JPH0418030A - Artificial blood - Google Patents

Abstract

PURPOSE:To obtain an artificial blood possessing both of sufficient oxygen carrying ability and effect capable of increasing an amount of blood platelet by suspending a liposome containing a hemoglobin in a solvent and controlling hemoglobin concentration and osmotic pressure to specific ranges. CONSTITUTION:A liposome membrane-forming substance is blended with an aqueous solution of hemoglobin, preferably a solution having 30-60wt.% hemoglobin concentration and the blend is stirred to prepare a suspension having 180-300nm average grain size of suspending grain and a glue osmotic pressure- controlling agent (e.g. albumin, acacia rubber or polyvinylpyrrolidone) is added thereto to each control hemoglobin concentration and glue osmotic pressure to 7-15% and 25-35mmHg. In the artificial blood, viscosity thereof is preferably controlled to <=4.5cp in 383 sec<-1> at 37 deg.C and the glue osmotic pressure is preferably controlled to 250-300mOsm.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to a method having an artificially regulated oxygen carrying capacity, which is used in the medical field for life-saving treatment of patients with massive bleeding.
Concerning life-saving infusions, namely artificial blood.

[Prior Art 1] It is known that plasma colloids, or plasma proteins, play an important role in maintaining the blood volume in the bile ducts of living organisms. For this reason, conventionally, by replenishing plasma with a plasma expander that has a colloid osmotic pressure equal to that of biological blood5,
It has been used as a method to recover from bleeding in patients. However, if more than 30% of the circulating blood volume bleeds, the oxygen supply to the peripheral tissues will be insufficient and additional oxygen delivery 141 will need to be administered.

Conventionally, natural blood containing natural red blood cells or concentrated red blood cells have been used as such oxygen carriers.
child. However, when using these natural red blood cells, the blood types of the donor and recipient must match in order to avoid blood clots due to antigen-antibody reactions. d) It is difficult to respond to emergencies because it is necessary to prepare -4, which requires an excessive amount of stock, and the cross-compatibility test is complicated and time-consuming. Moreover, such blood or red blood cell concentrates have a short effective storage period of 3 weeks (4°C), and frozen blood that can be stored for a long time by cryopreservation is also used, is expensive, and has a long shelf life. Due to storage, red blood cells become vulnerable to osmotic pressure during use, making them susceptible to hemolysis. Furthermore, if natural blood or concentrated blood cells are used, there is a risk of infection with hepatitis, AIDS, etc.

To solve these problems, polyethylene glycol, polypropylene glycol, or ethylene glycol-propylene glycol copolymers were modified by binding to hemoglobin to extend the residence time of hemoglobin in the circulating bloodstream. A blood substitute has also been disclosed (Japanese Patent Publication No. 2-6337), but it is clear that in this blood substitute, if the concentration of modified mogubin is increased,
It has been pointed out that the colloid osmotic pressure is extremely excessive.

That is, when the concentration of 5m hemoglobin is increased to 14-15%, which is the normal hemoglobin concentration in living organisms, the colloid osmotic pressure reaches 65-35 mmHg.

This is the normal oncotic pressure of natural blood, 25-35
It is considerably higher than mmHg, and when such artificial blood is administered to a living body, there is a risk that water will flow into the blood vessels or become excessive, causing cells outside the blood vessels to become dehydrated. This is dangerous as there is a risk that blood pressure will rise extremely due to the ability to increase plasma volume. Furthermore, increasing the hemoglobin concentration increases the viscosity of the blood substitute, making it higher than natural blood. Therefore, artificial blood using this type of modified hemoglohino is difficult to administer intravenously, and when administered to a living body, there is a risk that the artificial blood will cause poor circulation within the blood vessels. In addition, in order to maintain colloid osmotic pressure at 25 to 35 mmHg, which is the normal value of natural blood, the upper limit of the concentration of modified hemoglobin is 6.0 to 13.5%.
This is considerably lower than the hemoglobin concentration of natural blood, which is 14 to 15%, and therefore the oxygen carrying capacity cannot be said to be sufficient.

