Polymeric protein product as well as preparation method and application thereof

Abstract

The invention discloses a polymeric protein product as well as a preparation method and application thereof. The preparation method of the polymeric protein product comprises the following steps: enabling protein and a cross-linking agent to react in a reaction system with the viscosity being greater than or equal to 2.5 mPa.s; wherein the protein comprises hemoglobin and / or hemoglobin derivatives. The preparation method provided by the invention can improve the content of the polymeric protein in the polymeric protein product.

Description

Technical Field

[0001] The invention relates to a polymerized protein product and a preparation method and application thereof. Background Art

[0002] Hemoglobin without cell membrane (also known as "hemoglobin oxygen carrier") has been widely developed for use as red blood cell substitutes, also known as blood substitutes, due to its ability to carry and release oxygen. It can prolong or save the patient's life in the event of acute blood loss or hemorrhagic shock.

[0003] Natural hemoglobin is generally composed of four subunits, of which two α subunits and β subunits are anchored and relatively fixed, while the other two α subunits and β subunits are in sliding contact and are easily dissociated in solution. The molecular radius of natural hemoglobin is small, and it will leak from the renal tubules when injected into the body, blocking the renal tubules and causing nephrotoxicity. In order to avoid nephrotoxicity, polymerization or modification methods are usually used to increase the molecular weight or molecular radius of hemoglobin carriers.

[0004] CN114303463B discloses a method for preparing a red blood cell substitute using a polyaldehyde compound. A reaction of polymerizing hemoglobin using a polyaldehyde compound as a crosslinking agent. In the polymerization reaction of hemoglobin, the concentration of the hemoglobin solution is 5-30 mg / mL, the concentration of the polyaldehyde compound solution is 0.02-0.15 mol / L, and the molar ratio of hemoglobin to the polyaldehyde compound is 1 to 20-40. The polyaldehyde compound solution is quickly added to the hemoglobin solution under stirring conditions and reacted for 40-120 minutes. After the reaction is terminated, an ultrafiltration or gel filtration method is used to obtain a hemoglobin polymer with a molecular weight between 100 and 1500 kDa.

[0005] CN108148132B discloses a method for preparing dipolymerized hemoglobin. Under inert gas nitrogen and stirring, a di(2-hydroxyethyl)iminotri(hydroxymethyl)methane solution of diaspirin with a pH of 9.5 is slowly dripped into a matrix-free human placental hemoglobin aqueous liquid with a hemoglobin weight content of 10% to perform intramolecular crosslinking to obtain a first polymer product; under inert gas nitrogen conditions, glutaraldehyde is fed into the obtained first polymer product through a titanium rod filter, and the molar ratio of glutaraldehyde to the first polymer product is 8:1.

[0006] In the above method, the content of polymerized protein is low, and the content of protein not involved in cross-linking is high. Summary of the invention

[0007] In view of this, one object of the present invention is to provide a method for preparing a polymerized protein product, wherein the polymerized protein product obtained by the method has a high content of polymerized protein. Another object of the present invention is to provide a polymerized protein product. Still another object of the present invention is to provide a use of the polymerized protein product.

[0008] The above purpose is achieved through the following technical solutions.

[0009] In one aspect, the present invention provides a method for preparing a polymerized protein product, comprising the following steps: reacting a protein and a cross-linking agent in a reaction system having a viscosity of ≥2.5 mPa·s;

[0010] Wherein, the protein includes hemoglobin and / or hemoglobin derivatives.

[0011] According to the preparation method of the present invention, preferably, the hemoglobin derivative is selected from one or more of deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin and sulfhemoglobin.

[0012] According to the preparation method of the present invention, preferably, the protein further comprises one or more of albumin, superoxide dismutase, peroxidase, carbonic anhydrase or fibrinogen;

[0013] The total amount of hemoglobin and hemoglobin derivatives in the protein is more than 85 wt%.

[0014] According to the preparation method of the present invention, preferably, the protein and the cross-linking agent are reacted in the presence of water and a high-viscosity compound;

[0015] The viscosity of the high-viscosity compound is greater than that of water; the high-viscosity compound is soluble in water or forms a colloid with water; and the high-viscosity compound does not participate in chemical reactions.

[0016] According to the preparation method of the present invention, preferably, the high-viscosity compound is selected from one or more of small molecule polyols, polyether diols, cellulose or its derivatives, salt compounds formed by cellulose or its derivatives, and hyaluronic acid.

[0017] According to the preparation method of the present invention, preferably, the small molecule polyol contains 2 to 8 carbon atoms, and the number of hydroxyl groups of the small molecule polyol is 2 to 6;

[0018] The polyether diol is selected from one or more of polyethylene glycol and polypropylene glycol;

[0019] The cellulose derivative is selected from one or more of carboxymethyl cellulose and hydroxymethyl cellulose;

[0020] The salt compound formed by cellulose or its derivatives is sodium salt or potassium salt of cellulose or its derivatives.

