KR100316154B1 - Polyethylene glycol-hemoglobin conjugate - Google Patents

Abstract

The present invention is to provide a novel PEG-hemoglobin conjugate of the following formula (1) that can be used for medical purposes with the safety and efficacy as an oxygen carrier.

[Formula 1]

Description

Polyethylene glycol-hemoglobin conjugate {POLYETHYLENE GLYCOL-HEMOGLOBIN CONJUGATE}

The present invention relates to a novel PEG-hemoglobin conjugate, and more particularly to a PEG-hemoglobin conjugate of the formula (1) that can be used for medical purposes with safety and efficacy as an oxygen carrier.

Activated polyethylene glycol (hereinafter referred to as 'PEG') or PEG derivative is attached to the surface of proteins, enzymes and other biological agents, and may be used as a medicine. The changes that result from PEG attachment are that the molecular weight increases by the amount of PEG attached, and that the surface is covered with PEG to reduce antigen-antibody responses. In addition, this has the advantage of increasing the remaining time in the body and decrease the antigenic expression. In the same principle, PEG and hemoglobin conjugated PEG and hemoglobin can be used as an oxygen carrier using the oxygen transport role of hemoglobin itself, and its application fields include artificial blood, stroke treatment, and tromotransfusion. The hemoglobin molecule has a molecular weight of about 65,000, and the molecule itself is only about 4 to 6 hours of residual half-life, which may cause toxicity. On the other hand, PEG-hemoglobin conjugates with PEG increase the residual half-life of the body by about 20-40 hours and decrease antigenic expression.

The prior art concerning the preparation of PEG-hemoglobin is as follows.

The PEG-hemoglobin described in US Pat. No. 4,670,417 is a reaction of polyethylene glycol succinate-PEG (SS-PEG) with hemoglobin to form PEG-hemoglobin of formula (2).

In addition, PEG-hemoglobin described in US Pat. No. 5,234,903 is a reaction of polyethylene glycol succinimidyl carbonate (Succinimidyl carbonate-PEG; SC-PEG) and hemoglobin, which forms PEG-hemoglobin of the following formula (3).

On the other hand, various methods of attaching PEG to hemoglobin have been studied. When using different kinds of PEG derivatives, the form of covalent bonds formed between PEG and hemoglobin is different.

The present invention seeks to provide a novel form of PEG-hemoglobin conjugate that can be used for medical purposes with stability and efficacy as an oxygen carrier.

In order to achieve the above object, the present invention provides a PEG-hemoglobin conjugate represented by the following Chemical Formula 1. The PEG-hemoglobin conjugate of the present invention can be obtained by reacting polyethylene glycol succinimidyl propionate (Succinimidyl propionate-PEG; hereinafter SP-PEG) with hemoglobin, wherein SP-PEG is a mixture of PEG and ethyl acrylate. It can obtain by reacting with N-hydroxysuccinimide. Herein, the SP-PEG preferably has a molecular weight of 100 to 200,000 Daltons. Hereinafter, the structure and operation of the present invention will be described in detail with reference to Examples. However, these Examples are only illustrative of the present invention, the scope of the present invention is not limited to these. Example 1 Preparation of Polyethyleneglycol Succinimidyl Propionate (SP-PEG)

100 g (MW 5,000, 0.02 mol) of methoxy-PEG (m-PEG) and 1.36 g (1 eq) of sodium ethoxide were placed in a 250 ml round flask and dissolved completely by heating to 80 ° C. in an oil bath. . The PEG used at this time may be used a variety of molecular weights between 100 to 200,000. 21.28 ml (0.2 mol) of ethyl acrylate was slowly added to the reaction mixture, and the mixture was heated and stirred at 100 to 110 ° C. for about 15 hours. Distilled water was added to the reaction mixture, and the mixture was washed with diethyl ether and extracted with 600 and 500 ml of dichloromethane. The extracted organic layer was dried over magnesium sulfate, washed twice with brine, and then distilled under reduced pressure to remove the organic solvent. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the mixture was filtered under reduced pressure to obtain a solid. The precipitate obtained by filtration was recrystallized with 200 ml of ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain 93 g of m-PEG ethyl propionate compound in the form of a white powder.

This process can be represented by the following scheme 1. In Scheme 1, n is 113 to 114.

93 g of the m-PEG ethyl propionate was dissolved in 300 ml of 1 N aqueous sodium hydroxide solution, and stirred at room temperature for 17 hours. The reaction mixture was acidified to pH 2 with 2N aqueous hydrochloric acid solution, 30 g of sodium chloride was added, and then extracted with dichloromethane (1000, 800, 600 mL). The extracted organic layer was dried over magnesium sulfate, washed twice with brine, and then distilled under reduced pressure to remove the organic solvent. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain an m-PEG propionic acid compound in the form of a white powder.

