect in an existing machine or The reason then becomes apparent.” The court later upheld this principle in In re Sponnoble, 405 F.2d 579, 56 CCPA 823, 160 USPQ 237 (1969), and subsequent cases followed it. In Sponnoble, the court found, based on a substantial body of opinion evidence, that "the evidence clearly shows that he [Sponnoble] discovered the source of the problem." 405 F.2d at 585, 56 CCPA at 833, 100 USPQ at 243. Similarly, the applicant disclosed sufficient evidence in the examples and corresponding charts of this patent application to prove that replacing sodium bicarbonate with THAM at an appropriate concentration can solve the problem of generating particulate matter in Denido solution caused by heat sterilization, making the present invention be commercialized.

In one embodiment of the present invention, the cardioplegia solution contains potassium ions (K ⁺ ), magnesium ions (Mg ²⁺ ), sodium ions (Na ⁺ ), chloride ions (Cl ^- ), gluconate ions, acetate ions, Sulfate ion (SO ₄ ^2- ), THAM and mannitol. In one embodiment, fully oxygenated patient blood and/or lidocaine may optionally be added prior to administration of the cardioplegic solution. In one embodiment, the composition of the present invention comprises about 27 to about 33 mEq/L potassium ions (K ⁺ ), about 16 to about 20 mEq/L magnesium ions (Mg ²⁺ ), about 120 to about 146 mEq/L magnesium ions (Mg 2+ ) dissolved in water. L sodium ion (Na ⁺ ), about 106 to about 130 mEq/L chloride ion (Cl ^- ), about 20 to about 24 mmol/L gluconate ion, about 22 to about 28 mmol/L acetate ion, about 6 to about 9 mmol/L L sulfate ion (SO ₄ ²⁻ ), about 5 to about 30 mmol/L THAM, and about 2 to about 5 g/L mannitol. In another embodiment, the composition of the present invention comprises about 29.63 mEq/L potassium ions (K ⁺ ), about 18.39 mEq/L magnesium ions (Mg ²⁺ ), about 133.80 mEq/L sodium ions (Na ⁺ ), about 118.51mEq/L chloride ion (Cl ^- ), about 21.98mmol/L gluconate ion, about 25.81mmol/L acetate ion, about 7.76mmol/L sulfate ion (SO ₄ ^2- ), about 10mmol /L THAM or about 20mmol/L THAM, and about 3.116g/L mannitol. In one embodiment, the water used to prepare the cardioplegia solution is distilled water, possibly sterile water. In another embodiment, the cardioplegia solution of the present invention does not contain calcium ions (Ca ²⁺ ). In another embodiment, the cardioplegia solution of the present invention does not contain sodium bicarbonate. Any of the embodiments of the present invention may further comprise about 0.1 to about 0.14 mg/mL lidocaine in the final solution. In addition, any embodiment of the present invention may further comprise fully oxygenated patient blood, the volume ratio of the fully oxygenated patient blood to the WAW cardioplegia fluid of the present invention is about 1:1 to about 1:8. In another embodiment, lidocaine may be replaced by other polarizing agents, such as procaine. In another embodiment, glacial acetic acid can be replaced by other pharmaceutically acceptable acids, such as hydrochloric acid, phosphoric acid or lactic acid. Accordingly, a use of the cardioplegia solution of the present invention for the preparation of a composition for inducing temporary cardiac paralysis can be provided.

The disclosure also provides a method for preparing the cardioplegia solution of the present invention, comprising the steps of: (a) mixing about 75 to about 95 mmol/L sodium chloride (NaCl), about 20 to about 24 mmol/L sodium gluconate (C ₆ H ₁₁ NaO ₇ ), about 22 to about 28 mmol/L sodium acetate trihydrate USP (C ₂ H ₃ NaO ₂ .3H ₂ O), about 27 to about 33 mmol/L potassium chloride USP (KCl), about 1.2 to About 1.6mmol/L magnesium chloride hexahydrate USP (MgCl ₂ .6H ₂ O), about 6 to about 9 mmol/L magnesium sulfate heptahydrate USP (MgSO ₄ .7H ₂ O), and about 15 to about 20 mmol/L mannitol USP (C ₆ H ₁₄ O ₆ ). In another embodiment, the method for preparing the cardioplegia solution of the present invention is the following step (a): mixing about 86.02mmol/L sodium chloride (NaCl), about 21.99mmol/L sodium gluconate (C ₆ H ₁₁ ) in water NaO ₇ ), about 25.85mmol/L sodium acetate trihydrate USP (C ₂ H ₃ NaO ₂ .3H ₂ O), about 29.59mmol/L potassium chloride USP (KCl), about 1.41mmol/L magnesium chloride hexahydrate USP ( MgCl ₂ .6H ₂ O), about 7.76mmol/L magnesium sulfate heptahydrate USP (MgSO ₄ .7H ₂ O), and about 17.1mmol/L mannitol USP (C ₆ H ₁₄ O ₆ ). In one embodiment, the water used to prepare the cardioplegia solution is distilled water, possibly sterile water. The method for preparing the cardioplegia solution of the present invention further comprises the following step (b): THAM and a pharmaceutically acceptable acid are mixed into the solution prepared in step (a), and the solution is adjusted to a desired pH value of about 8.2 to about 8.6, and bring the THAM concentration to about 5 to about 30 mmol/L, about 10 to about 20 mmol/L, about 10 mmol/L, or about 20 mmol/L. In one embodiment, the pharmaceutically acceptable acid is glacial acetic acid. The composition can be prepared 365 days before use. In one embodiment, the preparation method of the cardioplegia solution of the present invention may optionally include another step: mixing lidocaine and/or Fully oxygenated patient blood.

In one embodiment, the method for preparing the cardioplegia solution of the present invention comprises the steps of: mixing about 15 to about 19 mmol of mannitol, about 5 to about 10 mmol of magnesium sulfate, and about 22 to about 27 mmol of potassium chloride into 1 liter of Plasma-Lyte A solution. In another embodiment, step (a) of the method for preparing the cardioplegia solution of the present invention comprises mixing about 17.10 mmol of mannitol, about 7.76 mmol of magnesium sulfate, and about 24.85 mmol of potassium chloride into 1 liter of Plasma-Lyte A solution. Each 1L of Plasma-Lyte A base solution contains about 140mEq sodium ion (Na ⁺ ), about 5mEq potassium ion (K ⁺ ), about 3mEq magnesium ion (Mg ²⁺ ), about 98mEq chloride ion (Cl ^- ), about 27mEq acetate ions, with approximately 23mEq gluconate ions. The method for preparing the cardioplegia solution of the present invention further comprises step (b): mixing THAM and a pharmaceutically acceptable acid into the solution prepared in step (a), and adjusting the solution to a desired pH value of about 8.2 to about 8.6, and bring the THAM concentration to about 5 to about 30 mmol/L, about 10 to about 20 mmol/L, about 10 mmol/L, or about 20 mmol/L. In another embodiment, lidocaine may be replaced by other polarizing agents, such as procaine.

