JPH045242A - Artificial blood - Google Patents

Abstract

PURPOSE:To obtain an artificial blood having oxygen-transporting activity as well as plasma-increasing effect, resistant to coagulation of liposome and safely applicable by intravenous injection by immobilizing an anticoagulant to the surface of a liposome including hemoglobin and suspending the liposome in an aqueous solution of a plasma expander. CONSTITUTION:An anticoagulant having a hydrophobic part at one end and a hydrophilic polymer chain at the other end (e.g. hydrogenated natural phospholipid bonded with polyethylene glycol) is fixed to a hemoglobin-containing liposome in such a manner as to fix the hydrophobic part to the lipid membrane and extend the hydrophilic polymer chain from the membrane surface outward. The hemoglobin-containing liposome modified by the above process is suspended in an aqueous solution of a plasma expander containing 0.3-40mum/ml of an artificially produced water-soluble polymer (preferably hydroxylated starch having a molecular weight of 20,000-70,000). The artificial blood is preferably adjusted to have a crystalloid osmotic pressure of 280-310 mOsm/l and a colloid osmotic pressure of 15-30 mmHg.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a life-saving infusion solution having an artificially adjusted oxygen carrying capacity, which is used in the life-saving treatment of patients with massive bleeding in the medical field. Regarding blood.

[Prior Art] It is known that plasma proteins, which are plasma colloids, play an important role in maintaining the blood volume in the blood vessels of living organisms. For this reason, conventional techniques have been used to recover the patient from hemostatic shock by replacing the plasma with a plasma expander that has a colloid osmotic pressure approximately equal to that of the body's blood. However, if 30% or more of the circulating blood volume is lost, the oxygen supply to peripheral tissues becomes insufficient, so administration of plasma expanders alone is insufficient, and it is necessary to administer additional oxygen carriers. arise.

Conventionally, natural blood containing natural red blood cells or concentrated red blood cells have been used as such oxygen carriers. However, when using these, the blood types of the donor and recipient must match in order to avoid blood clotting due to antigen-antibody reactions, and for this purpose it is necessary to store blood of various blood types. However, it may be difficult to respond to emergencies due to the need for excessive stock and the complicated cross-compatibility tests. Moreover, the effective storage period of such natural blood or red blood cell concentrate is as short as 3 weeks (4° C.). Frozen blood is also used, which can be stored for a long time by cryopreservation, but it is expensive and due to long storage, there are
The problem is that red blood cells are more vulnerable to osmotic shock, making them more susceptible to hemolysis. Furthermore, when administering these natural blood or concentrated red blood cells, there is a concern that infectious diseases such as hepatitis and AIDS may occur.

In order to solve these problems, research is being carried out on artificial oxygen carriers made of fluorocarbon emulsions (Japanese Patent Publication No. 33367/1983), but this artificial oxygen carrier has the ability to transfer oxygen to fluorocarbons. Because it relies solely on the physical dissolution phenomenon of oxygen, its oxygen carrying capacity is insufficient and its use is limited to situations where high partial pressures of oxygen can be applied. In addition, fluorocarbons may interfere with the innate immune system in vivo, and furthermore, fluorocarbons are not metabolized sufficiently in vivo, so they remain viable for a long time even after disappearing from the circulation. Problems that remain in the body have also been pointed out.

In addition, blood substitutes have been disclosed in which polyethylene glycol, polypropylene glycol, or ethylene glycol-propylene glycol copolymers are modified by bonding with hemoglobin to extend the residence time of hemoglobin in the circulating bloodstream (Japanese Patent Publication No. 2002-120012-1). Publication No. 6337).

However, even in this blood substitute, the retention time of hemoglobin is not sufficient, and furthermore, when the concentration of modified hemoglobin is increased, the viscosity of the aqueous solution increases dramatically.
Since the colloid osmotic pressure becomes extremely excessive, the upper limit of the concentration of modified hemoglobin is 6% at most, and at this concentration, it cannot be said that the oxygen transport ability is sufficient.