[0021] According to the preparation method of the present invention, preferably, the cross-linking agent is selected from one or more of polyaldehyde compounds, diformaldehyde, open-ring raffinose, diaspirin, activated forms of polyethylene oxide and dextran, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N,N'-phenylenedimaleimide, and diimide compounds;

[0022] The molar ratio of the total molar amount of hemoglobin and its derivatives to the cross-linking agent is 1:(150-300).

[0023] The preparation method according to the present invention preferably further comprises the following steps:

[0024] The cross-linked product obtained by the reaction of the protein and the cross-linking agent is reacted with a reducing agent to obtain a reaction product; small molecules in the reaction product are removed and then concentrated.

[0025] In another aspect, the present invention provides a polymeric protein product, which is prepared by the above-mentioned preparation method.

[0026] In another aspect, the present invention provides the use of the above-mentioned polymer protein product as a blood substitute; or in the preparation of a drug for treating and / or preventing liver damage, kidney damage or heart damage.

[0027] The present invention adds a compound with high viscosity and which does not react with the reaction system into the reaction system, thereby increasing the viscosity of the reaction system, reducing the thermal free motion of the reactants, reducing the probability of intermolecular collision, reducing the reaction rate, and increasing the content of polymerized protein in the polymerized protein product, thereby obtaining a polymerized protein product with higher uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the UV-visible spectrum of the gel filtration separation of the polymerized protein preparation obtained in Example 1.

[0029] Figure 2 This is the UV-visible spectrum of the gel filtration separation of the polymerized protein preparation obtained in Example 2.

[0030] Figure 3 This is the UV-visible spectrum of the gel filtration separation of the polymerized protein preparation obtained in Example 3.

[0031] Figure 4 This is a graph of ALT enzyme activity of mice in each group of Experimental Example 5.

[0032] Figure 5 This is a graph of AST enzyme activity of mice in each group of Experimental Example 5.

[0033] Figure 6This is a graph of ALT enzyme activity of mice in each group of Experimental Example 6.

[0034] Figure 7 This is a graph of AST enzyme activity of mice in each group of Experimental Example 6.

[0035] Figure 8 This is a graph of the LDH enzyme activity of each group of mice in Experimental Example 7.

[0036] Fig. 9 This is a graph of CK enzyme activity of mice in each group of Experimental Example 7.

[0037] Fig.10 This is a graph of ALT enzyme activity of mice in each group of Experimental Example 8.

[0038] Fig.11 This is a graph of AST enzyme activity of mice in each group of Experimental Example 8.

[0039] Fig.12 This is a graph of the CRE enzyme activity of each group of mice in Experimental Example 8.

[0040] Fig.13 This is a graph of the Urea enzyme activity of each group of mice in Experimental Example 8.

[0041] Fig.14 This is a graph of the CRE enzyme activity of each group of mice in Experimental Example 9.

[0042] Fig.15 This is a graph of the Urea enzyme activity of each group of mice in Experimental Example 9. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0044] <Method for preparing polymerized protein products>

[0045] The preparation method of the polymerized protein product of the present invention comprises the following steps: reacting the protein and the cross-linking agent in a reaction system with a viscosity of ≥2.5 mPa·s; wherein the protein comprises hemoglobin and / or a hemoglobin derivative.

[0046] The protein can be derived from humans, animals or recombinant proteins. The animal can be a mammal. Examples of animals include, but are not limited to, pigs, cows, etc.

[0047] The hemoglobin derivative can be selected from one or more of deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin, and sulfhemoglobin. Preferably, the hemoglobin derivative is selected from one or more of carboxyhemoglobin, deoxyhemoglobin, and oxyhemoglobin.

[0048] The protein may also include one or more of albumin, superoxide dismutase, peroxidase, carbonic anhydrase or fibrinogen. In certain embodiments, the protein may also include one or more of superoxide dismutase and catalase.

[0049] The total content of hemoglobin and hemoglobin derivatives in the protein is greater than 85wt%; preferably, the total content of hemoglobin and hemoglobin derivatives is greater than or equal to 90wt%; more preferably, the total content of hemoglobin and hemoglobin derivatives is greater than or equal to 95wt%.

[0050] The protein can be used in the form of a protein solution. The solvent of the protein solution can be water.

[0051] The concentration of the protein solution may be 5 to 60 mg / L. Preferably, the concentration of the protein solution is 8 to 50 mg / L. In certain embodiments, the concentration of the protein solution is 15 to 25 mg / L. In other embodiments, the concentration of the protein solution is 35 to 45 mg / L.

[0052] The pH of the protein solution may be between 7 and 9. In certain embodiments, the pH is between 7.5 and 8. In other embodiments, the pH is between 8.5 and 9.

