KR19980020773A - Gram-negative sepsis containing hemoglobin modified substance as an active ingredient - Google Patents

Abstract

The present invention relates to a therapeutic agent for Gram-negative sepsis (G (-) sepsis), and more particularly, a protein constituting hemoglobin in 4 to 12 aliphatic or aromatic hydrocarbon chains in which a linkage of hemoglobin subtissue may have a substituent. A hemoglobin modified body modified by a compound having a functional group capable of reacting with an amino acid or a polyethylene glycol or polypropylene adduct thereof and surface-treated with a polymer having a molecular weight of 1,000 to 10,000 is effective. By providing a gram-negative sepsis treatment ingredient, it neutralizes the properties of endotoxin and nitric oxide, which are the main causes of Gram-negative sepsis caused by Gram-negative bacteria (G (-) bacteria) infection. Prevents toxic manifestations and consequently lowers mortality from Gram-negative sepsis. The.

Description

A remedy for G (-) sepsis having a hemoglobin derivative as the main ingredients

The present invention relates to a therapeutic agent for Gram-negative sepsis (G (-) sepsis), and more particularly, endotoxin and nitrogen monoxide, which are the main causes of Gram-negative sepsis caused by Gram-negative bacteria (G (-) bacteria) infection. The present invention relates to a gram-negative sepsis treatment which neutralizes the properties of nitric oxide, thereby preventing clinical toxic expression and consequently lowering the death rate due to gram-negative sepsis.

Sepsis or septic shock is a series of clinical pathologies caused by bacteria penetrating into blood vessels or cells due to burns or visceral rupture, resulting in 35% mortality. Sepsis includes Gram-positive sepsis (G (+) sepsis) caused by Gram-positive bacteria and Gram-negative sepsis caused by Gram-negative bacteria, each accounting for 50% of the incidence of sepsis.

Gram-positive sepsis does not have pathological toxicity unless Gram-positive bacteria continue to live in the cell, but Gram-negative sepsis has a toxic effect even when Gram-negative bacteria die. The main cause of the toxicity is Gram-negative bacteria It has been found to be endotoxin, and when the endotoxin invades the bloodstream, it has several levels of toxicity, and its effect is amplified with time, making treatment difficult.

Endotoxins bind to endotoxin binding proteins in the bloodstream and then act on white blood cells to release various cytokines. This cytokine is mainly secreted in the shock state of the body, and in the case of Gram-negative sepsis, these cytokines have various harmful effects on the human body. Nitrogen monoxide acts as an intermediate carrier in the process by which endotoxins induce white blood cells to secrete cytokines. Clinical manifestations of Gram-negative sepsis include increased blood pressure, loss of autonomic control of blood vessels, and blood coagulation in blood vessels, resulting in cell edema and hypoxia. This can lead to multiple organ failure and death of the patient.

Therefore, the present invention is to treat the gram negative sepsis, and has a strong affinity for endotoxin and nitrogen monoxide, and once adhered to neutralize their toxicity and physiological action, and as a result It is an object of the present invention to provide Gram-negative sepsis treatment, which serves to stop clinical manifestations.

The present invention for achieving the above object has a functional group capable of reacting with the amino acid of the protein constituting hemoglobin to the 4 to 12 aliphatic or aromatic hydrocarbon chain which hemoglobin substructure may have a substituent. According to the present invention, there is provided a gram-negative sepsis treatment agent comprising a hemoglobin modified substance modified by a compound or a polyethylene glycol or polypropylene adduct thereof and whose surface is treated with a polymer having a molecular weight of 1,000 to 10,000 as an active ingredient. The method for producing the hemoglobin modified body is introduced in Patent Application Nos. 96-20739 filed on June 11, 1996 by the applicant, which will be described below.

The hemoglobin used as a raw material in the method for producing a hemoglobin modified body may be hemoglobin of humans or animals, or genetically engineered hemoglobin cultured by genetic engineering may be used.