In order to overcome these drawbacks, hemoglobin-containing liposomes, in which hemoglobin is encapsulated in the inner layer of liposomes made of a lipid bilayer membrane, are being investigated (Japanese Patent Laid-Open No. 1989-1995).
2-151718, JP-A-58-183625, JP-A-61-37735), such suspensions of hemoglobin-containing liposomes have extremely low colloid osmotic pressure, so they cannot be administered into the blood vessels of living bodies. However, little effect on maintaining or increasing circulating blood volume could be expected.

In order to overcome the above-mentioned drawbacks, artificial blood has been disclosed that has both oxygen-carrying ability and plasma volume expansion effect by suspending hemoglobin-containing liposomes in an aqueous solution of a plasma volume expander (U.S. Patent No. 4 However, in this artificial blood, hemoglobin-containing liposomes tend to aggregate in an aqueous solution of a plasma expander, so the viscosity of the suspension becomes high, making it difficult to administer intravenously. Ta. Furthermore, if liposomes are significantly aggregated, there is a risk that when administered into a living body, the liposome aggregates may become embolized within blood vessels, leading to death of the living body.

[Problems to be Solved by the Invention] The present invention has been made in view of the above-mentioned problems, and provides a hemoglobin-containing liposome that has both an oxygen transporting ability and a plasma volume increasing effect, and has an oxygen transporting ability. The purpose of the present invention is to provide artificial blood that does not aggregate in an aqueous bulking agent solution, is safe even when injected into a living body, and is effective in saving lives of patients with massive bleeding.

[Means for Solving the Problems 1] In order to solve the above problems, the artificial blood according to the present invention has the following configuration.

(1) An anti-aggregation agent that has a hydrophobic part at one end and a hydrophilic polymer chain part at the other end, the hydrophobic part is fixed to the lipid membrane, and the hydrophilic polymer chain part is on the membrane surface. A hemoglobin-containing liposome modified to extend outward from the hemoglobin is suspended in an aqueous solution of a plasma expander whose main component is an artificially produced water-soluble polymer compound. blood.

(2) The artificial blood according to (1) above, wherein the anti-aggregation agent is a polyethylene glycol-bonded hydrogenated natural phospholipid.

(3) The average molecular weight of the water-soluble polymer compound is 20,000 to 7.
The artificial blood according to (1) or (2) above.

(4) The artificial blood according to any one of (1) to 3) above, wherein the water-soluble polymer compound is hydroxyethyl starch.

(5) The artificial blood according to any one of (1) to 4), which is adjusted to a crystalloid osmotic pressure that is acceptable to the living body to which it is administered.

(6) The artificial blood according to any one of (1) to 5) above, which is adjusted to a colloid osmotic pressure that is acceptable to the organism to which it is administered. (7) The electrolyte composition is substantially the same as that of plasma. The artificial blood according to any one of (1) to 6) above.

(8) The artificial blood according to any one of (1) to 6), wherein the electrolyte composition is substantially equal to Ringer's solution, lactated Ringer's solution, or Taleps-Ringer's solution.

Hemoglobin-containing liposomes and methods for producing the same have already been disclosed in JP-A-52-151758 and JP-A-58-18362.
No. 5, No. 61-37735, No. 62-178521, No. 63-209746, No. 63-211230,
No. 63-275522, No. 64-61426, No. 6
No. 4-75418 and Japanese Unexamined Patent Publication No. 1-180245.

In the artificial blood of the present invention, various known plasma expanders can be used, but when natural plasma proteins are used, there is a risk of developing various infectious diseases such as AIDS, and plasma expanders may be used. There are concerns about drug shortages or economic problems. Therefore, if an artificial plasma substitute can be used, it is desirable to use it as much as possible, and therefore, it is desirable to use an artificially produced water-soluble polymer compound as the plasma expander. Examples of such water-soluble polymer compounds include gum acacia, modified gelatin, polyvinylpyrrolidone, dextran, polyethylene glycol, carboxymethyl cellulose, and hydroxyethyl starch.

The molecular weight of such a plasma expander must be sufficiently large because it is necessary to adjust the colloid osmotic pressure of artificial blood to an osmotic pressure that is acceptable to the organism to which it is administered. However, if the molecular weight of the plasma expander is too large, there is a problem that the viscosity of the artificial blood becomes so high that it cannot be easily administered intravenously, or that hemoglobin-containing liposomes tend to aggregate. Therefore, the average molecular weight of the plasma expander is 20,
000 to 70°, C100 is preferred, and more preferably 30.000 to 40°Ooo.