Problems to be Solved by the Invention The present invention has been made in view of the above-mentioned problems, and has the following features: - The moglobin concentration, colloid osmotic pressure and viscosity are adjusted to a range that can be tolerated by living organisms, and The aim is to provide artificial blood that has both sufficient oxygen carrying capacity and plasma volume expansion effects.

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

(1) Artificial blood made by suspending liposomes containing hemoglobin in a solvent, wherein the hemoglobin concentration in the solvent is 7 to 15%, and the colloid osmotic pressure is 25 to 35 m
Artificial blood characterized by being adjusted to +nHg.

(2) The viscosity of the artificial blood is such that the shear rate is 383 SeC.
-1, the above (
Artificial blood described in D.

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 injected blood of the present invention, since hemoglobin is encapsulated in liposomes, colloid osmotic pressure adjustment +1
Unless Q nl is added, the colloid osmotic pressure is substantially Q nl
I+, IIB. However, if the colloid osmotic pressure is extremely low compared to the normal colloid osmotic pressure of biological ml fluid, the ability to maintain circulating blood volume will be insufficient, and if it is too high, extravascular cells There is a risk that the body may become dehydrated or that blood pressure may rise dramatically. Therefore, the artificial blood of the present invention is one in which the osmotic pressure tS is adjusted by adding a colloid osmotic pressure regulator, and specifically, 25 to 3
It is desirable that the pressure be adjusted to 5 mmHg.

Examples of such protoplasmic θ permeability regulators include proteins derived from natural plasma such as albumin and globulin, or proteins such as aca, agol4, modified gelatin, polyvinylpyrrolidone, dextran, polyethylene glycol, calhoki/methyl cellulose, and hydroxyethyl starch. Examples include artificial water-soluble polymer compounds.

The average molecular weight of such a colloid osmotic pressure regulator is 20.0.
01J to 70,000 is preferable, more preferably 30
,000 to 40,000. If the average molecular weight is too large, the viscosity of the artificial blood will become high and it will not be possible to smoothly inject it intravenously, which is not preferable.

In addition, the concentration of such a colloid osmotic pressure regulator in artificial blood is 0°3 to 4.0 at the above-mentioned molecular weight.
Approximately mol/μQ is preferable. If it exceeds this range, the viscosity of the artificial blood will become high and it will not be possible to stagnate it smoothly, which is not preferable.

Further, below this range, it becomes difficult to substantially adjust the colloid osmotic pressure to a range acceptable to living organisms.

The liposome-forming lipid in the present invention is not particularly limited;
Natural or synthetic lipids can be used as long as they form liposomes. In particular, phospholipids are preferably used,
For example, to obtain the artificial blood of the present invention, phospholipids such as phosphatidylethanolamine, phosphatidylserine, sphingomyelin, phosphatidylserine, etc. derived from egg yolk, soybeans, and other natural materials, or 1rlft & chemical Those obtained by conventional synthetic means can 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 electrically conductive substances.

In the artificial blood of the present invention, the hemoglobin concentration is adjusted to 7 to 15%. As mentioned above, in the artificial blood of the present invention, hemoglobin is encapsulated in liposomes, so the colloid osmotic pressure is not affected by the globin concentration.

Therefore, it is possible to increase the hexaglobin concentration by 7-15%, preferably a concentration equivalent to that of native plasma14.
It can be increased up to ~15%. Moreover, encapsulation has the advantage that the rate of disappearance in the bloodstream can be effectively suppressed. The hemoglobin concentration U can be adjusted at the step of dissolving and dispersing the hemoglobin-containing liposome suspension in an aqueous solution of a colloid osmotic pressure regulator.

Inside the liposome! =The concentration and viscosity of the hemoglobin solution to be taken in are not particularly limited, but the viscosity is preferably 10 to 3.0 OOcp (4°C) and the hemoglobin concentration is 30 to 60% (W/W).

In addition, in the present invention, the ratio (L/H) between the weight of the lipid forming the liposome membrane (L) and the weight (H) of the solute in the aqueous solution taken into the liposome is determined by the encapsulation efficiency and the impact on living organisms. From a safety point of view, 0.40 to 1.6
7 is desired. If this value is greater than 0.40, the lipid membrane has sufficient thickness without the risk of solute leakage, while if it is less than 1.67, the lipid dosage is reduced;
Since the viscosity of the hemoglobin-containing liposome is also low, it is advantageous for circulating moving bodies to which artificial blood is administered.