In one embodiment of the present invention, the cardioplegia solution prepared by any method of the present disclosure comprises about 29.63 mEq/L K ^{+ , about 18.39 mEq/L Mg 2+ ,} about 133.80 mEq/L dissolved in water Na ⁺ , about 118.51mEq/L Cl ^- , about 21.98mmol/L gluconate ion, about 25.81mmol/L acetate ion, about 7.76mmol/L sulfate ion, about 5 to about 30mmol/L THAM, and about 3.116 g/L Mannitol. In one embodiment, the cardioplegia solution prepared by the method of the present disclosure does not contain sodium bicarbonate. In one embodiment, the cardioplegia solution prepared by the method of the present disclosure does not contain calcium ions.

The present invention also provides a method for administering any cardioplegia solution disclosed herein to a patient's heart during heart surgery to induce cardioplegia, the method comprising any existing administration method for inducing cardioplegia with Denidol solution. In one embodiment, the method of administering any cardioplegic solution of the present invention to a patient during cardiac surgery to induce cardioplegia comprises rapidly perfusing the patient's heart with any cardioplegic solution of the present invention. In one embodiment, 20 to 30 mL/kg of the cardioplegia solution of the present invention can be perfused into the patient's heart. The cardioplegic solution may be stored at a temperature of from about 4°C to about room temperature.

In one embodiment, the cardioplegia solution is infused into the root of the cross-clamped aorta, and/or directly into the coronary sinus. In one embodiment, a balloon-cuffed catheter can be used as a catheter for cardioplegia fluid through the right atrium into the coronary sinus, and the cardioplegia fluid can be infused into the coronary circulation through venous circulation. ). The advantage of this method is that when used in patients with diffuse coronary artery disease (diffuse coronary artery disease), it can be more evenly distributed, and its delivery does not depend on a functioning aortic valve (aortic valve).

In one embodiment of the present invention, the method of using the cardioplegia solution of the present invention in cardiac surgery to induce temporary cardiac paralysis comprises infusing the heart with the cardioplegia solution of the present invention at about 4°C to about 35°C Inject the patient's heart, preferably at about 10°C to about 21°C, most preferably at about 13°C. Those skilled in the art know that using the present invention to induce cardioplegia in a patient to preserve cardiac function involves perfusing the heart with the aqueous solution under moderate hypothermia. The administration procedure of the present invention further comprises perfusing the heart with about 20 to about 30 mL/kg of cardioplegic solution. Another embodiment of the present invention comprises perfusing the patient's heart with the aqueous solution of the present invention at moderately low temperature every 20 to 40 minutes to induce cardioplegia and preserve cardiac function. The method of use of the present invention may comprise perfusing the heart with the aqueous solution of the present invention in a cycle of about 20 to 40 minutes, at moderate hypothermia for at least about 24 cycles. In another embodiment, the method of use of the present invention comprises continuous delivery of a cardioplegic solution.

In any of the embodiments of the present disclosure, the cardioplegic solution of the present invention maintains a pH of about 8.2 to about 8.6, or a pH of about 8.3 to about 8.5, or a pH of about 8.4. In any embodiment of the present disclosure, the THAM concentration of the cardioplegic solution of the present invention is maintained at about 5 mM to about 30 mM, or about 5 mM to about 20 mM, or about 10 mM to about 20 mM. In one embodiment, the composition of the present invention contains 20 mM THAM, and its storage temperature is about 25°C.

In one embodiment, the storage temperature of the cardioplegic solution of the present invention is about 4°C to about 25°C in order to ensure stable pH value, stable osmotic pressure and prevent the formation of particulate matter. In one embodiment, any cardioplegic solution of the present disclosure is stored at a temperature of about 4°C to about 16°C to ensure stable pH, stable osmotic pressure and prevent the formation of particulate matter. In another embodiment, in order to ensure stable pH, stable osmotic pressure and prevent the formation of particulate matter, the storage temperature of any cardioplegic solution of the present disclosure is about 16°C.

[Example]

Stability test

In order to compare the stability of pH value, particulate matter formation and osmotic pressure between the cardioplegic solution of the present invention and Denido cardioplegic solution stored at 25°C, 16°C and 4°C for up to 365 days, the inventors implemented three stabilization measures sex experiment. The four cardioplegic fluids studied in this experiment included two examples of the WAW cardioplegic fluid of the present invention whose ingredients are shown in Table 1, and two Denido cardioplegic fluids. More specifically, two embodiments of the present invention used in this experiment, one contains about 29.63 mEq/L K ⁺ , about 18.39 mEq/L Mg ²⁺ , about 133.80 mEq/L Na ⁺ , about 118.51 mEq/L Cl ^- , about 21.98mmol/L gluconate ion, about 25.81mmol/L acetate ion, about 7.76mmol/L sulfate ion, about 3.116g/L mannitol, and an aqueous solution of about 10mM THAM, and the other is an aqueous solution with The former is the same aqueous solution, the only difference is that the concentration of THAM is 20 mM instead of 10 mM. The two cardioplegic solutions of the present invention are all sterilized by heat. The first Denido cardioplegic solution used in the stability test was also heat sterilized, and the second Denido cardioplegic solution used in the stability test was filter sterilized instead of heat sterilized.

The above four cardioplegia solutions were stored at 4°C, 16°C and 25°C after sterilization, and stored on the 1st day, 7th day, 14th day, 28th day, 90th day, 120th day and 365th day Days to collect experimental data. The heat sterilization method is to heat the solution to 121°C in a sterilizer for 15 minutes.