When using human or animal blood, only red blood cells are first isolated and then hemoglobin is extracted from red blood cells.

After several stages of purification, impurities are removed and hemoglobin is at least 99.9% pure.

In the case of genetically engineered hemoglobin, it is extracted from the mother and then purified through a multi-stage separation and purification process.

Pure hemoglobin is a structure in which four substructures of two α-chains and two β-chains (molecular weight of 16,000 each) are combined and have a molecular weight of about 64,000, and the non-covalent bonds between substructures are hydrogen bonds and van der Waals bonds. Each substructure can be combined with one oxygen molecule. This type of binding between substructures of hemoglobin is fluid, and hemoglobin experiences structural changes in the process of binding and desorption of oxygen molecules.

Preferably, the hemoglobin used herein is purified as described above and then exposed to nitrogen to gradually reduce the oxygenated hemoglobin concentration so that the reaction starts when the oxygenated hemoglobin concentration decreases to 15-35%. .

This is because, as shown in Table 1 below, when the oxygen saturation concentration of hemoglobin before the reaction is higher than 30%, p50 is also high as a result, which is not suitable for supplying oxygen to hypoxic cells.

Therefore, if the oxygen saturation concentration of hemoglobin is adjusted to 15-35% prior to the reaction of the substructure connective structure, which is the first step in the preparation of hemoglobin denaturation, p50 is about 9-11 mmHg. It will be superior.

Oxygen saturation concentration of hemoglobin before reaction (%) p50 (mmHg) 15 9.5 30 11.2 45 16.3 60 22.3 75 24.8 90 24.5

After the oxygenated hemoglobin concentration is 15-35%, the bond between these substructures of hemoglobin is first modified by chemical reactions, ie by changing the structure of the subtissue linkages, thus altering the original oxygenation capacity of hemoglobin. do.

This chemical reaction is a compound having a functional group (halogen or succinimide group) capable of reacting with 4 to 12 aliphatic or aromatic hydrocarbon chains which may have a substituent by reacting with amino acids of proteins constituting hemoglobin, or Polyethylene glycol or polypropylene adduct (preferably having a molecular weight of 1,000 to 5,000) is reacted with hemoglobin to modify the structure of the linkage between the substructures of hemoglobin. Once this reaction occurs between the hemoglobin sub-tissues, the structure remains permanent.

As a specific example of the compound used for the structural modification of such a substructure, bis-3,5-dibromosalicylic acid, bis-3,5-dibromosuccinic acid, bis-3,5-dibromofumaric acid , Bis-3,5-dibromomuconic acid, disuccinimidyl glutarate, dithiobis-succinimidyl propionate, disuccinimidyl suverate (the above compounds form internal linkages of subtissues); Bis-succinimidyl succinate polypropylene glycol, bis-succinimidyl carbonate polyethylene glycol, bis-succinimidyl succinate propylene glycol and bis-succinimidyl carbonate polypropylene glycol (the above compounds are used to Forming).

Once the connections between the subtissues are completed, the next step is to surface the hemoglobin. Linear polymers having a molecular weight of about 1,000 to 10,000, preferably 1,000 to 5,000, are used, and at one end of the polymer, a reactor capable of selectively binding to lysine of hemoglobin to form a covalent bond.

Preferred examples of the polymer used in the present invention include succinates such as polyethylene glycol-succinimidyl succinate, polyethylene glycol-succinimidyl carbonate, polypropylene glycol-succinimidyl succinate, or polypropylene glycol-succinimidyl carbonate. Mention may be made of polyethyleneglycol or polypropylene glycol having imidyl succinate or succinimidyl carbonate groups at the ends.

When the polymer and hemoglobin are mixed with each other in an optimal reaction environment, the polymers bind to amino acids located on the surface of hemoglobin, or lysine, and each hemoglobin molecule attaches 5 to 20 polymers to the surface.