In addition, the concentration of such an aqueous polymer compound in an aqueous solution is 0.3 to 4.0μ in the above-mentioned molecular weight.
About m/mff is preferable. If it exceeds this range, there is a problem that the viscosity of the artificial blood becomes high and it cannot be easily administered intravenously, or that hemoglobin-containing liposomes tend to aggregate. In addition, when we compare various water-soluble polymer compounds, below this range, it becomes difficult to adjust the colloid osmotic pressure to a range acceptable to the living body. In order to obtain the preferred artificial blood of the present invention which is least likely to aggregate hemoglobin-containing liposomes and is therefore most safe for living organisms, an aqueous solution of a plasma expander is added to and mixed with an aqueous suspension of hemoglobin-containing liposomes. Alternatively, raw material powder for the plasma expander may be added and dissolved in an aqueous suspension of hemoglobin-containing liposomes.

In general, ribosomes tend to aggregate in natural plasma and in artificial plasma expanders. Although this aggregation reaction is reversible, the suspension of liposomes aggregated has a high viscosity and is difficult to administer intravenously, and if aggregated liposomes are administered into the blood vessels of a living body, the aggregates of liposomes may form in blood vessels. There is a risk that it will become clogged inside the body and cause death. Therefore,
Hemoglobin-containing liposomes, which constitute artificial blood with oxygen-carrying ability used for patients with massive bleeding, are required not to aggregate in plasma expanders. Such an aggregation reaction of liposomes in an aqueous solution of a plasma expander is thought to be caused by the adsorption of plasma expander molecules onto the liposome surface due to interaction between the surface of the lipid membrane constituting the liposome and the plasma expander molecules. Guessed.

The present inventors first immobilized an anti-aggregation agent (protein adsorption inhibitor) having a hydrophobic part at one end and a hydrophilic polymer chain part at the other end on the liposome surface. We have applied for technology to suppress the aggregation of liposomes when administered to patients (Japanese Patent Application No. 01-63507, Japanese Patent Application No. 01-284912).
The present invention was completed based on the finding that aggregation of liposomes in an aqueous solution of an artificial plasma expander can be effectively prevented.

Any of the anti-aggregation agents disclosed in these publications can be used in the artificial blood according to the present invention. It is preferable to use natural phospholipids).

PEG-bonded phospholipids have a structure in which polyethylene glycol (PEG) is covalently bonded to the parent xylem of a hydrogenated phospholipid, and each molecule contains one or more PEG chains. The end of the PEG chain that is not bound to the phospholipid is
It may be a hydroxyl group, a short chain ether such as methyl or ethyl, or a short chain ester such as acetic acid or lactic acid.

Here, it is preferable to use soybean lecithin, egg yolk lecithin, phosphatidylethanolamine, etc. as the natural phospholipid.

For the purposes of the present invention, the PEG chain length in the PEG-conjugated natural phospholipid molecule is preferably from 5 to 1000 mol, more preferably from 40 to 200 mol, with an average degree of polymerization. If it is below this range, the aggregation prevention effect of hemoglobin-containing liposomes in artificial blood is difficult to be expressed, and if it is above this range, the water solubility of the PEG-bonded natural phospholipid becomes high, and the liposome membrane is It becomes difficult to be fixed.

In order to covalently bond PEG and phospholipid, a functional group having reactive activity is required in the polar part of the phospholipid.

Examples of this functional group include the amino group of phosphatidylethanolamine, the hydroxyl group of phosphatidylglycerol, and the carboxyl group of phosphatidylserine, and the amino group of phosphatidylethanolamine is preferably used.