This process is shown in Scheme 2 below. In Scheme 2, n is 113 to 114.

86.5 g of the m-PEG propionic acid was dissolved in 250 ml of dichloromethane and stirred under the condition of 0 to 5 ° C. To this mixture was added 3.9 g (2 equivalents) of N-hydroxy succinimide (NHS) and then 7.0 g (2 equivalents) of dicyclohexylcarbodiimide was added to 50 ml of dichloromethane. It melt | dissolved and it added slowly on 0-5 degreeC conditions. The reaction mixture was stirred at room temperature for about 15 hours. The reaction mixture was filtered under reduced pressure to remove byproduct dicyclohexylurea and distilled under reduced pressure to remove the organic solvent. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain an m-PEG succinimidyl propionate compound in the form of a white powder.

This process is shown in Scheme 3 below. In Scheme 3, n is 113-114.

Example 2 Preparation of a Combination of SP-PEG and Hemoglobin

Blood was collected from mammalian animals to separate only red blood cells, and hemoglobin was isolated and purified from red blood cells. Then, hemoglobin was dissolved in an aqueous solution of 0.15 M sodium chloride, 0.01 M Na-phosphate pH 8.0, and SP-PEG obtained in Example 1 was dissolved. Hemoglobin: SP-PEG = 1: 20 equivalent ratio was added. The reaction proceeded at room temperature with vigorous stirring and kept the pH at 8.0. The reaction was completed within 1 to 2 hours, after which ultrafiltration or diafiltration was performed to remove unreacted PEG.

This process is shown in Scheme 4 below. In Scheme 4, n is 113 to 114.

Example 3 Properties of SP-PEG and Hemoglobin Binders

(1) purity

The purity of the PEG-hemoglobin conjugate prepared in Example 2 was confirmed by HPLC, isoelectrofocusing (IEF), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Size exclusion HLPC showed a single peak at the retention time of a material having a molecular weight of 100,000 to 130,000 at 280 nm, confirming that no macromolecule other than hemoglobin or other impurities exist. IEF showed that the pI (isoelectric point) of this PEG-hemoglobin was the same as the pI (6.8) of bovine hemoglobin. This indicates that the chemical modification did not affect the pI of hemoglobin. SDS-PAGE shows three different bands resulting from the breakdown of hemoglobin, demonstrating the absence of foreign proteins. will be.

(2) phospholipid and impurities removal

Hemoglobin from which phospholipids were removed through a polyethylene imine (PEI) column was confirmed to be free of phospholipids remaining in the hemoglobin solution through Size Exclusion HLPC.

(3) endotoxin level

Quantitative analysis of the endotoxin content using Kinetic Turbidimetric Assay of LAL (Endosafe) showed 0.03 to 0.5 EU / ml, and the rabbit pyrogen test also proved safe to use.

(4) free iron concentration

Since iron atoms are known to be toxic as hemoglobin is released as they are oxidized, it is desirable to have as few iron atoms in the PEG-hemoglobin conjugate as the final product. As a result of analysis, the amount of iron ions in the PEG-hemoglobin conjugate of the present invention was mild and the toxicity was not considered.

(5) oncotic pressure and viscosity

Pulsation (called oncotic pressure or colloid osmotic pressure) is measured with an oncometer. The viscosity of the liquid at 37 ° C. was measured using an Ubbeholde calibrated viscometer (Fisher Scientific), and the swelling pressure was 22-23 mmHg and the viscosity was 3.9-4.1 cpp.

(6) electrolytes and pH

Consisting of 110 mM NaCl and 5 mM Na-phosphate, similar to normal saline, pH 7.2-7.6.

(7) p50

P50 was measured to assess the affinity for the PEG-hemoglobin conjugates of the invention for oxygen. As a result, p50 was 9.6-11.1 mmHg, and Hill coefficient was 1.5. This means that 70% of the PEG-hemoglobin conjugate is bound to oxygen at pO ₂ 20 mmHg and 25% to oxygen in the blood. In other words, it can be said that it has the ability to efficiently deliver oxygen at a low oxygen partial pressure. Therefore, when the PEG-hemoglobin conjugate of the present invention is used as a long-term preservation solution, it is possible to prevent ischemia due to lack of oxygen and to prevent cell death by stopping metabolism due to lack of oxygen.

The components of the PEG-hemoglobin conjugate of the present invention are shown in Table 1 below.