[0053] The present invention reacts the protein and the cross-linking agent in a reaction system with a viscosity of ≥2.5 mPa·s. Preferably, the viscosity of the reaction system is 3.5 to 500 mPa·s; more preferably, the viscosity of the reaction system is 4.5 to 200 mPa·s. In some embodiments, the viscosity of the reaction system is 2.5 to 100 mPa·s. In other embodiments, the viscosity of the reaction system is 3.5 to 20 mPa·s. This helps to increase the content of polymerized protein.

[0054] The reaction system can be made to reach the above viscosity by adding a high viscosity compound to the reaction system. Specifically, the protein and the cross-linking agent are reacted in the presence of water and the high viscosity compound.

[0055] The viscosity of the high-viscosity compound is greater than the viscosity of water. The high-viscosity compound dissolves in water or forms a colloid with water. The high-viscosity compound does not participate in chemical reactions. The high-viscosity compound can be selected from one or more of small molecule polyols, polyether diols, cellulose or its derivatives, salt compounds formed by cellulose or its derivatives, and hyaluronic acid.

[0056] The small molecule polyol may contain 2 to 8 carbon atoms and 2 to 6 hydroxyl groups. Preferably, the number of carbon atoms is 3 to 6; more preferably, the number of carbon atoms is 3 to 4. Preferably, the number of hydroxyl groups is 3 to 5; more preferably, the number of hydroxyl groups is 3 to 4. Examples of small molecule polyols include, but are not limited to, ethylene glycol, propylene glycol, glycerol, butylene glycol, and butanetriol.

[0057] The polyether diol can be selected from one or more of polyethylene glycol and polypropylene glycol.

[0058] The cellulose derivative may be selected from one or more of carboxymethyl cellulose and hydroxymethyl cellulose.

[0059] The salt compound formed by cellulose or its derivatives may be a sodium salt or a potassium salt of cellulose or its derivatives.

[0060] Examples of salt compounds formed by cellulose and its derivatives include, but are not limited to, sodium cellulose, sodium carboxymethyl cellulose, potassium carboxymethyl cellulose, and sodium hydroxymethyl cellulose.

[0061] The high-viscosity compound can be used directly or in the form of an aqueous solution. The concentration of the aqueous solution of the high-viscosity compound can be 5 to 20 wt %, or 8 to 15 wt %.

[0062] The volume ratio of the high viscosity compound to the protein solution may be (0.3-5):1, preferably (0.5-3):1. In certain embodiments, the volume ratio of the high viscosity compound to the protein solution is (1.5-2):1.

[0063] The above-mentioned high-viscosity compounds can effectively increase the content of polymerized protein.

[0064] The crosslinking agent can be selected from one or more of polyaldehyde compounds, diformaldehyde, open-ring raffinose, diaspirin, activated forms of polyethylene oxide and dextran, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-phenylenedimaleimide, and diimide compounds.

[0065] The polyaldehyde compound refers to a compound containing at least two aldehyde groups in the molecule. The polyaldehyde compound can be selected from one or more of glyoxal, malondialdehyde, succindialdehyde, glutaraldehyde, adipaldehyde, and phthalaldehyde.

[0066] The molar ratio of hemoglobin and its derivatives to the cross-linking agent can be 1:(150-300); preferably 1:(170-260). In certain embodiments, the molar ratio of hemoglobin and its derivatives to the cross-linking agent is 1:(200-230). This can increase the content of polymerized protein.

[0067] The crosslinking agent can be used in the form of an aqueous solution. The concentration of the crosslinking agent solution can be 20 to 150 mM, preferably 30 to 120 mM. In certain embodiments, the concentration of the crosslinking agent solution is 60 to 90 mM.

[0068] In certain embodiments, a protein, a cross-linking agent, water, and a high viscosity compound are formed into a reactant; and the reactant is then polymerized.

[0069] At the polymerization reaction temperature, the viscosity of the reactant is ≥2.5 mPa·s; preferably, the viscosity of the reactant is 3.5 to 500 mPa·s; more preferably, the viscosity of the reactant is 4.5 to 200 mPa·s. In some embodiments, at the polymerization reaction temperature, the viscosity of the reactant is 2.5 to 100 mPa·s. In other embodiments, at the polymerization reaction temperature, the viscosity of the reactant is 3.5 to 20 mPa·s.

[0070] Specifically, a highly viscous compound or its aqueous solution is mixed with a protein solution to obtain a mixed solution. The highly viscous compound or its aqueous solution can be mixed with a protein solution in a rotary shaker. The mixed solution is mixed with a crosslinking agent aqueous solution to obtain a product to be reacted. The crosslinking agent aqueous solution can be added to the mixed solution once or several times, and then the mixed solution is mixed with the crosslinking agent aqueous solution. For example, it can be divided into 2 times, 4 times, 8 times, etc.

[0071] The polymerization reaction time may be 8 to 48 hours, preferably 10 to 24 hours, and more preferably 12 to 13 hours.