The finished hemoglobin modified product thus increases the total molecular weight to 100,000 to 200,000.

That is, according to the present invention, hemoglobin molecules undergo a two-step reaction of first, binding denaturation between substructures, and second, surface treatment using a polymer.

The binding between the subtissues is carried out under a special reaction environment, and after completion of the process, the oxygen separation ability of hemoglobin changes, which plays a role of delivering oxygen only to hypoxic cells.

The surface treatment process using polymers has several benefits, one of which is that polymers disguise antigens located on the surface of hemoglobin so that the patient's immune system does not produce an antibody response. Another benefit is a significant increase in the molecular weight of SB1, which increases blood flow residence time to about 60 hours, which was only about 4 hours for hemoglobin without polymer surface treatment.

On the other hand, the present invention is the invention, according to the discovery that the hemoglobin modified product prepared according to the above process as an active ingredient can be used as a therapeutic agent for gram negative sepsis.

The reason why the hemoglobin modified product can be used as a Gram-negative sepsis treatment agent is as follows.

Hemoglobin, the main part of the hemoglobin modified substance, has a part on the surface that shows a strong affinity for endotoxin, so that it is difficult to separate once adhered. Endotoxins are polysaccharides, and their active moieties adhere to the surface of hemoglobin, so once they form a conjugate with hemoglobin, they lose the toxic effects of endotoxin. The heme pocket located inside the hemoglobin, the part where oxygen is bound and released, is bound by affinity 1,000 times stronger than oxygen when nitrogen monoxide is around, so that the combination of nitrogen monoxide and heme pocket When this happens, it is also almost impossible to separate again, which can disable the physiological action of nitric oxide.

Thus, by disabling the effects of endotoxin and nitrogen monoxide, blood vessels and visceral organ cells are also protected from them, thereby increasing the survival rate.

Hereinafter, the action of the hemoglobin denaturant for gram-negative sepsis treatment according to the present invention will be described as specific examples and experimental examples.

Herein, the hemoglobin modified product for the treatment of Gram-negative sepsis used as a test solution was prepared according to the example described in the Patent Application Nos. 96-20739 of the present applicant.

Example 1

Purified hemoglobin in human brain is dissolved in 0.05M Tris solution at room temperature and adjusted to pH7.0-7.2.

At this time, hemoglobin exposed to air is 90 to 95% of oxygenated hemoglobin, so when exposed to nitrogen while passing it through a gas exchange device, the concentration of oxygenated hemoglobin can be gradually reduced. Turn off the gas exchanger when the oxygenated hemoglobin concentration is reduced by 15-35%, and do not expose the hemoglobin solution to air.

Maintaining this state, bis-3,5-dibromosalicylic acid is added as a subtissue linkage (connector: hemoglobin ratio = 4: 1). This reaction takes about 2 to 4 hours. After the reaction is completed, change the solution to Tris solution to remove all unreacted materials.

The pH is then adjusted to 7.6-7.8 while changing the solution to 0.5 M sodium chloride solution and 0.05 M sodium phosphate solution. Then, as a polymer for surface treatment, polyethylene glycol-succinimidyl succinate having a molecular weight of 5,000 is mixed with the solution to cause a reaction (polymer: hemoglobin ratio = 20: 1).

This surface treatment reaction takes about 1 to 2 hours, and stored in a solution that can be physiologically injected through solution replacement after completion of the reaction, that is, 0.15 M sodium chloride, 0.03 N sodium bicarbonate, and 0.05 M sodium phosphate solution. Do it.

Example 2

Purified hemoglobin in bovine blood is added to 0.1M Tris solution at room temperature and the pH is adjusted to 7.6-7.8. Hemoglobin lowers the oxygenated hemoglobin concentration to 15-35% through a gas exchanger. Dissuccinimidyl glutarate is mixed with the solution as a subtissue linkage (the ratio of subtissue linkage to hemoglobin is 6-8: 1).