Ingredient amount PEG-hemoglobin binder concentration (g / dl) 5 to 6 Hemoglobin concentration (g / dl) 2.5 to 3 Oxyhemoglobin (%) 80.0-90.0 Methemoglobin (%) 0-15 Carboxyhemoglobin (%) 4 to 6 p50 (mmHg) 9.6 to 11.1 Hill coefficient 1.5 Viscosity (cp) 3.9 to 4.1 Swelling pressure (mmHg) 22-23 Osmolarity (mOsm) 310 to 320 Phospholipids and Other Impurities Not detected Endotoxin (EU / mL) 0.05 to 0.5 Free Iron (mg / ㎗) 1.1 to 2.21 Sterility pass Storage Stability 24 hours at room temperature-2 years at -20 ℃

Example 4 Preclinical Toxicity Testing to Verify the Safety of PEG-hemoglobin Conjugates

(1) Single dose toxicity test using rat

Experiments were conducted to determine the toxicity seen when a single dose of the PEG-hemoglobin conjugate of the present invention was administered to rats. PEG-hemoglobin conjugates were divided into three groups, T1, T2, and T3, as shown in Table 2 below, and the negative control group was injected with Ringer's solution.

Test group Male and female The number of animals Dose Rate (ml / kg / hr) Dosage (Ml / kg) (Mg / kg) Negative Control cock 5 25 50 0 female 5 25 50 0 T1 cock 5 25 12.5 875 female 5 25 12.5 875 T2 cock 5 25 25 1750 female 5 25 25 1750 T3 cock 5 25 50 3500 female 5 25 50 3500 General symptoms and the presence of dead animals were observed at least once every hour from 1 to 6 hours after administration and from the next day to 7 days after administration. Body weights were measured before initiation and 1, 3 and 7 days after administration, respectively. All surviving animals were euthanized with CO ₂ gas and then opened and visually observed all internal organs.

No deaths were observed throughout the test period in both the negative control and test substance administered male and female. Therefore, the LD ₅₀ value of this test substance was judged to be 50 ml / kg or more in both male and female animals. No abnormalities were observed in the negative control group and the test substance administration group. In weight change, no abnormalities were observed in all male and female test groups compared to the negative control group during the test period. However, on the 1st day after the administration, weight gain inhibition or reduction was observed regardless of the administration of the test substance in all the administration groups, including the negative control group. In autopsy findings, no changes related to the administration of test substance were observed in all groups of male and female. Renal extension of renal pelvis was observed in one case of the female negative control group and one case of the 25 mg / kg administration group.

In other words, as a result of intravenous single-dose administration of the PEG-hemoglobin conjugate of the present invention to male and female rats, no abnormalities were observed in the presence or absence of death animals, general symptoms and weight change, and autopsy findings. The LD ₅₀ value was determined to exceed 50 ml / kg (3500 mg / kg in the amount of PEG-hemoglobin conjugate) in both sexes.

(2) Single dose toxicity test in dogs

Experiments were conducted to determine the toxicity that occurs when a single dose of PEG-hemoglobin conjugate of the present invention is applied to a beagle dog. PEG-hemoglobin conjugates were divided into three groups: T1, T2, and T3, and negative control was injected with Ringer's solution.

Test group Male and female The number of animals Dose Rate (ml / kg / hr) Dosage (Ml / kg) (Mg / kg) Negative Control cock 2 25 25 0 female 2 25 25 0 T1 cock 2 25 6.25 875 female 2 25 6.25 875 T2 cock 2 25 12.5 1750 female 2 25 12.5 1750 T3 cock 2 25 25 3500 female 2 25 25 3500

General symptoms and the presence of dead animals were observed at least once every hour until 1-6 hours after administration, and every day from the next day to 7 days after administration. Body weights were measured before administration and 1, 3, 7 and 14 days after administration. All surviving animals were euthanized with pentobarbital sodium, and then opened and visually observed internal organs.

No deaths were observed throughout the trial in both negative controls and male and female subjects. No abnormalities were observed in the negative control group and the test substance administration group. No change in body weight was observed in all study males and females compared to the negative control during the trial.

Therefore, the LD ₅₀ of the PEG-hemoglobin conjugate of the present invention was judged to be higher than 50 ml / kg or 3500 mg / kg.

Example 5 Pharmacology of PEG-hemoglobin conjugates

(1) impact on general behavior

Animals were male and female of ICR mice (20-25 g body weight) in each group. The experimental method was a modified Irwin method, and general behavior observation was carried out using an experimental observation cage at 0, 0.25, 0.5, 1, 2 and 4 hours after the test material was administered intravenously.

In the general behavior shown by the animals up to 5 hours after intravenous administration of the PEG-hemoglobin conjugate of the present invention, no abnormal symptoms were observed in comparison with the control animals, and the test results are shown in Table 4 below.