[0072] The polymerization reaction temperature may be 2 to 50° C., preferably 4 to 40° C. In certain embodiments, the polymerization reaction temperature is 15 to 30° C.

[0073] The polymerization can be carried out on a rotary shaker.

[0074] In certain embodiments, the following steps may also be included: reacting the cross-linked product obtained by the polymerization reaction with a reducing agent to obtain a reaction product; removing small molecules from the reaction product, and then concentrating it to obtain a polymerized protein product.

[0075] The reducing agent can be selected from one or more of dimethylaminoborane, trimethylaminoborane, triethylaminoborane, sodium borohydride. Preferably, the reducing agent is selected from one or more of dimethylaminoborane, sodium borohydride, trimethylaminoborane. The reducing agent can be used in the form of a solution.

[0076] The molar ratio of the crosslinking agent to the reducing agent may be 1:(3-20); preferably 1:(5-18); more preferably 1:(8-12).

[0077] The reduction reaction time may be 2 to 30 hours, preferably 4 to 25 hours. In certain embodiments, the reduction reaction time is 8 to 12 hours.

[0078] The tangential flow method can be used to remove small molecules in the reaction product. These small molecules include high viscosity compounds, crosslinking agents, reducing agents and their reaction products. Specifically, ultrafiltration fibers and ultrafiltration flat membranes can be used. The pore size of the ultrafiltration fiber or ultrafiltration flat membrane can be 10 to 120 kDa; preferably 30 to 100 kDa; more preferably 40 to 70 kDa.

[0079] The counteracting solution can be selected from one or more of physiological saline, Ringer's solution, and lactated Ringer's solution.

[0080] Concentration increases the concentration of protein in the polymerized protein preparation so that the concentration of protein in the obtained polymerized protein preparation is 80 to 250 mg / mL. In certain embodiments, the concentration of protein in the protein preparation is 90 to 210 mg / mL. In certain embodiments, the concentration of protein in the protein preparation is 100 to 150 mg / mL.

[0081] <Polymerized protein products and uses>

[0082] The polymerized protein product of the present invention is prepared by the above method.

[0083] In the polymerized protein preparation, the content of polymerized protein in the total protein is greater than 95wt%; preferably, the content of polymerized protein in the total protein is greater than 96wt%; more preferably, the content of polymerized protein in the total protein is greater than 98wt%.

[0084] The oxygen affinity (P50) of the polymerized protein product can be 6-40 mmHg, preferably 20-28 mmHg, so that it can be used as a hemoglobin oxygen carrier or a red blood cell substitute.

[0085] The polymerized protein product of the present invention can be used as a blood substitute and has the functions of expanding blood volume and increasing blood oxygen content.

[0086] The polymeric protein preparation of the present invention has a certain effect on treating and / or preventing liver damage, kidney damage and heart damage. Therefore, the present invention also provides the use of the polymeric protein preparation in preparing a drug for treating and / or preventing liver damage, kidney damage and heart damage.

[0087] Liver damage can be caused by chemical drugs such as triptolide, 5-fluorouracil, and cisplatin.

[0088] Heart damage can be caused by chemotherapy drugs such as doxorubicin, 5-fluorouracil, and cisplatin.

[0089] Kidney damage can be caused by chemotherapy drugs such as doxorubicin, 5-fluorouracil, and cisplatin.

[0090] Example 1

[0091] 20 mL of 10 wt% polyethylene glycol solution was added to 20 mL of bovine carboxyhemoglobin solution, and then mixed on a rotary shaker for 5 minutes to obtain a mixed solution. The concentration of the bovine carboxyhemoglobin solution was 40 mg / mL and the pH was 8.0.

[0092] A 100 mM glutaraldehyde solution was added to the mixed solution three times to obtain a product to be reacted. The molar ratio of glutaraldehyde to bovine carboxyhemoglobin was 210:1. At the polymerization reaction temperature (20°C), the viscosity of the product to be reacted was 4.5 mPa·s.

[0093] The reactants were polymerized on a rotary shaker at 20° C. for 10 h to obtain a cross-linked product. A 10 wt % dimethylaminoborane solution was added to the cross-linked product for a reduction reaction for 10 h to obtain a reaction product. The molar ratio of glutaraldehyde to dimethylaminoborane was 1:10.

[0094] The reaction product was filtered through an ultrafiltration fiber column with a pore size of 30 kDa to remove small molecules in the reaction product, and the counter solution was normal saline; then the product was concentrated to a total protein concentration of 200±5 mg / mL to obtain a polymerized protein product.

[0095] Example 2

[0096] Add 60 mL of glycerol to 20 mL of protein solution, and then shake on a rotary shaker to obtain a material to be treated. The solute of the protein solution consists of porcine hemoglobin, superoxide dismutase and catalase. In the protein solution, the concentration of porcine hemoglobin is 20 mg / mL, the concentration of superoxide dismutase is 0.5 mg / mL, and the concentration of catalase is 0.3 mg / mL. The pH of the protein solution is 7.5. Nitrogen is introduced into the material to be treated to convert more than 90% of the porcine hemoglobin into deoxyhemoglobin to obtain a mixed solution.