In order to covalently bond the functional group of phospholipid and PEG, there are methods using cyanuric chloride, methods using carbodiimide,
There are methods using acid anhydrides, methods using glutaraldehyde, etc. To bond the amino group of phos-7 atidylethanolamine with PEG, cyanuric chloride (2,4
, 6-trichloro-s-triazine) is preferred. For example, by combining monomethoxypolyethylene glycol and cyanuric chloride using a known reaction procedure, 2-0-methoxypolyethylene glycol-4,6
-dichloro S-triazine (activated PEGI) or 2
．． 4bis(○-methoxypolyethylene glycol)6-
Chloro-5-triazine (activated PEG2) is obtained. By bonding these with an amine group by a dehydrochloric acid condensation reaction, a phospholipid in which PEG is covalently bonded to the polar head of phosphatidylethanolamine can be obtained. here,
When activated PEG1 is used, the phospholipid in the molecule contains one PEGWA chain, and when activated PEG2 is used, it contains two PEG chains. Furthermore, by reacting monomethoxy PEG with succinic anhydride to introduce a carboxyl group into the PEG terminal, and reacting this with phosphatidylethanolamine in the presence of carbodiimide, a PEG-bonded natural phospholipid can be obtained through an amide bond. .

To produce a liposome in which a PEG-bonded natural phospholipid is fixed to a lipid layer, the PEG-bonded natural phospholipid is uniformly mixed with a liposome-forming lipid in advance, and the resulting mixed lipid is used to form a liposome by a conventional method. That's fine. In this case, the mixing ratio of the liposome-forming lipid and the PEG-bonded natural phospholipid is 0 in terms of molar ratio to the phospholipid that is the main component.
．． The content is 1 to 50 mol%, preferably 0.5 to 20 mol%, and more preferably 1 to 5 mol%. If it is below this range, the effect of preventing liposome aggregation in artificial blood will be insufficient, and if it is above this range, hemoglobin-containing liposomes will become unstable due to the solubilization ability of PEG-bonded natural phospholipids. Become. In addition, to form a liposome containing hemoglobin in the inner layer using the obtained mixed lipid, it can be carried out according to the liposome formation method usually used for boat fishing. Also, known liposome-forming lipids are used, such as phospholipids derived from egg yolk, soybeans, and other natural materials, such as phos-7 atidylethanolamine, phos-7 atidylserine, sphingomyelin, and phosphatidylcholine; Alternatively, those obtained by organic chemical synthesis means may be used alone or in combination as the main component. Furthermore, sterols such as cholesterol and cholestanol may be added as membrane stabilizers, and phosphatidic acid, dicetyl phos-7ate, higher fatty acids, etc. may be added as charged substances.

The state of existence of PEG-bonded natural phospholipids in the liposome membrane is not clear, but the hydrophobic part of the PEG-bonded natural phospholipid is within the hydrophobic region of the liposome membrane, and the hydrophilic PEG chain is separated from the hydrophilic region. It is presumed that it exists in the aqueous medium outside the membrane.

The concentration of the PEG-bonded natural phospholipid thus obtained in artificial blood is preferably 0.1 to 1.0 μmol/m 4 .

In the artificial blood of the present invention, if the crystalloid osmotic pressure is too low or too high compared to the normal crystalloid osmotic pressure of the living body, there is a risk of destroying and hemolyzing the natural red blood cells of the living body. . Even in natural blood, the crystalloid osmotic pressure is not constant and there are individual differences, but in the artificial blood of the present invention,
It is desirable that the crystalloid osmotic pressure is adjusted to a level that does not cause harm to living organisms when administered to healthy individuals, that is, to a crystalloid osmotic pressure that is tolerated by living organisms, specifically 250 to 350. mosm/12, more preferably in the range of 280-310 mosm/Q.

In the artificial blood of the present invention, if the colloid osmotic pressure is too low compared to the normal colloid osmotic pressure of the living body, the effect of maintaining or increasing circulating blood volume will be insufficient, and if it is too high, the effect of increasing the circulating blood volume will be insufficient. There is a risk that excessive water will flow into the blood vessel, causing cells outside the blood vessel to become dehydrated.

Even in natural blood, the colloid osmotic pressure is not constant and there are individual differences, but in the artificial blood of the present invention, the colloid osmotic pressure will not cause harm to living organisms when administered to healthy people. It is desirable that the colloid osmotic pressure be adjusted to a level that is acceptable to the living body, specifically 10 to 40 mmHg, more preferably 15 to 30 mm.
Selected within the Hg range.