Abbreviation Dosage Route of administration The number of animals The number of abnormal expression animals 0 min 15 minutes 30 minutes 60 minutes 120 minutes 240 minutes Vehicle 20 ml / kg i.v. 8 0 0 0 0 0 0 PEG-hemoglobin conjugate 5 ml / kg i.v. 8 0 0 0 0 0 0 10 ml / kg i.v. 8 0 0 0 0 0 0 20 ml / kg i.v. 8 0 0 0 0 0 0 Diazepam 6 mg / kg i.p. 8 0 7 7 5 2 0

(2) effect on spontaneous movement

The animals used eight male ICR mice (20-25 g body weight) in each group. After 30 minutes of intravenous administration of the test substance, the animals were placed in a plastic box (260 × 250 × 400 mm), and the movements of the animals for 5 minutes were measured using a mortity meter (PAS, San Diego) at 0, 15, 30 At 60, 120 and 240 minutes. The result of measuring the spontaneous momentum did not show any effect compared to the control group.

(3) Rotarod test

The animals used 8 male ICR mice in each group. The experiment used the Rotarod method developed by Dunhum and Miya. The animals were trained for 3 minutes at rotarod (16 rpm) the day before the experiment, then only animals that did not fall off the rotarod for 3 minutes were used for the test. The animals were counted from the rotarod (16 rpm) for 1 minute at 0, 15, 30, 60, 120 and 240 minutes after intravenous administration of the test substance. The test showed no effect compared to the media control group.

(4) Effects on Hexobarbital Sleep Time

The animals used 8 male ICR mice in each group. After 30 minutes of intravenous administration of the test substance to the animals, hexobarbital sodium (70 mg / kg) was intraperitoneally administered, and the time from the disappearance of the righting reflex to the animal was measured.

As shown in Table 5 below, no effect was observed in the test substance-administered group compared to the medium control group.

Abbreviation Dosage Route of administration The number of animals Sleep time Deviation(%) Excipient 20 ml / kg i.v. 8 49.8 ± 5.0 100 PEG-hemoglobin conjugate 5 ml / kg i.v. 8 57.6 ± 8.8 116 10 ml / kg i.v. 8 56.1 ± 10.5 113 20 ml / kg i.v. 8 60.3 ± 12.9 121 Chloropromazine 6 mg / kg i.p. 8 189.4 ± 43.1 380

(5) effect on normal body temperature

The animals used 8 male ICR mice in each group. The test substance was administered intravenously to the animals and body temperature was measured at 0, 15, 30, 60, 120 and 240 minutes.

Table 6 shows the results. As a result of measuring the temperature of the animals from just after administration of the test substance to 240 minutes, there was a significant increase in the 10, 20 ㎖ / kg administration group at 10 minutes after administration, and a slight significant increase in the 20 ㎖ / kg administration group even at 240 minutes. Indicated.

Abbreviation Dosage (ml / kg) Route of administration The number of animals The number of abnormal expression animals 0 min 15 minutes 30 minutes 60 minutes 120 minutes 240 minutes Vehicle 20 i.v. 8 36.6 ± 0.50 37.5 ± 0.3 37.3 ± 0.2 37.3 ± 0.3 37.0 ± 0.3 36.6 ± 0.4 PEG-hemoglobin conjugate 5 i.v. 8 36.8 ± 0.4 36.9 ± 0.5 37.1 ± 0.4 37.2 ± 0.5 37.4 ± 0.3 36.9 ± 0.3 10 i.v. 8 37.0 ± 0.5 36.9 ± 0.5 37.1 ± 0.4 37.3 ± 0.4 37.6 ± 0.5 37.0 ± 0.4 20 i.v. 8 36.8 ± 0.3 37.3 ± 0.4 37.3 ± 0.4 37.3 ± 0.4 37.2 ± 0.2 37.1 ± 0.4

(6) Analgesic action: inhibition of the riding reaction caused by acetic acid

The test substance was intravenously administered to the animal, and after 30 minutes, 0.1 ml / 10 g of acetic acid (1%) was intraperitoneally administered, and the number of riding, which is the movement of the animal to stretch the whole body for 5 minutes, was measured. As a result, the test substance had no effect compared to the control group. These results are shown in Table 7 below.

Abbreviation Dosage Route of administration The number of animals Number of rides (No.) Excipient 20 ml / kg iv 8 13.5 ± 3.4 PEG-hemoglobin conjugate 5 ml / kg iv 8 14.3 ± 3.5 10 ml / kg iv 8 15.0 ± 3.4 20 ml / kg iv 8 14.9 ± 3.8 Ketoprofen 10 mg / kg ip 8 6.1 ± 3.6 (7) Analgesic action: suppressing licking reaction caused by hot plate

Thirty minutes after the test material was administered to the animal, the animal was placed on a heating plate (57 ° C.) and the time at which the animal began to lick the forefoot was measured. As a result, the test substance had no effect compared to the control group. These results are shown in Table 8 below.