Once this reaction is complete, the solution is replaced with 0.25M sodium chloride solution, 0.05M sodium phosphate solution and the pH is adjusted to 7.6-7.8. Subsequently, the surface treatment step was carried out according to the procedure of Example 1 to prepare a hemoglobin modified body.

Example 3

Hemoglobin purified from bovine blood is added to 0.1-0.3 M sodium chloride and 0.05 M sodium phosphate solution at room temperature to adjust the pH to 7.4 to 7.6. Again, the gas exchange device is used to reduce the oxygenated hemoglobin concentration to 15-35%.

Next, bis-succinimidyl succinate polyethylene glycol is added as a subtissue linker. The molecular weight of the polymer linkage is 1,000 to 5,000, wherein the ratio of the substructure linkage to hemoglobin is 5 to 10: 1. After this reaction was completed, the surface treatment process was repeated as in Example 1.

At the end of this reaction, the solution is replaced with a physiological solution.

On the other hand, the oxygen separation ability of the blood or hemoglobin can be expressed as a measure of p50, which indicates the surrounding oxygen concentration (pO ₂ , mmHg) when the oxygen saturation concentration of hemoglobin reaches 50%. The higher it is, the more difficult the oxygen bonds, and the easier the oxygen separation can be analyzed.

Thus, p50 of the samples prepared according to the above example is shown in Table 2, where it can be seen that the p50 of human blood is 26-28mmHg and the samples of Examples 1-3 are 9-11mmHg.

sample Average temperature during measurement (℃) p50 Human red blood cells 36.5 27.2 Example 1 Sample 1 36.5 9.7 Example 1 Sample 2 36.8 10.6 Example 2 Sample 1 36.8 9.6 Example 2 Sample 2 36.8 10.6 Example 3 Sample 1 37.2 10.3 Example 3 Sample 2 37.4 11.1

Experimental Example

In order to test whether the hemoglobin denatured body contains the gram-negative sepsis efficacy as an active ingredient, endotoxin was directly injected into experimental animals to simulate the symptoms of gram-negative sepsis. Sprague-Dawley rats weighing 150-250 g were used as experimental animals, and the oxygen cell concentrations, muscle arterial pressure changes, and survival rates up to 3 days after the experiment were measured. It was set as.

Oxygen concentration was measured by measuring the oxygen concentration of the abdominal muscles, the measuring instrument was used oxyspot phosphorimeter (manufacturer: Medical Systems Corp., Greenvale, NY), the method is a decrease in phosphorescence Measured by the method, the injected phosphor (palladium-porphyrin) is a phenomenon that the intensity of phosphorescence decreases when combined with oxygen, indirectly determine the intracellular oxygen concentration by measuring the intracellular phosphorescence using this property (Ref. Wilson et. Al, J. Appl. Physiol., 70, 2691-2696 [1992]).

For the artificial induction of Gram-negative sepsis, the experimental animals were injected intravenously with endotoxin and the dose was 20 mg / Kg or 30 mg / Kg.

LD ₉₀ of endotoxin is known to be 20-30 mg / Kg, so the expected mortality at the dose is 90%.

After 30 minutes of endotoxin injection, the test solution was injected. At this time, the experimental animals were divided into four groups assigned to each group, the first group was not injected with any test solution, the second group was injected with Ringer's solution 20mg / Kg, the third group was Gram-negative sepsis treatment 10mg / Kg, and the fourth group was 20 mg / Kg of Gram-negative sepsis treatment was injected.

In this experiment, muscle cell oxygen concentration and blood pressure were measured before endotoxin injection (default), 30 minutes after endotoxin injection, and 30 minutes after test solution injection.

Survival experiments were conducted separately, divided into four groups as described above and assigned to each group 20 experimental animals. At this time, the endotoxin injection amount was 30 mg / Kg, and the survival rate was observed at 1, 2, 24, 48 and 72 hours after the test solution injection.