In the artificial blood of the present invention, if its electrolyte composition is significantly different from the normal electrolyte composition in biological blood,
Since the normal physiological functions of the living body may be inhibited, the electrolyte composition should be adjusted to be substantially equal to the normal electrolyte composition in the plasma of the living body, or substantially equal to Ringer's solution, lactated Ringer's solution, or Taleb-Ringer's solution. It is preferable.

Next, the present invention will be explained in more detail with reference to Examples and Comparative Examples.

[Example] Monomethoxypolyethylene glycol 5.000 (PE
G5, 100g (manufactured by Union Carbide) of 1.2-
The mixture was dissolved in 500 mff of dichloroethane, and logs of succinic anhydride and 8 mQ of pyridine were added thereto, followed by boiling under reflux for 3 days under a nitrogen stream. After filtering and evaporation, 20
After dissolving in 0 mQ of distilled water and washing the aqueous phase with ether, it was extracted with 200 mff of chloroform. After evaporation, dissolve in 400 mQ of ethanol and 9Q of hexane.
It was reprecipitated and purified. The residue was collected by filtration and dried under vacuum to obtain 85.6 g of one-end carboxy PEG 5K. 30 g of this, 7 g of hydrogenated soybean phosphatidylethanolamine, and 18 g of synchhexylcarbodiimide were heated and dissolved in 50 mQ of chloroform immediately after distillation, and reacted overnight at 50°C. After filtering, evaporate and dissolve in ethanol.
Insoluble materials were removed by filtration, and the solution was reprecipitated into hexane.

The filter leaves were vacuum dried to obtain 34 g of PEG-bonded hydrogenated soybean phospholipid (H5PE-PEG5K). The obtained H5
When a physiological saline solution of PE-PEG5 was administered intravenously to mice and its LD50 was determined, it was lIg/kg body weight, indicating extremely low toxicity.

Hydrogenated soybean lecithin 126g, cholesterol 64g
10 g of 1 myristin M was dissolved in 400 mff of dichloromethane, and the organic solvent was removed by evaporation.

Add 50% hemoglobin aqueous solution 2000 to the obtained mixed lipid.
After shaking and mixing, the French press treatment was repeated 10 times at a pressure of 500 kg/am''.The resulting French press treatment solution was diluted 10 times with physiological saline and centrifuged (17.0 QQr, p , m, 30 minutes) and centrifugal washing of the precipitated liposomes was repeated twice with physiological saline.

The precipitated liposomes after washing were suspended in physiological saline at a hemoglobin concentration of 10% (Sample 1: Comparative Example). The average particle size of the obtained liposomes was 0.2 μm. This liposome suspension was centrifuged (17,
000 r, p, ml for 30 minutes), and the precipitated liposomes were diluted with an average molecular weight of 30,
6 of hydroxyethyl starch from 000 to 40.000
% physiological saline solution (Salin Hess, manufactured by Kyorin Pharmaceutical Co., Ltd.) (Sample 2: Comparative Example). When this suspension was observed under an optical microscope (400x magnification), the liposomes were completely aggregated and the size of the aggregates exceeded 50 μm. Add 12mβ of physiological saline containing 5% H5PEPEG5K to 300ml of sample l,
After being left at room temperature for 3 hours, diluted 10 times with physiological saline and centrifuged (17,00 or p, m, 30 minutes), the precipitated liposomes were diluted with an average molecular weight of 30 so that the hemoglobin concentration was 10%. ,000 to 40,000 hydroxyethyl starch was resuspended in a 6% physiological saline solution (Salin Hess, manufactured by Kyorin Pharmaceutical Co., Ltd.). When this liposome suspension was observed using an optical microscope (400x magnification), no liposome aggregates exceeding 1 μm were observed (Sample 3: Example).

The crystalloid osmotic pressure and colloid osmotic pressure of Sample 1.2.3 were measured and the results are shown in Table 1.

Table 1 The crystalloid osmotic pressure and the colloid osmotic pressure were measured using an osmometer Model 13G2 (manufactured by Advance Inc.) and a colloid osmometer 4400 (manufactured by Wesco Inc., USA), respectively.