[0097] A 40 mM succinic dialdehyde solution was added to the mixed solution in 5 portions to obtain a product to be reacted. The molar ratio of succinic dialdehyde to porcine hemoglobin was 250:1. At the polymerization reaction temperature (10°C), the viscosity of the product to be reacted was 67 mPa·s.

[0098] The reactants were polymerized on a rotary shaker at 10°C for 12 hours to obtain a cross-linked product. Sodium borohydride was added to the cross-linked product and reduced for 24 hours to obtain a reaction product. The molar ratio of succinaldehyde to sodium borohydride was 1:15.

[0099] The reaction product was filtered through an ultrafiltration fiber column with a pore size of 100 kDa to remove small molecules in the reaction product, and the counter solution was Ringer's solution; then the product was concentrated to a total protein concentration of 130±5 mg / mL to obtain a polymerized protein product.

[0100] Example 3

[0101] 10 mL of glycerol was added to 20 mL of the human oxygenated hemoglobin solution, and then mixed on a rotary shaker to obtain a mixed solution. The concentration of the human oxygenated hemoglobin solution was 10 mg / mL and the pH was 8.5.

[0102] Add 80 mM malondialdehyde solution to the mixed solution in 6 portions to obtain a product to be reacted. The molar ratio of malondialdehyde to human oxyhemoglobin is 180:1. At the polymerization reaction temperature (5°C), the viscosity of the product to be reacted is 4.5 mPa·s.

[0103] The reactants were polymerized on a rotary shaker at 5°C for 15 hours to obtain a cross-linked product. Trimethylaminoborane was added to the cross-linked product and reduced for 4 hours to obtain a reaction product. The molar ratio of malondialdehyde to trimethylaminoborane was 1:6.

[0104] The reaction product was filtered through an ultrafiltration fiber column with a pore size of 50 kDa to remove small molecules in the reaction product, and the counter solution was lactated Ringer's solution; then the total protein concentration was concentrated to 100±5 mg / mL to obtain a polymerized protein product.

[0105] Experimental Example 1

[0106] Polymer protein content: The polymer protein content of the polymer protein preparations of Examples 1 to 3 was obtained by separation through a gel filtration column and analysis by a multi-angle laser light scattering instrument. The polymer protein content of each polymer protein preparation is shown in Table 1.

[0107] The UV-visible spectra of the gel filtration separation of the polymerized protein preparations of Examples 1-3 are as follows: Figure 1-3 As shown. The wavelength of ultraviolet light is 280nm.

[0108] Table 1

[0109]

[0110] Experimental Example 2

[0111] Colloidal osmotic pressure: The colloidal osmotic pressure of the polymerized protein product was measured by a colloidal osmotic pressure meter. The osmotic pressure of the polymerized protein product obtained in Example 1 was 35 mmHg, indicating that it has a strong capacity for volume expansion.

[0112] Oxygen affinity: This experiment uses a blood oxygen analyzer (Hemox-analyzer) to measure the oxygen dissociation curve of the polymer protein product. According to the instrument operating instructions, 5mL of HEMOX-Solution, 20μL ADDITIVE-A, 10μL ANTI-FOAMING AGENT, and 10μL ADDITIVE-B are transferred, and a quantitative test sample is added to prepare a test solution with a concentration of 0.5mg / mL. The test solution is added to the sample pool. After the instrument is heated to 37°C and stabilized, oxygen is introduced until the value is stable, and the pO2 value is adjusted to 142.0, and the S1 and S2 values ​​are adjusted to 2.50, and S1 / S2 is 0.00±0.01. Oxygen is introduced again until the pO2 reading is 140.0 or above, and the P50 value at this time is recorded. Repeat the above steps and measure in parallel. The oxygen affinity (P50) of the polymer protein product obtained in Example 2 is 22±3mmHg.

[0113] Experimental Example 3

[0114] A rat weighing 250±10g was taken, anesthetized with sodium pentobarbital, and monitored by a physiological recorder connected to the left femoral artery cannula, at which time the blood pressure was 115±10mmHg; then blood was slowly bled through the right femoral artery, and the blood volume of the bled blood was 15% of its total blood volume, at which time the blood pressure dropped to 90±10mmHg; an equal volume of the polymerized protein product of Example 1 was slowly infused through the tail vein, and the blood pressure gradually recovered to 120±12mmHg. This shows that the polymerized protein product of the present invention has a volume expansion function.