The standard pH value of the cardioplegia solution in this experiment is about 8.2 to about 8.6, which is the pH range for reducing metabolic acidosis during cardioplegia. The stability standard of particulate matter in this experiment is that the concentration of >=10 μm particulate matter must be equal to or lower than 25/mL, and the concentration of >=25 μm particulate matter must be equal to or lower than 3/mL. Pharmacopeia, USP) defines acceptable particulate matter concentrations for pharmaceutical solutions. The osmotic pressure stability standard of this experiment is that the osmotic pressure should be less than 388mOsm/kg. This is the osmotic pressure limit for the safety of the human body obtained from the observation of THAM-containing products. Take THAM Hospira as an example, the osmotic pressure of its 0.3M solution It is 389mOsm/L.

pH stability test

Fig. 1, Fig. 2 and Fig. 3 compare two kinds of embodiments of the cardioplegia solution of the present invention, the Denido cardioplegia solution through heat sterilization and the Denido cardioplegia solution through filter sterilization respectively. The pH value stability test results after 365 days of storage at ℃ and 4℃.

Fig. 1 is the test result of the pH value stability of the above four kinds of cardioplegia solutions stored at 25°C. As shown in FIG. 1 , only the WAW cardioplegia solution of the present invention containing about 20 mM THAM maintained a pH value of 8.2 to 8.6 during the 365-day experiment, meeting the pH stability criteria. The pH of the WAW cardioplegia solution of the present invention containing about 10 mM THAM dropped below 8.2 between the 80th day and the 310th day. Nevertheless, the pH stability of the two cardioplegic solutions of the present invention is substantially better than that of the two Denido cardioplegic solutions during the 365-day experiment. For example, the pH of the heat-sterilized Denido cardioplegic solution The pH value was continuously higher than 8.6 during the experiment period, and the pH value of the filter-sterilized Denido cardioplegia solution was only maintained in the range of 8.2 to 8.6 in the first 90 days, and the pH value continued to exceed the high standard of 8.6 after 90 days.

Fig. 2 is the test result of the pH value stability of the above four cardioplegic solutions stored at 16°C. As shown in FIG. 2 , the two embodiments of the WAW cardioplegia solution of the present invention maintained a pH value of 8.2 to 8.6 during the 365-day experiment. The two Denido cardioplegic solutions used in this experiment did not maintain a pH value of 8.2 to 8.6. More specifically, the pH of the heat-sterilized Denido cardioplegic solution was continuously higher than 8.6 during the experiment. The Denido Cardioplegic Solution was only maintained in the range of 8.2 to 8.6 for the first 90 days, and its pH value continued to exceed the high standard of 8.6 after 90 days.

Fig. 3 is the test result of the pH value stability of the above four kinds of cardioplegia solutions stored at 4°C. As shown in Figure 3, for the WAW cardioplegia solution of the present invention containing 10mM THAM, the pH value dropped below the low mark of 8.2 from the 150th day to the 300th day, and for the WAW cardioplegia solution of the present invention containing 20mM THAM, the pH The value is above the high mark of 8.6 from day 0 to about day 30. The pH stability of the two Denido cardioplegic solutions used in this experiment was similar to that when stored at 16°C, and the pH values were inconsistent most or all of the time during the experiment Meet the standards from 8.2 to 8.6. For example, the pH value of the heat-sterilized Denido cardioplegic solution was consistently higher than the high standard of 8.6 during the experiment, and the filter-sterilized Denido cardioplegic solution only met the pH in the first 90 days. A pH value of 8.2 to 8.6 is standard, and its pH value is continuously higher than 8.6 after the 90th day.

Therefore, as shown in Figure 1, Figure 2 and Figure 3, at the three storage temperatures of 4°C, 16°C and 25°C, the pH stability of the two embodiments of the WAW cardioplegia solution of the present invention is substantially better than that after heat sterilization With filter sterilized Denido cardioplegic solution. As shown in FIG. 3 , the WAW cardioplegia solution of the present invention with a THAM concentration of 20 mM had the most stable pH value during the 365-day experiment. Therefore, in one embodiment, the WAW cardioplegia solution containing 20 mM THAM with the ingredients shown in Table 1 was stored at 25°C. In addition, as shown in FIG. 2 , the WAW cardioplegia solution of the present invention containing 10 mM THAM and the WAW cardioplegia solution of the present invention containing 20 mM THAM maintained a pH of 8.2 to 8.6 during the experiment. Therefore, in another embodiment, the WAW cardioplegia solution containing 10 mM THAM containing the ingredients in Table 1 of the present invention was stored at 16°C. In another embodiment, the WAW cardioplegia solution containing 20 mM THAM containing the ingredients in Table 1 of the present invention was stored at 16°C.

Particulate Matter Stability Test

4A and 4B are experimental results of particulate matter stability experiments. As shown in Figures 4A and 4B, the results of the particle matter stability test of the heat-sterilized Denido cardioplegia solution did not meet the standard at all during the 365-day test period, while the other three cardioplegic solutions all met the standard. More specifically, the heat-sterilized Denido cardioplegic solution exceeded the concentration standards for particulate matter >= 10 μm in volume and >= 25 μm in volume on day 0. Although the experimental data of the 7th, 15th, and 28th days in Figure 4A showed that the concentration of particulate matter >=10 μm decreased to an acceptable range during the experimental observation period, it exceeded the acceptable range again after the 90th day concentration range. Similarly, as shown in the experimental data of the 15th and 28th days in Figure 4B, although the concentration of particulate matter >= 25 μm only dropped to the acceptable range during part of the experimental observation period, it exceeded the acceptable range again after the 90th day. Accepted concentration range. As mentioned above, the importance of this experimental result lies in the fact that heat sterilization is a preferred and common process for commercialization, but it makes it difficult for Denido cardioplegic solution to be commercialized, but the cardioplegic solution of the present invention has solved this problem and can commoditization. Therefore, one embodiment of the present invention is about 29.63 mEq/L K ⁺ , about 18.39 mEq/L Mg ²⁺ , about 133.80 mEq/L Na ⁺ , about 118.51 mEq/L Cl ^- , about 21.98 mmol/L dissolved in water Gluconate ion, about 25.81mmol/L acetate ion, about 7.76mmol/L sulfate ion, about 3.116g/L mannitol, and cardioplegia solution with THAM concentration between about 10mM and about 20mM, and the sterilization is Sterilized by heat.

Osmotic pressure stability test

Fig. 5 is the experimental result of the osmotic pressure stability experiment. As shown in Figure 5, the four cardioplegia solutions in this experiment all meet the standard of osmotic pressure lower than 388mOsm/kg. In addition, the osmotic pressure of the WAW cardioplegia solution of the present invention containing 20 mM THAM is the highest, and the osmotic pressure of the WAW cardioplegia solution of the present invention containing 10 mM THAM is similar to that of Denido cardioplegia solution sterilized by filtration.