Abbreviation Dosage Route of administration The number of animals Liking time (sec) Excipient 20 ml / kg i.v. 8 4.2 ± 0.6 PEG-hemoglobin conjugate 5 ml / kg i.v. 8 4.2 ± 1.0 10 ml / kg i.v. 8 4.5 ± 1.4 20 ml / kg i.v. 8 4.4 ± 0.8 Codeine 100 mg / kg i.p. 8 13.2 ± 6.1

(8) anticonvulsant effect: effects on pentylenetetrazole-induced spasms

After 30 minutes of intravenous administration of the test substance, the effect of pentylenetetrazole (100 mg / kg) on the liver was measured. As a result, the test substance was found to have no anticonvulsant effect. These results are shown in Table 9 below.

Abbreviation Dosage Route of administration Cramp (No) TE time (sec) protect(%) Excipient 20 ml / kg i.v. 8/8 57.6 ± 8.8 0 PEG-hemoglobin conjugate 5 ml / kg i.v. 8/8 59.9 ± 21.1 0 10 ml / kg i.v. 8/8 61.5 ± 20.8 0 20 ml / kg i.v. 8/8 67.6 ± 42.1 0 Codeine 100 mg / kg i.p. 0/8 - 100

(9) Anticonvulsant effect: effect on strychnine-induced spasms

After 30 minutes of intravenous administration of the test substance, the effect of strikinin (2 mg / kg) on the abdominal cavity was measured. As a result, the test substance was found to have no anticonvulsant effect. These results are shown in Table 10 below.

Abbreviation Dosage Route of administration Cramp (No) TE time (sec) protect(%) Excipient 20 ml / kg i.v. 8/8 164.5 ± 39.5 0 PEG-hemoglobin conjugate 5 ml / kg i.v. 8/8 148.0 ± 30.6 0 10 ml / kg i.v. 8/8 169.9 ± 27.1 0 20 ml / kg i.v. 8/8 170.4 ± 27.1 0 Diazepam 10 mg / kg i.p. 4/8 286.8 ± 32.8 50

(10) Anticonvulsant effect: effect on electroshock induced spasms

After 30 minutes of intravenous administration of the test substance, the effects of cramps on the animals were measured after applying electric shock (60 ㎃, 0.3 sec) to both ears of the animal. As a result, the test substance was found to have no anticonvulsant effect. These results are shown in Table 11 below.

Abbreviation Dosage Route of administration Cramp (No) protect(%) Excipient 20 ml / kg i.v. 8/8 0 PEG-hemoglobin conjugate 5 ml / kg i.v. 8/8 0 10 ml / kg i.v. 8/8 0 20 ml / kg i.v. 8/8 0 Diazepam 20 mg / kg i.p. 8/8 0

(11) Effects on the cardiovascular system: Effects on blood pressure and heart rate of non-anesthetic rats

SD rats weighing 350-450 g were anesthetized with thiopental (50 mg / kg, ip), and then a cannula was inserted into the femoral artery and the femoral vein, respectively, and the other end of the cannula was subcutaneously. Followed by the back of the neck was fixed. After surgery, the animals were allowed to stabilize overnight and the catheter inserted in the femoral artery was connected to a pressure transducer and a physiograph on the day, and the blood pressure and heart rate were converted into a data acquisition system (Biopak, Model). MP100). After confirming that the blood pressure was stabilized about 1 hour, the test substance was administered intravenously, and blood pressure and heart rate were measured up to 6 hours after administration at regular time intervals, and the change in blood pressure and heart rate before drug administration was expressed. As a result, no significant effect was observed in comparison with the medium control animal group. These results are shown in Tables 12 and 13 below. Table 12 shows the change rate of the average blood pressure, Table 13 shows the change rate of the heart rate.