[0115] Experimental Example 4

[0116] Six 250±10g SD rats were taken and the following operations were performed respectively: after anesthesia with sodium pentobarbital, the left femoral artery was cannulated and connected to a physiological recorder for monitoring, and the physiological parameters at this time were recorded as the basic physiological parameters; then the right femoral artery was slowly bled, and the blood volume of the bled blood was 60% of its total blood volume, causing the animal to be in deep shock, and the physiological parameters at this time were recorded as the physiological parameters at the end of shock; then the same volume of the polymerized protein product of Example 2 was slowly infused through the tail vein, and the blood pressure gradually recovered, and the physiological parameters at this time were recorded as the physiological parameters after resuscitation. The physiological parameters of each stage are specifically shown in Table 2.

[0117] Table 2

[0118]

[0119] Note: The data in Table 2 are the average values ​​of 6 groups of experiments.

[0120] As shown in Table 2, after the rats were infused with the polymerized protein product of the present invention after shock, the blood pressure and pH value were basically restored to the basic stage, and the base excess and lactic acid also recovered significantly, which indicates that the polymerized protein product of the present invention has an anti-shock effect.

[0121] Experimental Example 5

[0122] Thirty healthy male KM mice aged 6-8 weeks with a body weight of 28-32 g were randomly divided into three groups, with 10 mice in each group, namely the control group, the triptolide group and the treatment group.

[0123] On the 1st to 6th day of the experiment, the mice in the control group were intraperitoneally injected with 0.9% saline in the same volume as the polymerized protein product solution given to the treated group.

[0124] On the 1st and 2nd days of the experiment, the mice in the triptolide group were intraperitoneally injected with 0.9% normal saline, which was the same volume of the polymerized protein product as that in the treatment group; on the 3rd to 5th days of the experiment, the mice in the triptolide group were intraperitoneally injected with 0.4 mg / kg of triptolide solution (the triptolide solution was formed by dissolving triptolide in 0.9% normal saline, and the injection dose was calculated based on triptolide); on the 6th day of the experiment, the mice in the triptolide group were intraperitoneally injected with 0.9% normal saline, which was the same volume of the polymerized protein product as that in the treatment group.

[0125] On days 1 to 6 of the experiment, the mice in the treatment group were given intraperitoneal injections of 50 mg / kg of a polymerized protein preparation solution (formed by dissolving the polymerized protein preparation of Example 2 in 0.9% saline); at the same time, on days 3 to 6 of the experiment, the mice in the treatment group were given intraperitoneal injections of 0.4 mg / kg of a triptolide solution (the triptolide solution was formed by dissolving triptolide in 0.9% saline, and the injection dose was calculated as triptolide).

[0126] On the 7th day of the experiment, peripheral blood samples were collected from each group of mice using the eyeball blood sampling method. The mice were fasted but not watered for 12 hours before sampling.

[0127] Biochemical index detection: The collected blood of each group of mice was centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was taken. The serum was tested for ALT and AST enzyme activities using an automatic biochemical analyzer. The results were as follows Figure 4-5 shown.

[0128] Depend on Figure 4 and 5It can be seen that compared with the control group, the levels of ALT and AST in the triptolide group were significantly increased, and the data between the two groups were statistically different. Compared with the triptolide group, the AST and ALT enzyme activities in the treatment group were significantly reduced, and the data between the two groups were statistically different. Moreover, after pre-protection and treatment, the ALT and AST of mice basically returned to the level of the control group, and there was no statistical difference in the data between the two groups.

[0129] Based on the above results, it is shown that the polymerized protein preparation of the present invention can significantly inhibit the increase of ALT and AST, the liver function indicators of mice induced by triptolide, and has a good protective and therapeutic effect on drug-induced liver injury.

[0130] Experimental Example 6

[0131] SPF grade ICR mice, male, 6-8 weeks old, weighing about 28 g, a total of 24 mice were randomly divided into three groups, namely, control group, CP group, and polymerized protein group, each with 8 mice. The polymerized protein preparation solution was prepared by dissolving the polymerized protein preparation of Example 3 in physiological saline.

[0132] Control group: From day 1 to day 6, the mice were injected with normal saline of the same volume as the polymerized protein product solution of the polymerized protein group.

[0133] CP group: On days 1-6, mice were injected with an equal volume of normal saline to that of the polymerized protein product in the polymerized protein group; in addition, mice in this group were intraperitoneally injected with 5 mg / kg cisplatin injection (the dosage was calculated as cisplatin) on days 1-3.

[0134] Polymer protein group: mice in this group were intraperitoneally injected with 50 mg / kg of polymer protein solution every day from day 1 to day 6 (the dosage was calculated based on the polymer protein solution); in addition, mice in this group were intraperitoneally injected with 5 mg / kg of cisplatin solution for three consecutive days from day 1 to day 3 (the dosage was calculated based on cisplatin).

[0135] On the 7th day of the experiment, peripheral blood samples were collected from each group of mice using the eyeball blood sampling method. The mice were fasted but not watered for 12 hours before sampling.