Cardiac paralysis experiment

The subjects of this experiment were male Sprague-Dawley rats with a body weight of about 350 to about 450 grams. The rats were divided into three groups according to the three types of cardioplegia to be administered, namely Denido cardioplegia group (D), For the WAW cardioplegia solution group (W) and the HTK cardioplegia solution group (H) of the present invention, the number of rats in each group is n=5+1. The WAW cardioplegia solution of the present invention used for cardioplegia experiment contains about 29.63mEq/LK ⁺ , about 18.39mEq/L Mg ²⁺ , about 133.80mEq/L Na ⁺ , about 118.51mEq/L Cl ^- , about 21.98mmol/ L gluconate ion, about 25.81mmol/L acetate ion, about 7.76mmol/L sulfate ion, about 20mM THAM, and about 3.116g/L mannitol.

Rats were given isoflurane via an anesthesia vaporizer machine (Isoflurane) anesthesia, 3,000 U/kg heparin (heparin) was administered by intraperitoneal injection (intraperitoneal injection) after complete anesthesia. The researcher isolated the rat heart, placed it in a container containing a small amount of ice-cold KB buffer (Krebs-Henseleit buffer, KHB), and then intubated the aorta and left atrium of the heart. Perfuse the heart with the corresponding cardioplegia solution. Groups W and D were injected with 20 mL/kg of cardioplegia solution, and group H was injected with 30 mL/kg of cardioplegia solution. The infusion time was 10 minutes, and the temperature of the cardioplegia solution was about 8°C. After infusion of cardioplegia solution, the rat heart was placed in a container filled with a small amount of KH buffer and placed on ice. Samples were taken at 0, 2, 4, 6 and 12 hours after infusion of cardioplegia solution, and histopathological sections were taken at Samples were taken at 0, 4 and 12 hours, and all samples were immediately stored at -80°C. The samples in this experiment were used to detect Bcl-2 and Bax/Bcl-2 ratio parameters.

Bcl-2

The Bcl-2 protein family has anti-apoptotic functions, and these proteins can directly interact with effectors and activators to inhibit their ability to promote apoptosis ^3,4,5 . In preclinical models, Bcl-2 binds to and sequesters BH3-only activators, preventing the activators from interacting with the pore-forming effector6 ^{, 7,8} . Likewise, Bcl-2 can directly affect the effector, preventing mitochondrial perforation. The dynamic balance between anti-apoptotic substances such as Bcl-2 and substances that promote apoptosis can determine whether cells are apoptotic.

³ Shamas-Din A, Kale J, Leber B, Andrews DW. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb Perspect Biol. 2013;5(4) ⁴ Portt, L.; Norman, G.; Clapp, C.; Greenwood, M.; Greenwood, T.M. Anti-apoptosis and cell survival: A review. Biochim. Biophys. Acta 2011, 1813, 238-259 ⁵ Hata AN, Engelman JA, Faber AC. The BCL2 Family: Key Mediators of the Apoptotic Response to Targeted Anticancer Therapeutics. Cancer Discov. 2015;5(5):475-87 ⁶ Shamas-Din A, Kale J, Leber B, Andrews DW. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb Perspect Biol. 2013;5(4) ⁷ Portt, L.; Norman, G.; Clapp, C.; Greenwood, M.; Greenwood, T.M. Anti-apoptosis and cell survival: A review. Biochim. Biophys. Acta 2011, 1813, 238-259 ⁸ Hata AN, Engelman JA, Faber AC. The BCL2 Family: Key Mediators of the Apoptotic Response to Targeted Anticancer Therapeutics. Cancer Discov. 2015;5(5):475-87

³ Shamas-Din A, Kale J, Leber B, Andrews DW. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb Perspect Biol. 2013;5(4) ⁴ Portt, L.; Norman, G.; Clapp, C.; Greenwood, M.; Greenwood, TM Anti-apoptosis and cell survival: A review. Biochim. Biophys. Acta 2011, 1813, 238-259 ⁵ Hata AN, Engelman JA, Faber AC. The BCL2 Family: Key Mediators of the Apoptotic Response to Targeted Anticancer Therapeutics. Cancer Discov. 2015;5(5):475-87 ⁶ Shamas-Din A, Kale J, Leber B, Andrews DW. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb Perspect Biol. 2013;5(4) ⁷ Portt, L.; Norman, G.; Clapp, C.; Greenwood, M.; Greenwood, TM Anti-apoptosis and cell survival: A review. Biochim. Biophys. Acta 2011, 1813 , 238-259 ⁸ Hata AN, Engelman JA, Faber AC. The BCL2 Family: Key Mediators of the Apoptotic Response to Targeted Anticancer Therapeutics. Cancer Discov. 2015;5(5):475-87

The results of Bcl-2 western blotting in Figure 6A and Figure 6B show that after administering glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is both a participant in glycolysis and a promoter of apoptosis, the present invention was administered The expression level of Bcl-2 in the W group given the cardioplegia solution was higher than that in the D group given the Denido cardioplegia solution. It can be seen that the ability of the WAW cardioplegia solution of the present invention to inhibit apoptosis is better than that of the Denido cardioplegia solution. Accordingly, the present invention provides methods comprising a method of cardioplegia that inhibits apoptosis.

Bax

According to research, Bax can interact with mitochondrial voltage-dependent anion channel (voltage-dependent anion channel, VDAC) and promote its opening, resulting in the loss of mitochondrial membrane potential and the release of cytochrome c (cytochrome c) ⁹ . The expression of this gene is regulated by the tumor suppressor protein P53, and Bax is also involved in the apoptosis regulated by P53.

Bax/Bcl-2 ratio

The apoptosis pathway regulated by mitochondria is regulated by anti-apoptotic proteins (Bcl-2, Bcl-XL, Mcl-1) and pro-apoptotic proteins (Bax, Bad, Bak) of the Bcl-2 protein family, among which Bcl-2 can interact with Bax-Bak and form an inactivating heterodimer to inhibit apoptosis. Some researchers believe that the ratio of Bax/Bcl-2 may be more important than the individual expression of Bax and Bcl-2 in determining the process of ^{apoptosis10,11} . The Bax/Bcl-2 ratio is an index for judging cell apoptosis. A higher Bax/Bcl-2 ratio may be associated with a higher apoptosis program initiation, while a higher caspase-3 concentration Often associated with the process of apoptosis ¹² .