Minutes Excipient 5 ml / kg 10 ml / kg 20 ml / kg Average(%) S.E.M. Average(%) S.E.M. Average(%) S.E.M. Average(%) S.E.M. 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10 2.13 2.89 -4.48 3.76 7.76 4.76 1.26 2.06 20 1.96 3.61 0.88 2.77 9.05 6.57 -0.05 3.95 30 3.45 5.24 1.09 3.34 14.04 4.62 4.44 4.71 60 -5.02 2.29 -1.96 2.68 12.49 4.76 6.41 2.01 90 -4.44 3.76 -3.69 1.54 10.05 5.50 4.95 4.73 120 -4.34 3.72 -5.51 2.46 6.72 5.37 8.81 2.48 240 -5.93 5.54 -8.55 2.97 7.44 4.76 -2.66 3.15 360 -2.67 6.95 -7.93 1.84 7.95 5.08 -3.72 3.38 Baseline (mmHg) 88.0 4.80 103.1 3.9 90.3 6.9 101.3 3.5 Minutes Excipient 5 ml / kg 10 ml / kg 20 ml / kg Average(%) S.E.M. Average(%) S.E.M. Average(%) S.E.M. Average(%) S.E.M. 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10 0.65 3.85 0.16 3.80 -0.82 3.11 3.39 1.50 20 1.30 3.46 -5.64 3.99 0.48 2.75 -0.46 1.73 30 0.85 5.89 1.80 7.02 0.01 1.97 -0.02 2.62 60 2.97 3.55 -3.34 2.43 3.45 1.66 0.74 1.48 90 4.10 3.78 -6.68 4.61 5.25 2.74 1.99 2.28 120 2.66 3.24 1.86 2.84 4.46 2.43 2.96 1.85 240 2.97 5.11 0.31 3.11 2.64 2.67 6.11 1.67 360 3.53 2.68 -1.30 3.99 -2.34 4.08 1.08 2.52 Baseline (mmHg) 294.9 6.53 294.9 15.73 275.8 14.29 269.8 13.84

(12) effects on breathing

Animals were used Hartley guinea pig (340 ~ 350 g) of 5 in each group. Intravenous administration of the test substance to the animal, then placed in the breeding box for respiration measurement and connected to a plethysmometer to test the effect of the test substance on the respiration rate and tidal volume of the animal Measurements were taken at 0, 15, 30, 60 and 120 minutes after dosing. Compared to the control animal group, there was no effect on breathing. The results are shown in Table 14 and Table 15 below.

Abbreviation Dosage (ml / kg) Respiratory rate (rate / minute) 0 min 15 minutes 30 minutes 60 minutes 120 minutes Excipient 20 105.2 ± 5.9 117.0 ± 16.9 102.3 ± 16.6 103.9 ± 8.9 98.8 ± 9.4 PEG-hemoglobin conjugate 5 102.7 ± 4.2 99.6 ± 5.5 98.9 ± 3.3 101.8 ± 4.5 96.3 ± 1.6 10 106.7 ± 7.3 114.4 ± 11.1 107.0 ± 4.8 111.8 ± 8.0 102.5 ± 4.6 20 109.3 ± 9.1 112.2 ± 11.4 99.2 ± 6.2 99.1 ± 4.9 96.8 ± 6.6 Abbreviation Dosage (ml / kg) Respiratory volume (ml) 0 min 15 minutes 30 minutes 60 minutes 120 minutes Excipient 20 2.9 ± 0.2 2.5 ± 0.2 2.9 ± 0.4 2.8 ± 0.4 2.9 ± 0.2 PEG-hemoglobin conjugate 5 2.4 ± 0.7 2.1 ± 0.3 2.6 ± 0.6 2.2 ± 0.4 2.2 ± 0.3 10 2.5 ± 0.5 2.1 ± 0.5 2.0 ± 0.2 2.5 ± 0.3 2.2 ± 0.2 20 2.8 ± 0.7 2.1 ± 0.5 2.4 ± 0.2 2.3 ± 0.6 2.4 ± 0.4

(13) Effects of Extraction Organs

Extracted ileum specimens were prepared as follows: struck the back of the guinea pig and stunned, cut the carotid artery, and then died of blood loss. Dip and soak in cold phosphate buffer (pH 7.4), wash the contents of the intestine with a syringe, and then follow the method of HP Rnag for longitudinal muscle-myenteric plexus. An organ bath containing the Krebs-Henseleit bicarbonate solution, which was cut into 2-2.5 cm lengths of the ileum and saturated with a 95% O ₂ -5% CO ₂ mixed gas. ) And isotonic contraction was measured. After stabilizing the specimen, direct action on the PEG-hemoglobin conjugate (10 ^-7 , 10 ^-6 , 10 ^-5 M) and pretreatment of the test material for 5 minutes was performed with acetylcholine (5 × 10 ^-7 M). The effect on the contractile action of, histamine (2 × 10 ^-6 M) and barium chloride (2 × 10 ^-3 M) was measured. The change in contractile force that appeared at this time was expressed as a percentage of the contractile force before drug administration of each of the constriction agents.