[0136] Biochemical index detection: The collected blood of each group of mice was centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was taken. The serum was tested for ALT and AST enzyme activities using an automatic biochemical analyzer. The results were as follows Figure 6-7 shown.

[0137] Depend on Figure 6-7 It can be seen that on the 7th day, ALT and AST in the CP group increased significantly, and liver damage in the polymer protein group was improved after intervention with polymer protein products.

[0138] Experimental Example 7

[0139] Six-week-old male ICR mice were selected, numbered and randomly divided into five groups, namely, a control group, an adriamycin group, a high-dose polymer protein group, a medium-dose polymer protein group, and a low-dose polymer protein group, with six mice in each group. The polymer protein preparation of Example 2 was dissolved in physiological saline to form a polymer protein preparation solution.

[0140] Control group (A1): During the experiment, the mice were intraperitoneally injected with the same volume of normal saline as the polymer protein product solution given to the medium-dose polymer protein group.

[0141] Adriamycin group (A2): Adriamycin 5 mg / kg was intraperitoneally injected on the first day; thereafter, adriamycin was administered every other day according to the administration method of the first day until the fifth day.

[0142] Polymer protein low-dose group (A3): 5 mg / kg of doxorubicin was intraperitoneally injected on the first day; doxorubicin was administered every other day according to the administration method of the first day until day 5. During the administration of doxorubicin, 10 mg / kg of polymer protein product solution was also administered every day (the dosage was calculated based on the polymer protein product).

[0143] Polymer protein medium dose group (A4): 5 mg / kg of doxorubicin was intraperitoneally injected on the first day; doxorubicin was administered every other day according to the administration method of the first day until day 5. During the administration of doxorubicin, 50 mg / kg of polymer protein product solution was also administered every day (the dosage was calculated based on the polymer protein product).

[0144] Polymer protein high-dose group (A5): 5 mg / kg of doxorubicin was intraperitoneally injected on the first day; doxorubicin was administered every other day according to the administration method of the first day until day 5. During the administration of doxorubicin, 100 mg / kg of polymer protein product solution was also administered every day (the dosage was calculated based on the polymer protein product).

[0145] The mice in each group were killed on the second day after the last administration. Blood was collected from the eyeball into a separation tube and allowed to stand for 30 minutes. The supernatant (serum) was collected and used to detect cardiac injury indicators.

[0146] Depend on Figure 8 and 9 It can be seen that the LDH and CK of mice in the medium-dose group of polyprotein decreased significantly compared with the doxorubicin group, and there was no significant difference compared with the control group. The LDH and CK of the low-dose group and high-dose group of polyprotein also decreased significantly compared with the doxorubicin group. After intervention with 50mg / kg polyprotein products, the heart damage of mice was effectively improved.

[0147] Experimental Example 8

[0148] SPF grade ICR mice, male, 6 weeks old, weighing about 28g were selected as experimental animals. The experimental animals were divided into three groups, namely control group (B1), model group (B2) and polymerized protein group (B3), with 8 mice in each group. 5-Fluorouracil (5-FU) was dissolved in 7wt% dimethyl sulfoxide to form a 5-fluorouracil solution. The polymerized protein product of Example 3 was dissolved in physiological saline to form a polymerized protein product liquid.

[0149] Control group: On days 1-5, the rats were intraperitoneally injected with 7wt% dimethyl sulfoxide, which was equal to the 5-fluorouracil solution injected in the model group, and with saline, which was equal to the polymerized protein solution injected in the polymerized protein group. On days 6-12, the rats were intraperitoneally injected with saline, which was equal to the polymerized protein solution injected in the polymerized protein group.

[0150] Model group: On days 1-5, 50 mg / kg of 5-fluorouracil solution (dosage is based on 5-fluorouracil) was intraperitoneally injected, and at the same time, a volume of normal saline equal to that of the polymerized protein product solution injected into the polymerized protein group was intraperitoneally injected. On days 6-12, a volume of normal saline equal to that of the polymerized protein product solution injected into the polymerized protein group was intraperitoneally injected.

[0151] Polymer protein group: On days 1-5, 50 mg / kg of 5-fluorouracil solution (dosage based on 5-fluorouracil) and 50 mg / kg of polymer protein product solution (dosage based on polymer protein product) were intraperitoneally injected. On days 6-12, 50 mg / kg of polymer protein product solution (dosage based on polymer protein product) were intraperitoneally injected.

[0152] On the 13th day, the whole blood and organs of mice in each group were collected, the serum was separated to measure the biochemical indexes of liver and kidney, and the tissues were preserved for subsequent histopathological examination. Figure 10-13 shown.

[0153] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are indicators of liver parenchymal damage; among them, ALT is a more sensitive indicator and is known as the clinical chemistry gold standard for hepatotoxicity. When the enzyme activity of ALT and AST in the serum increases, it indicates that the liver may be damaged. Urea and creatinine (CRE) are two important indicators of renal function; among them, CRE can more accurately predict renal damage than Urea, and when the levels of both increase, it indicates that the kidneys are damaged.