⁹ V. Shoshan-Barmatz, D. Ben-Hail, L. Admoni, Y. Krelin, S.S. Tripathi. The mitochondrial voltage-dependent anion channel 1 in tumor cells. Biochim. Biophys Acta, 1848 (10) (2015), pp. 2547-2575 ¹⁰ Raisova M, Hossini AM, Eberle J, Riebeling C, Weider T, Sturm I, et al. The bax/bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J Invest Dermatol 2001; 117 (2): 333-340 ¹¹ Del P.G., Venditti A., del P.M., Maurillo L., Buccisano F., Tamburini A., Cox M.C., Franchi A., Bruno A., Mazzone C. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood. 2003; 101: 2125-2131. 10.1182/blood-2002-06-1714

⁹ V. Shoshan-Barmatz, D. Ben-Hail, L. Admoni, Y. Krelin, SS Tripathi. The mitochondrial voltage-dependent anion channel 1 in tumor cells. Biochim. Biophys Acta, 1848 (10) (2015), pp 2547-2575 ¹⁰ Raisova M, Hossini AM, Eberle J, Riebeling C, Weider T, Sturm I, et al. The bax/bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J Invest Dermatol 2001; 117 (2): 333-340 ¹¹ Del PG, Venditti A., del PM, Maurillo L., Buccisano F., Tamburini A., Cox MC, Franchi A., Bruno A., Mazzone C. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood. 2003; 101: 2125-2131. 10.1182/blood-2002-06-1714

As shown in FIG. 7 , the ratio of Bax/Bcl-2 at each data point in group W administered WAW cardioplegia of the present invention was substantially lower than that in group N administered denidol cardioplegia. Therefore, the present invention provides a method for inducing cardioplegia and inhibiting cell apoptosis using the cardioplegia solution of the present invention.

Cardioplegic solution for maintenance of heart function in isolated rats

Experimental animals: the experimental subjects of this experiment are male Sprague-Dawley rats with a body weight of 300 to 400 g, and there are 6 rats in each group. The heart isolation and in vitro culture procedures in this experiment were approved by the Institutional Animal Care and Use Committee.

Cardioplegia solution: WAW cardioplegia solution, Denido cardioplegia solution, Plegisol ^® cardioplegia solution, HTK cardioplegia solution, KH buffer solution and normal saline were used in this experiment. The WAW cardioplegia solution of the present invention used in this experiment contains about 29.63mEq/LK ⁺ , about 18.39mEq/L Mg ²⁺ , about 133.80mEq/L Na ⁺ , about 118.51mEq/L Cl ^- , about 21.98mmol/L glucose Acid ion, about 25.81mmol/L acetate ion, about 7.76mmol/L sulfate ion, about 20mM THAM, and about 3.116g/L mannitol.

Isolation and perfusion of the heart: Before the heart was isolated, the rats were anesthetized with sodium pentobarbital (60 mg/kg) by intraperitoneal injection, as described above. The researchers cut the abdomen of the rats and injected heparin (500 U/mL) intravascularly into the vena cava 3 minutes before removing the heart. After the rats were opened, the researchers quickly cut off the hearts, immersed them in KH buffer solution at 10°C to 15°C or the cardioplegia solution corresponding to the experimental group, and rinsed the aorta with the same solution 5 times to remove residuals. Blood. The researcher weighed the heart of the rat and intubated the aorta through the aorta, and used a roller pump to perfuse the KH buffer solution or cardioplegia solution at 10°C to 15°C, the WAW cardioplegia solution of the present invention and Denido cardioplegia 20 mL/kg of cardioplegia solution, 30 mL/kg of HTK cardioplegia solution, 20 mL/kg of Plegisol ^® cardioplegia solution, and continuous infusion of KH buffer solution at a rate of 10-20 mL/min. After 2 hours of perfusion, the researchers treated the isolated heart model (Langendorff model) ¹³ , this method was reverse perfusion (retrograde) with KH buffer (Sigma, St. )heart. Thereafter, the heart of the rat was perfused with KH buffer solution for 30 minutes without circulation to remove the cardioplegia solution, and then the heart was perfused with 100 mL of perfusate with a fixed volume for 20 minutes, and the benchmark data were obtained as above. The KH buffer was continuously injected with 95% O ₂ /5% CO ₂ bubbles, and the temperature was maintained at 37°C with a hot water jacket. After the rat hearts were treated with the above-mentioned cardioplegic solutions, the researchers evaluated the heart function according to the experimental method shown in the figure below.

¹² Jarskog LF, Selinger ES, Lieberman JA, Gilmore JH. Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation. Am J Psychiatry 2004; 161: 109-115

¹² Jarskog LF, Selinger ES, Lieberman JA, Gilmore JH. Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation. Am J Psychiatry 2004; 161: 109-115

In order to observe the survival rate of cardiac cells and the size of myocardial infarction after cardiac ischemia, the researchers conducted another experiment of inducing acute myocardial ischemia-reperfusion injury, and the experimental results are shown in Figure 8B. After the 30-minute reference period, the researchers administered 30 minutes of global no-flow ischemia to the rat hearts at 37°C, and then reversed the corresponding cardioplegia solution for 4 hours as a reverse perfusion injury. The hearts of the control group were treated with KH buffer was perfused for 290 minutes.

Evaluation of cardiac function: A pressure transducer was connected to the arm of the aortic cannula in the rat heart to monitor the coronary perfusion pressure (CPP). A pressure-volume catheter (PV catheter) (ADVantage Pressure-Volume System, Transonic, Netherlands) is inserted into the left ventricle through the same arm of the aortic catheter, and the left ventricular pressure-volume loop (left ventricular pressure-volume) is measured in real time. loops). The measurement of coronary blood flow is to collect the perfusion fluid for 30 seconds, and calculate its mL/min/g after normalizing the heart wet weight. The investigators collected an additional 0.1 mL of perfusate for cardiac enzyme analysis, as described below. Blood pressure signals were recorded in the MP36 four-channel MP36 data acquisition system (Biopac Systems, Inc., CA), as described above.

¹³ Bøtker, H.E., Hausenloy, D., Andreadou, I. et al. Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection. Basic Res Cardiol (2018) 113: 39

¹³ Bøtker, HE, Hausenloy, D., Andreadou, I. et al. Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection. Basic Res Cardiol (2018) 113: 39

Cardiac enzyme analysis: centrifuge the perfusate or left ventricular tissue homogenate as described below at 620g, collect the supernatant and store it at -70°C, and analyze its B-type sodium excretion with a commercial reagent group (Roche, Tokyo, Japan) Diuretic peptide (B-type natriuretic peptide), cardiac troponin I (troponin I), creatine phosphatase-MB subtype (creatine kinase MB) and lactate dehydrogenase activity to assess the degree of myocardial damage, as above mentioned.