The PEG-hemoglobin conjugate of the present invention, which is a test substance for the jong-geun-janggeun sample of the guinea pig extraction chair, did not show any significant direct contraction and relaxation action, and when the sample was pretreated for 5 minutes, There was no effect on the contraction caused by barium. The results are shown in Table 16 below.

Abbreviation log [M] Contractile Responses (%) No pretreatment Acetylcholine Histamine Barium chloride Average (S.E.M.) Average (S.E.M.) Average (S.E.M.) Average (S.E.M.) Excipient 0 0.00 (0.0) 146.5 (6.1) 148.3 (7.8) 124.8 (6.0) PEG-hemoglobin conjugate 5 0.00 (0.0) 152.3 (3.8) 138.6 (10.1) 133.9 (5.8) 10 0.00 (0.0) 156.4 (3.8) 129.1 (7.7) 134.2 (2.9) 20 0.00 (0.0) 161.5 (5.1) 132.9 (7.7) 131.5 (7.3) N 8 8 8 8

(14) effect on intestinal transport

Thirty minutes after the test substance was administered to the animal, 0.1 ml orally was administered orally with a solution of 5% activated carbon suspended in 10% Arabic gum. After 30 minutes, the intestines were removed to measure the distance of carbon transport and the total small intestine length from the pylorus and calculate the percentage.

After intravenous administration of the test substance, the ability of transporting activated carbon by the test substance was measured. The test substance had no effect compared to the control animals.

(15) Effect on gastric juice secretion

Feed and water were freely ingested, fasted 24 hours before the start of the test, and water was saved 10 hours before the start of the test. Fasted animals were anesthetized with ether, and then dissected the pyloric region by the method of Shay et al., And immediately after the test substance was administered into the vein and left for 5 hours to collect gastric juice. The collected gastric juice was centrifuged at 3000 rpm for 10 minutes and then gastric juice, pH, and total acidity were measured. As a result, the test substance had no effect compared to the control. Table 17 shows these results.

Abbreviation Dosage Route of administration The number of animals Volume (ml) pH Total acidity (μEq) Excipient 20 ml / kg i.v. 5 11.9 ± 1.9 1.44 ± 0.24 108.6 ± 111.7 PEG-hemoglobin conjugate 5 ml / kg i.v. 5 10.7 ± 0.7 1.10 ± 0.07 893.1 ± 87.8 10 ml / kg i.v. 5 10.5 ± 1.1 1.24 ± 0.17 929.2 ± 145.0 20 ml / kg i.v. 5 12.5 ± 1.1 1.12 ± 0.04 1209.2 ± 136.6 Atropine 1 mg / kg i.p. 5 5.8 ± 1.2 1.20 ± 0.00 587.9 ± 149.2

(16) Effect on urine volume and electrolyte

The saline solution equivalent to 2.5% of the weight of the overnight fasted animal was first administered, and two hours later, oral administration of distilled water equivalent to 2.5% of the weight of the animal was administered intravenously. Thereafter, the mouse was placed in a metabolism cage for mice to collect urine up to 5 hours, and pH, volume, K ⁺ , Na ⁺ , and Cl ⁻ were measured. As a result, no effect was observed with this test substance as compared to the control animal group. Table 18 shows these results.

Abbreviation Dosage Route of administration pH Urine volume (ml) Na ⁺ (mmol / L) Cl ^- (m㏖ / ℓ) K ⁺ (mmol / L) Excipient 20 ml / kg i.v. 7.0 ± 0.0 2.8 ± 0.3 80.7 ± 3.2 105.38.1 44.3 ± 9.6 PEG-hemoglobin conjugate 5 ml / kg i.v. 7.0 ± 0.0 2.3 ± 0.5 54.7 ± 10.1 71.03.6 37.0 ± 13.1 10 ml / kg i.v. 7.0 ± 0.0 2.7 ± 0.3 64.7 ± 28.1 84.032.2 28.0 ± 2.6 20 ml / kg i.v. 7.0 ± 0.0 3.9 ± 1.2 55.3 ± 31.1 88.312.7 30.3 ± 10.2 Purosemide 1 mg / kg i.p. 7.0 ± 0.0 6.0 ± 1.8 133.0 ± 4.0 161.722.2 30.3 ± 6.5

Therefore, the PEG-hemoglobin conjugate of the present invention was determined to have no effect on the central nervous system, the cardiovascular system, the autonomic nervous system, and other organs except for the effect of temporarily causing an increase in body temperature after administration.