According to Table 3 and Table 4, which are the results of the animal efficacy test, muscle cell oxygen concentration and blood pressure decreased after 30 minutes after endotoxin injection, indicating the general symptoms of Gram-negative sepsis. Muscle cell oxygen concentration and blood pressure in the Ringer's fluid group remained almost the same as those in the control group, with no signs of recovery. On the other hand, the Gram-negative sepsis treatment group has proven its efficacy as a Gram-negative sepsis treatment by restoring muscle tissue oxygen concentration and blood pressure to almost normal levels.

When the endotoxin dose is 20 mg / Kg division Muscle Cell Oxygen Concentration (mg / Kg) Blood pressure (mmHg) Default After endotoxin injection After injection of test solution Default After endotoxin injection After injection of test solution Control group 22 ± 3 15 ± 4 12 ± 3 84 ± 6 65 ± 7 58 ± 9 Ringer's Liquid Group 21 ± 4 14 ± 3 11 ± 4 80 ± 5 62 ± 5 68 ± 7 Gram-negative sepsis group (10 mg / Kg) 23 ± 5 12 ± 4 19 ± 3 87 ± 7 63 ± 4 76 ± 6 Gram-negative sepsis treatment group (20 mg / Kg) 21 ± 3 14 ± 5 18 ± 4 82 ± 6 68 ± 6 72 ± 7

When the endotoxin dose is 20 mg / Kg division Muscle Cell Oxygen Concentration (mg / Kg) Blood pressure (mmHg) Default After endotoxin injection After injection of test solution Default After endotoxin injection After injection of test solution Control group 23 ± 4 17 ± 3 13 ± 4 82 ± 10 62 ± 8 63 ± 7 Ringer's Liquid Group 25 ± 3 19 ± 4 12 ± 5 84 ± 8 60 ± 7 61 ± 8 Gram-negative sepsis group (10 mg / Kg) 20 ± 5 13 ± 4 18 ± 4 85 ± 8 66 ± 7 78 ± 10 Gram-negative sepsis treatment group (20 mg / Kg) 22 ± 4 16 ± 5 19 ± 4 81 ± 6 70 ± 5 75 ± 8

When the endotoxin dose is 30 mg / Kg Default After endotoxin injection 1 hour after injection of test solution 4 hours 24 hours 48 hours 72 hours Control group 100 100 80 65 15 0 0 Ringer's Liquid Group 100 100 75 55 20 0 0 Gram-negative sepsis group (10 mg / Kg) 100 100 100 100 85 85 75 Gram-negative sepsis treatment group (20 mg / Kg) 100 100 100 100 90 85 70

According to Table 4, which is the result of the survival experiment, the control group and the Ringer's solution group decreased the survival rate to 15-20% after 24 hours, and recorded 0% after 48 hours.

On the other hand, in the Gram-negative sepsis group, the survival rate was 85-90% after 24 hours and 70-75% after 72 hours, demonstrating that the hemoglobin variant had efficacy as a therapeutic for Gram-negative sepsis. It was.

The hemoglobin denatured substance can be used as a therapeutic agent for gram negative sepsis by eliminating the toxicity of endotoxin which is the cause of gram negative sepsis due to its molecular structure.

Claims (2)

A compound having a functional group capable of reacting with an amino acid of a protein constituting hemoglobin to bind to 4 to 12 aliphatic or aromatic hydrocarbon chains in which the hemoglobin substructure may have a substituent, or a polyethylene glycol or polypropylene addition thereof By water, denatured; A Gram-negative sepsis treatment agent whose surface contains a hemoglobin modified body surface-treated with a polymer having a molecular weight of 1,000 to 10,000 as an active ingredient. The method of claim 1, wherein the hemoglobin modified gram negative sepsis treatment, characterized in that the p50 value indicating the oxygen separation ability is less than 20mmHg.