Triphenyltetrazolium chloride (TTC) staining was used to determine the size of myocardial infarction: after perfusion and weighing, the rat heart was cut into 2-mm slices from the base to the tip, and the heart model of isolated perfusion due to global ischemia This results in a homogeneous response, requiring only the middle section with the most cardiac tissue to be used for infarct size analysis. For analysis, staining with 1% 2,3,5-triphenyltetrazolium chloride (Sigma) for 20 minutes at 37°C differentiates pale infarcts from red viable myocardial blocks, as described above. The remaining tissue sections were stored at -80°C for subsequent biochemical analysis, or fixed with 4% paraformaldehyde for indirect immunofluorescent staining.

Western blotting analysis: After sampling the heart tissue of the left ventricle, the whole protein, cytoplasmic protein and mitochondrial protein samples were prepared with commercial reagent kits (BioVision, Milpitas, CA), and the protein samples were prepared with commercial reagent kits (Bio-Rad, Hercules, CA) were separated after assay and electrophoretically transferred to a polyvinylidene difluoride membrane as described above. After blocking with 5% skimmed milk, anti-cytochrome c antibody, anti-BCL02 antibody, anti-Bax antibody, anti-Bcl-X _L antibody, anti-caspase-9 (caspase-9) were added to the polyvinylidene fluoride membrane Antibody, anti-Apaf-1 antibody, anti-caspase-3 antibody or anti-inflammatorium NLRP-3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), at 4°C overnight. After washing, the polyvinylidene fluoride membrane was added with the corresponding immunoglobulin G (horseradish peroxidase-conjugated IgG) linked to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) and left at room temperature for 1 hour. After washing the polyvinylidene fluoride membrane, the researchers used a commercial chemical luminescence kit (Thermo Scientific, Rockford, IL) to detect complexes formed by antibody binding. Luminescence band densities of appropriate molecular weights were semi-quantified by densitometry using an image analysis system (Diagnostic Instruments, Sterling Heights, MI).

The researchers examined the geometric localization of protein expression in the heart with indirect immunofluorescent staining, as described above. More specifically, 5-μm heart sections were fixed in 4% formaldehyde. After blocking endogenous peroxidase, the researchers added specific antibodies, and the samples were left overnight at 4°C. Anti-rabbit or anti-goat immunoglobulin G (Jackson ImmunoResearch) were used as negative controls. The next day, the heart slices were added with immunoglobulin G linked to horseradish peroxidase, left at room temperature for 1 hour, and developed with tyramide signal amplification kit (PerkinElmer, MA). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (4'6-diamidino-2-phenylindole, DAPI).

Experimental results of administration of cardioplegia solution to isolated rat hearts

Figure 8A and Figure 8B are the survival rate and myocardial infarction size in the rat heart experiment, respectively. As shown in FIG. 8A , the group with the highest cardiac survival rate in the experiment was the positive control group administered only with KH buffer solution, followed by the W group administered with the cardioplegia solution of the present invention. As shown in Figure 8B, compared with the groups administered with ^Plegisol® cardioplegia, HTK cardioplegia, and Denido cardioplegia, myocardial infarction in the hearts of rats administered the cardioplegia solution of the present invention was substantially smaller . The results of the above two experiments show that the cardioplegia solution of the present invention is better than the other three cardioplegia solutions in maintaining the function of the heart.

Electrocardiogram voltage: As shown in Figure 9, compared with the groups given the other three cardioplegia solutions, the heart voltage of the rats given the cardioplegia solution of the present invention was higher, which was closest to that of only KH buffer solution The control group means that the heart of the rats in the W group is healthier and more functional. At 0, 30, 60, 90 and 120 minutes, when P<0.05, compared with the voltage of the heart of rats given Plegisol ^® cardioplegia solution, the voltage of the heart of the rat given the cardioplegia solution of the present invention high and have significant differences. At 0, 30 and 60 minutes, when P<0.05, compared with the voltage of the heart of rats given HTK cardioplegia solution, the voltage of the heart of the rat given the cardioplegia solution of the present invention was higher and significantly difference. More importantly, at 0, 30, 60, 90, and 120 minutes, when P<0.05, compared with the heart voltage of rats administered with Denido cardioplegia, the cardioplegia solution of the present invention was administered The voltage of the heart of the rats was higher and there was a significant difference. In addition, at 0, 30, 60, 90 and 120 minutes, when P<0.05, compared with the voltage of the heart of rats only given KH buffer solution, the heart voltage of rats given cardioplegia solution of the present invention There was no significant difference in voltage. The above experimental results show that the cardioplegic solution of the present invention can maintain a higher voltage to the heart given the cardioplegic solution, and the effect is better than the other three cardioplegic solutions.

Left ventricular systolic pressure: Similarly, as shown in Figure 10, the left ventricular systolic pressure of the heart of the rat given the cardioplegia solution of the present invention was substantially higher than that of the rat heart given the other three cardioplegia solutions, and the experiment The results were most similar to the positive control group given only KH buffer. Statistical tests performed at P<0.05 showed that at 0, 30, 60, 90 and 120 minutes, heart rate of rats administered Plegisol ^® cardioplegia, HTK cardioplegia and more importantly Denido cardioplegia The systolic pressure of the left ventricle is significantly different from the systolic pressure of the rat heart given the cardioplegia solution of the present invention. The systolic pressure of the left ventricle of the heart of the rats given the cardioplegia solution of the present invention is more similar to the systolic pressure of the left ventricle of the hearts of the rats of the positive control group given only the KH buffer solution, indicating that the cardioplegia solution is superior in maintaining blood pressure In Plegisol ^® Cardioplegic Solution, HTK Cardioplegic Solution and Denido Cardioplegic Solution.