Example 6 Langendorff Cardiac Extraction Test of PEG-hemoglobin Conjugates

SD rats of 250-300 g body weight were anesthetized with sodium pentobarbital (50 mg / kg, ip), followed by administration of heparin (1000 U / kg, iv), and grover. The heart was taken out according to the method of the back. In other words, retrograde perfusion (physiological fluid, modified Krebs-Henseleit bicarbonate) through aortic cannula in vivo while inserting a cannula (PE 240) into the trachea and artificial respiration using a rodent ventilator. Buffer: mM: 112 Sodium Chloride, Potassium Chloride, 1.2 Magnesium Sulfate, Potassium Hydrogen Phosphate, 25 Sodium Hydrocarbonate, 1.25 Calcium Chloride, 11.5 glucose, 2.0 pyruvate) quickly hung in a Langendorf vessel and removed unnecessary tissue attached to the heart . A rubber balloon suspended from a mixture of ethanol and distilled water (1: 1 vol / vol) was inserted through the pulmonary vein into the left ventricle and connected to a pressure transducer to measure the left ventricular pressure delivered to the balloon under isolumetric conditions. . End diastolic pressure (EDP) is maintained at 10 mmHg and stabilized within 10-15 minutes by constant pressure perfusion (75 mmHg) using a Gottlieb valve, allowing LVDP to fall within the range of 70-120 mmHg. Only the holding heart was used. From the moment the heart was fixed to the Langendorf device, the heart was immersed sufficiently in the chamber containing the physiological fluid throughout the entire experiment, so that the temperature was maintained thoroughly at 37 ° C. systolic pressure (LVEDP), left ventricular end diastolic pressure (LVEDP), left ventricular developing pressure (LVDP), and double product (DP) are the parameters used to evaluate coronary blood vessel function and coronary flow (CF) and other heart rates. (HR, heart rate) and the like were measured. DP is an important parameter indirectly determining the function of the heart in the Langendorf heart where cardiac output cannot be measured unlike cardiac output. HR was calculated by multiplying the HR by LVDP according to Watts' method. LVDP is calculated by subtracting LVEDP from LVP. Total coronary blood flow was measured with a flow meter via a coronary flow probe (1.0 mm diameter) fixed on an aortic cannula.

Modeled on the Langendorf tester, which maintains a constant perfusion pressure proposed by Curtis and Heerserk, the extracted heart is placed on the Langendorf tester and the physiological fluid at an appropriate pressure (65 mmHg, 80 cm H ₂ 0). Perfusion of the Krebs-Hanslite solution causes the aortic valves of the heart to close and the left and right ventricles remain empty. Perfusion flows into the coronary artery of the heart through coronary ostium on the left and right sides of the aorta, and then through the coronary sinus and the coronary vein, through the open right atrium (vena cava). It flows out and falls off.

Thus, the heart maintains its normal rhythm by its automaticity. In this state, left ventricular pressure, heart rate, and coronary blood flow were measured. After anesthetizing with pentobarbital 30 mg / kg, heparin was administered at 5000 USP-unit / kg, and the heart was quickly removed by dissecting the chest while maintaining the airway. The extracted heart is suspended in a Langendorf apparatus and perfused with Krebs-Henseleit bicarbonate (KHB) through the ascending aorta to the retrograde, filled with 50% ethanol. The filled balloon was placed in the left ventricle to fix the left ventricular EDP at 10 mmHg, and then stabilized for about 30 minutes. PEG-hemoglobin conjugates of the present invention were injected in the order of 10 ⁻⁷ , 10 ⁻⁶ , 10 ⁻⁵ to detect drug response. The retrolog perfusion was maintained at a pressure of 80 cm H ₂ 0 and the KHB solution was used by saturating with 95% O ₂ -5% CO ₂ in advance.

The PEG-hemoglobin conjugate of the present invention, a test substance, was treated in the extracted heart at concentrations of 10 ⁻⁷ , 10 ⁻⁶ , and 10 ⁻⁵ M, and then LVP, LVEDP, LVDP, DP, coronary blood flow, and heart rate were measured. As a result, the test substance did not have a negative effect on heart function and coronary vascular function.

As described above, the novel PEG-hemoglobin conjugate of the present invention has stability and efficacy as an oxygen carrier and thus can be used for medical purposes.

Claims (4)

Polyethylene glycol-hemoglobin conjugate [Formula 1] The polyethylene glycol-hemoglobin conjugate according to claim 1, wherein the polyethylene glycol-hemoglobin conjugate is obtained by reacting polyethylene glycol succinimidyl propionate and hemoglobin together. The polyethylene glycol-hemoglobin conjugate according to claim 2, wherein the polyethylene glycol succinimidyl propionate is obtained by reacting polyethylene glycol with ethyl acrylate and N-hydroxy succinimide. The polyethylene glycol -hemoglobin conjugate according to claim 2, wherein the polyethylene glycol succinimidyl propionate has a molecular weight of 100 to 200,000 Daltons.