Blood Exchange Experiment> Further, using Samples 1 and 2.3, an advanced blood exchange experiment was conducted on domestic rabbits. In the blood exchange experiment, 25 mff/kg was administered from a catheter inserted into the abdominal aorta I from the femoral artery of a domestic rabbit.
, 20m (2/kg blood was removed, and the same amount of artificial blood was administered each time.Finally, 20m (2/kg blood was removed, 40
More than 85% of the blood was replaced with artificial blood by administering mQ/kg of artificial blood. After observing the condition of the rabbit for 24 hours thereafter, planned sacrifice, autopsy, and histopathological examination were performed.

In cases where sample I, in which hemoglobin-containing liposomes were dispersed in physiological saline, was administered, symptoms of dehydration and uria, likely caused by insufficient colloid osmotic pressure, occurred immediately after replacement, and the condition worsened over time, resulting in death approximately 8 hours later. did. In addition, in sample 2, in which aggregation of hemoglobin-containing liposomes in the plasma expander was not avoided, the condition did not worsen even after administration.
Although he survived for 4 hours, autopsy revealed hemorrhage in the lungs, which was thought to have been caused by vascular embolism due to aggregation of hemoglobin-containing liposomes, and pathological examination revealed vasotropic coagulation (Intravital coagulation) in the lungs and kidneys due to liposome aggregates.
Ascular-Coagulation, I
C) fl! was observed. In addition, in the case of 3 administrations of samples that did not cause agglutination even in plasma expanders, 24 hours after administration.
The stiffness remained stable over time, and no significant changes were observed upon autopsy and pathological examination.

Furthermore, in the artificial blood of the present invention, the crystalloid osmotic pressure is adjusted to a range acceptable to the living body, so hemolytic toxicity can be avoided, and the colloid osmotic pressure is adjusted to the range acceptable to the living body. By this, a moderate plasma volume increase effect can be imparted.

[Effects of the Invention] As detailed above, the artificial blood of the present invention has both an oxygen transporting ability and a plasma volume increasing effect, and moreover, hemoglobin-containing liposomes having oxygen transporting ability aggregate in a plasma volume expander. It has a low viscosity and is easy to administer in vivo, and it is safe because aggregates do not clog in capillaries, and is effective in saving lives of patients suffering from massive bleeding.

In addition, when PEG-bonded hydrogenated natural phospholipids are used as an anti-aggregation agent, their intravenous toxicity is extremely low, making it safe even when administered in large amounts, and also preventing aggregation of hemoglobin-containing liposomes in natural plasma. Therefore, there is no risk that hemoglobin-containing liposomes will aggregate within blood vessels even after administration.

Applicant Terumo Co., Ltd. Company Representative Patent Attorney Masatoshi Iwade

Claims (8)

[Claims] (1) An anti-aggregation agent having a hydrophobic part at one end and a hydrophilic polymer chain part at the other end, the hydrophobic part is fixed to the lipid membrane, and the hydrophilic polymer chain part is on the membrane surface. A hemoglobin-containing liposome modified to extend outward from the hemoglobin is suspended in an aqueous solution of a plasma expander whose main component is an artificially produced water-soluble polymer compound. blood. (2) The artificial blood according to claim 1, wherein the anti-aggregation agent is a polyethylene glycol-bonded hydrogenated natural phospholipid. (3) The average molecular weight of the water-soluble polymer compound is 20,000 to 7.
The artificial blood according to claim 1 or 2, wherein the artificial blood is 10,000. (4) The artificial blood according to any one of claims 1 to 3, wherein the water-soluble polymer compound is hydroxyethyl starch. (5) The artificial blood according to any one of claims 1 to 4, which is adjusted to have a crystalloid osmotic pressure acceptable to the living body to which it is administered. (6) The artificial blood according to any one of claims 1 to 5, which is adjusted to have a colloid osmotic pressure that is acceptable to the living body to which it is administered. (7) The artificial blood according to any one of claims 1 to 6, wherein the electrolyte composition is substantially the same as that of plasma. (8) The artificial blood according to any one of claims 1 to 6, wherein the electrolyte composition is substantially equal to Ringer's solution, lactated Ringer's solution, or Kreps-Ringer's solution.