Left ventricular diastolic pressure: the real-time pressure measured by the pressure-volume catheter (ADVantage Pressure-Volume System, Transonic, Netherlands) of the rat heart given the cardioplegia solution of the present invention, which is made into the left ventricle through the arm of the aortic cannula The measured left ventricular blood pressure-volume loop was higher than the real-time measurement of the left ventricular blood pressure-volume loop in rat hearts given the other three cardioplegic solutions. As shown in Figure 11, the left ventricular diastolic pressure of rat hearts administered with the cardioplegic solution of the present invention was substantially higher than that of rat hearts administered with the other three cardioplegic solutions, and was closest to that of rats administered only KH buffer solution. Left ventricular diastolic pressure in positive control group. When P<0.05, at 0, 30, 60, 90 and 120 minutes, the diastolic pressure of the left ventricle of the heart of rats administered the cardioplegia solution of the present invention was compared with that administered with ^Plegisol® cardioplegia solution and HTK cardioplegia solution, There was a significant difference in the diastolic pressure of the left ventricle of rat hearts compared with the more important Denido Cardioplegic Solution. The left ventricular diastolic pressure of the heart of the rats administered the cardioplegia solution of the present invention is the closest to the left ventricular diastolic pressure of the positive control group administered only KH buffer solution, indicating that compared with the other three cardioplegic solutions, the cardioplegia solution of the present invention fluid to maintain a healthy left ventricular diastolic pressure is better.

Heart rate: As shown in Figure 12, the heart rate of the rats given the cardioplegia solution of the present invention is substantially higher than that of the rats given the other three cardioplegia solutions, and the closest approximation is only KH buffer solution The positive control group's heart rate. When P<0.05, at 0, 30, 60, and 90 minutes, compared with the heart rate of rats given Plegisol ^® Cardioplegic Solution, HTK Cardioplegic Solution, and more importantly Denido Cardioplegic Solution, administration The heart rate of the rats given the cardioplegia solution of the present invention was higher and significantly different. The heart rate of rat hearts administered with the cardioplegia solution of the present invention was the closest to the heart rate of the positive control group administered only with KH buffer solution, indicating that compared with ^Plegisol® cardioplegia solution, HTK cardioplegia solution and Denido cardioplegia solution, The cardioplegia solution of the present invention has better efficacy in maintaining a healthy heart rate.

Coronary blood flow: Coronary blood flow is the blood circulation in the blood vessels that supply the myocardium. The coronary arteries supply oxygenated blood to the myocardium. After the oxygen in the blood is exhausted, the cardiac veins transport the blood away. Since the rest of the body, especially the brain, requires an almost constant supply of oxygenated blood, the heart has to work all the time, sometimes quite hard. Therefore, the blood circulation of the heart is not only very important to the heart tissue, but also very important to the perception of the whole body and even the brain. As shown in FIG. 13 , the coronary blood flow of rat hearts administered with the cardioplegic solution of the present invention was substantially higher than that of rat hearts administered with the other three cardioplegic solutions. When P<0.05, at 0 and 30 minutes, compared with the coronary blood flow of rat hearts given ^Plegisol® cardioplegia and HTK cardioplegia, the cardioplegia solution of the present invention was administered to a greater extent. Coronary blood flow in rat hearts was higher and significantly different. More importantly, when P<0.05, at 0, 30, 60, 90 and 120 minutes, compared with the coronary blood flow of the heart of rats given Denidol cardioplegia, the hearts given the present invention Coronary blood flow in the hearts of rats treated with paralytic solution was higher and had significant differences. The coronary blood flow of the rat hearts given the cardioplegia solution of the present invention is the closest to the coronary blood flow of the positive control group only given the KH buffer solution, indicating that compared with the other three cardioplegic solutions, the cardioplegia solution of the present invention It is more effective in maintaining healthy coronary blood flow.

Lactate dehydrogenase: Lactate dehydrogenase is widely expressed in body tissues and is a non-specific biomarker of organ damage. Since this experiment uses a single organ (isolated heart model), the expression of lactate dehydrogenase can be used to estimate the degree of heart damage . As shown in FIG. 14 , among the four cardioplegia solutions used in this experiment, the group administered with the cardioplegia solution of the present invention had the lowest expression of lactate dehydrogenase. When P<0.05, at 0, 30, 60, 90 and 120 minutes, compared with the concentration of lactate dehydrogenase in the heart of rats administered with Plegisol ^® cardioplegia and HTK cardioplegia, administering the present invention The concentration of lactate dehydrogenase in the hearts of rats treated with the cardioplegia solution was lower and significantly different. More importantly, when P<0.05, at 30, 60, 90, and 120 minutes, compared with the concentration of lactate dehydrogenase in the hearts of rats administered with denido cardioplegia, the cardioplegia of the present invention The concentration of lactate dehydrogenase in the hearts of rats with the same fluid was lower and significantly different. The concentration of lactate dehydrogenase released from the hearts of rats given the cardioplegia solution of the present invention was lower than that of the hearts of rats given the other three cardioplegia solutions, indicating that compared with the other three Cardioplegia solution, the cardioplegia solution of the present invention has better efficacy in preventing tissue damage.

Those skilled in the art can understand that changes can be made to the above embodiments without departing from the concept of the present invention. It is understood, therefore, that this invention is not limited to the disclosed embodiments, but covers modifications within the spirit and scope of the invention as defined by the claims.

It should be understood that both the foregoing general description and the following detailed description are illustrative and illustrative only, and not intended to limit the scope of the claimed invention.

The above and other variations can be made in light of the detailed description. In general, terms used in the following disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description clearly defines such term. Accordingly, the actual scope of the technology includes the disclosed embodiments and all equivalent ways of implementing the technology.

Table 4 Table 4A

Table 7Table 7A

Claims (24)