[0154] Depend on Figure 10-13 It can be seen that the model group showed obvious liver and kidney damage on the 13th day, while the polymerized protein group showed that the liver and kidney damage of mice were effectively improved after intervention with 50 mg / kg of polymerized protein products.

[0155] The results of histopathological examination showed that the liver and kidney tissue structures of mice in the model group were significantly abnormal, with obvious necrotic foci in the liver tissue, hyperplasia of renal capsule epithelial cells, and deformation of renal tubular epithelial cells; the liver and kidney tissue structures of mice in the polymer protein group were significantly alleviated compared with those in the model group.

[0156] Experimental Example 9

[0157] SPF grade ICR mice, male, 6-8 weeks old, weighing about 28 g, were randomly divided into three groups, namely, control group (C1), CP group (C2), and polymerized protein group (C3), with 8 mice in each group. The polymerized protein preparation of Example 3 was dissolved in physiological saline to form a polymerized protein preparation liquid.

[0158] Control group: From day 1 to day 7, the mice were injected with normal saline of the same volume as the polymer protein product solution injected into the polymer protein group.

[0159] CP group: The mice in this group were injected with physiological saline of the same volume as the polymer protein product solution injected in the polymer protein group every day from day 1 to day 7. At the same time, the mice in this group were intraperitoneally injected with 4 mg / kg cisplatin injection (dosage is calculated as cisplatin) every day from day 1 to day 4.

[0160] Polymer protein group: 50 mg / kg of polymer protein product solution was intraperitoneally injected into the mice of this group every day from day 1 to day 7; meanwhile, 4 mg / kg of cisplatin injection solution (dosage is calculated as cisplatin) was also intraperitoneally injected into the mice of this group every day from day 1 to day 4.

[0161] Blood was collected from mice in each group on the 8th day, and the serum was separated to determine the activity of CRE and Urea.

[0162] Depend on Figure 14-15 It can be seen that on the 8th day, the CRE and Urea levels in the CP group increased significantly, indicating that the mouse renal injury model was successfully established. After the intervention of 50 mg / kg polymer protein products in the polymer protein group, the CRE and Urea levels decreased compared with the model group, indicating that the polymer protein products have a certain protective effect on mice with renal injury.

[0163] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. A method for preparing a polymerized protein product, characterized in that: The method comprises the following steps: reacting a protein and a cross-linking agent in a reaction system with a viscosity of ≥2.5 mPa·s; Wherein, the protein includes hemoglobin and / or hemoglobin derivatives.

2. The preparation method according to claim 1, characterized in that: The hemoglobin derivative is selected from one or more of deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin, and sulfhemoglobin.

3. The preparation method according to claim 1, characterized in that: The protein further comprises one or more of albumin, superoxide dismutase, peroxidase, carbonic anhydrase or fibrinogen; The total amount of hemoglobin and hemoglobin derivatives in the protein is more than 85 wt%.

4. The preparation method according to claim 1, characterized in that: reacting the protein and the cross-linking agent in the presence of water and a highly viscous compound; The viscosity of the high-viscosity compound is greater than that of water; the high-viscosity compound is soluble in water or forms a colloid with water; and the high-viscosity compound does not participate in chemical reactions.

5. The preparation method according to claim 4, characterized in that: The high-viscosity compound is selected from one or more of small molecule polyols, polyether diols, cellulose or its derivatives, salt compounds formed by cellulose or its derivatives, and hyaluronic acid.

6. The preparation method according to claim 5, characterized in that: The small molecule polyol contains 2 to 8 carbon atoms, and the number of hydroxyl groups of the small molecule polyol is 2 to 6; The polyether diol is selected from one or more of polyethylene glycol and polypropylene glycol; The cellulose derivative is selected from one or more of carboxymethyl cellulose and hydroxymethyl cellulose; The salt compound formed by cellulose or its derivatives is sodium salt or potassium salt of cellulose or its derivatives.

7. The preparation method according to claim 1, characterized in that: The cross-linking agent is selected from one or more of polyaldehyde compounds, diformaldehyde, open-ring raffinose, diaspirin, activated forms of polyethylene oxide and dextran, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N,N'-phenylenedimaleimide, and diimide compounds; The molar ratio of the total molar amount of hemoglobin and its derivatives to the cross-linking agent is 1:(150-300).

8. The preparation method according to claim 1, characterized in that: The following steps are also included: The cross-linked product obtained by the reaction of the protein and the cross-linking agent is reacted with a reducing agent to obtain a reaction product; small molecules in the reaction product are removed and then concentrated.

9. A polymerized protein product, characterized in that: The polymerized protein product is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the polymerized protein product according to claim 9 as a blood substitute; or use in the preparation of a drug for treating and / or preventing liver damage, kidney damage or heart damage.

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