A cardioplegic solution containing potassium (K ⁺ ), magnesium (Mg ²⁺ ), sodium (Na ⁺ ), chloride (Cl ^- ), gluconate, acetate, sulfate ions dissolved in water (SO ₄ ^2- ), THAM and mannitol, wherein the concentration of potassium ion (K ⁺ ) is about 27 to about 33 mEq/L, the concentration of magnesium ion (Mg ²⁺ ) is about 16 to about 20 mEq/L, and the concentration of sodium ion (Na ⁺ ) concentration is about 120 to about 146 mEq/L, chloride ion (Cl ⁻ ) concentration is about 106 to about 130 mEq/L, gluconate ion concentration is about 20 to about 24 mmol/L, acetate ion concentration is about 22 to about 28mmol/L, the sulfate ion (SO ₄ ²⁻ ) concentration is about 6 to about 9mmol/L, the THAM concentration is about 5 to about 30mmol/L, and the mannitol concentration is about 2 to about 5g/L. The cardioplegia solution described in item 1 of the scope of the patent application, wherein the concentration of potassium ions (K ⁺ ) is about 29.63 mEq/L, the concentration of magnesium ions (Mg ²⁺ ) is about 18.39 mEq/L, and the concentration of sodium ions (Na ⁺ ) The concentration is about 133.80mEq/L, the chloride ion (Cl ^- ) concentration is about 118.51mEq/L, the gluconate ion concentration is about 21.98mmol/L, the acetate ion concentration is about 25.81mmol/L, the sulfate ion (SO ₄ ^2- ) The concentration is about 7.76mmol/L, the THAM concentration is about 20mmol/L, and the mannitol concentration is about 3.116g/L. The cardioplegia solution described in claim 1 further comprises lidocaine, wherein the concentration of the lidocaine in the cardioplegia solution is about 0.1 to about 0.14 mg/mL. The cardioplegia solution described in Item 1 of the scope of the patent application also includes fully oxygenated patient blood, and the volume ratio of the fully oxygenated patient blood to the cardioplegia fluid described in Item 1 of the scope of application is 1:1 to 1:8. The cardioplegia solution described in claim 1 of the patent application, wherein the cardioplegia solution maintains a pH value of 8.2 to 8.6 for 365 days. The cardioplegia solution as described in claim 1, wherein the cardioplegia solution does not contain calcium. The cardioplegia solution as described in claim 1, wherein the cardioplegia solution does not contain sodium bicarbonate. A method for preparing the cardioplegia solution described in Item 1 of the scope of the patent application, comprising the following steps: A, adding sodium chloride (NaCl), sodium gluconate (C ₆ H ₁₁ NaO ₇ ), sodium acetate trihydrate to water USP (C ₂ H ₃ NaO ₂ .3H ₂ O), potassium chloride USP (KCl), magnesium chloride hexahydrate USP (MgCl ₂ .6H ₂ O), magnesium sulfate heptahydrate USP (MgSO ₄ .7H ₂ O) and manna Alcohol USP (C ₆ H ₁₄ O ₆ ); and second, THAM and glacial acetic acid are mixed in the solution obtained from step (a); wherein the sodium chloride (NaCl) concentration in water is about 75 to about 95mmol/L, Sodium gluconate (C ₆ H ₁₁ NaO ₇ ) concentration is about 20 to about 24mmol/L, sodium acetate trihydrate USP (C ₂ H ₃ NaO ₂ .3H ₂ O) concentration is about 22 to about 28mmol/L, chloride The concentration of potassium USP (KCl) is about 27 to about 33 mmol/L, the concentration of magnesium chloride hexahydrate USP (MgCl ₂ .6H ₂ O) is about 1.2 to about 1.6 mmol/L, the concentration of magnesium sulfate heptahydrate USP (MgSO ₄ .7H ₂ O ) concentration is about 6 to about 9mmol/L, mannitol USP (C ₆ H ₁₄ O ₆ ) concentration is about 15 to about 20mmol/L; wherein by mixing THAM and glacial acetic acid into the solution obtained from step (a) , adjust the solution to the desired pH range of 8.2 to 8.6, and the THAM concentration to reach about 5 to about 25 mmol/L. The method described in item 8 of the scope of patent application, wherein the concentration of sodium chloride (NaCl) in water is about 86.02mmol/L, the concentration of sodium gluconate (C ₆ H ₁₁ NaO ₇ ) is about 21.99mmol/L, three Sodium acetate hydrate USP (C ₂ H ₃ NaO ₂ .3H ₂ O) concentration is about 25.85mmol/L, potassium chloride USP (KCl) concentration is about 29.59mmol/L, magnesium chloride hexahydrate USP (MgCl ₂ .6H ₂ O ) concentration is about 1.41mmol/L, magnesium sulfate heptahydrate USP (MgSO ₄ .7H ₂ O) concentration is about 7.76mmol/L, mannitol USP (C ₆ H ₁₄ O ₆ ) concentration is about 17.1mmol/L; , by mixing THAM and glacial acetic acid in the solution obtained from step (a), adjust the solution to the desired pH range of 8.2 to 8.6, and the THAM concentration reaches about 20mmol/L or about 10mmol/L. The method described in item 8 of the patent claims further includes the step of adding lidocaine so that the concentration of the lidocaine in the cardioplegia solution is about 0.1 to about 0.14 mg/mL. The method described in item 8 of the scope of application for patents also includes the step of adding fully oxygenated patient's blood, so that the volume ratio of fully oxygenated patient's blood to the cardioplegia as described in item 1 of the scope of application for patents is 1:1 to 1:8. The method as described in claim 7, wherein the cardioplegia solution does not contain calcium. The method as described in claim 8, wherein the cardioplegia solution does not contain sodium bicarbonate. The method described in item 8 of the patent application, wherein this step can be completed 365 days before using the cardioplegia solution. A method for preparing a cardioplegia solution as in item 1 of the scope of the patent application, comprising the following steps: A, mixing mannitol, magnesium sulfate, THAM and potassium chloride into the Plasma-Lyte A base solution; and B, mixing THAM and ice Acetic acid to adjust the pH of the solution to 8.2 to 8.6; wherein about 15 to about 19 mmol of mannitol, about 5 to about 10 mmol of magnesium sulfate, and about 22 to about 27 mmol of potassium chloride are added to 1L of the Plasma-Lyte A base solution; wherein Each 1L of Plasma-Lyte A base solution contains about 140mEq sodium ion (Na ⁺ ), about 5mEq potassium ion (K ⁺ ), about 3mEq magnesium ion (Mg ²⁺ ), about 98mEq chloride ion (Cl ^- ), about 27mEq acetate ions, with approximately 23mEq gluconate ions. The method described in item 15 of the scope of the patent application, wherein mannitol, magnesium sulfate, THAM and potassium chloride can be added to the Plasma-Lyte A basic solution 365 days before using the cardioplegia solution. The method described in item 13 of the patent claims further includes the step of adding lidocaine so that the concentration of lidocaine in the cardioplegia solution is about 0.1 to about 0.14 mg/mL. The method described in item 13 of the scope of application, further comprising the step of adding fully oxygenated patient blood, so that the volume ratio of fully oxygenated patient blood to the cardioplegia described in item 1 of the scope of application is 1:1 to 1:8. The method as described in claim 15, wherein the cardioplegia solution is stored at room temperature. A use of the cardioplegia solution described in item 1 of the patent application for preparing a composition that causes temporary cardiac paralysis, which includes perfusing the patient's heart with the cardioplegia solution as described in item 1 of the patent application. The use as described in claim 18, wherein the cardioplegia solution is administered to the patient at low temperature. The use as described in claim 20, wherein the cardioplegia solution is administered to the patient at normal temperature. The use as described in item 20 of the patent application, wherein the cardioplegia solution is administered only once during the operation. The use as described in claim 20, wherein the cardioplegia solution is administered more than once during the operation.

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