The viscosity of such artificial blood is that the shear rate is 383
5. -, knee 1, at a temperature of 37°C, it is desirable that the viscosity be adjusted to be equal to or lower than that of natural blood, that is, 4.5 cp or less.

In addition, in the artificial blood of the present invention, if the quality osmotic pressure is too high or low compared to the normal quality osmotic pressure of the living body, there is a risk of hemolyzing the natural red blood cells of the living body. It is preferable that the osmotic pressure is 250 to 250 b, which is the quality osmotic pressure that the living body can tolerate. Further, the electrolyte composition is preferably adjusted to be substantially equal to that of natural blood, Su7 gel solution, or lactated Ringer's solution.

Next, the method for producing artificial blood of the present invention will be explained.

Although known methods can be used to produce hemoglobin-containing liposomes, it is preferable to add distilled water to a homogeneous mixed powder obtained by freeze-drying liposome membrane-forming lipids,
Hydrate the lipids by heating to ~80°C, add an aqueous hemoglobin solution to the hydrate, and use a 5~l Q'CC high speed stirrer to adjust the average particle size of the suspended particles in the mixture to 180~300.
This is achieved by stirring until the temperature reaches 100 nm.

Mixing and stirring of the liposome membrane-forming lipid and hemoglobin aqueous solution (performed by stirring using a stirring type cell disrupter (Waring blender) until the outer diameter of the suspended particles becomes 180 to 300 nm.The stirring is Number of revolutions 10
,000 to 20.00 rpm for 6 to +a minutes.

The liposome suspension thus obtained is washed and concentrated according to a conventional method.

The washed and concentrated liposome suspension has a colloid osmotic pressure of 25-35 mmHg and a hemoglobin concentration of 7-15%.
A colloid osmotic pressure regulator is added and mixed so that the artificial blood of the present invention is obtained. The colloid osmotic pressure regulator may be added and dissolved in the water-suspended iEW of liposomes in the form of raw material powder.

Next, the present invention will be explained in more detail by showing examples and comparative examples.

[Example 11 Equal weight of purified soybean phosphatidylcholine, cholesterol, myristic acid, and vitamin E with a hydrogenation rate of 90%.
rLyophilized powder [Presome, manufactured by Nihon Taide) 1 (Jag
An equal amount of distilled water was added to perform hydration treatment at 60°C for 160 minutes.

Add 60 m (+-i) of a red blood cell membrane-removed hemoglobin solution with a hemoglobin concentration of 50% (W・'w) to the hydrated hydric lipids, and mix with a high-speed stirrer while cooling to 5°C.
I using Ajiho Seven Mixer [Tokushu Kika Kogyo Co., Ltd.]
O, (100 rpm, 60 W + in) was performed. The average particle size of the suspended particles was measured using a light scattering particle size measuring device (D
Measured with LS-700, manufactured by Ohita Denshi Co., Ltd. 2
It was 20 nm.

The obtained liposome suspension was diluted 10 times with physiological saline, and filter sterilization and particle size control were performed in a circulating filtration device using a 0.45μ filter. The obtained filtrate was treated with a plasma separator to remove hemoglobin and microparticles that were not incorporated into the liposomes. Physiological saline was added to the filtrate with soil until hemoglobin was no longer detected, and circulation washing was repeated until the hemoglobin concentration was finally concentrated to 5%. The volume of the liposome suspension obtained is l
The hemoglobin recovery rate was 200 nIQ, 20%, and the average particle size of the liposomes was 210 nm.

Moglopin concentration in the suspension: H (B/m+2) and liposome membrane-forming lipid concentration: L (mg/m12)
The ratio L/H was 0°70.

Centrifuge this liposome suspension (17,00 Orl)
ms 3 t1 min), and the precipitated liposomes were adjusted to have an average molecular weight of 30°000-4 so that the hemoglobin concentration was 15%.
Artificial blood (Example) was obtained by suspending 0,000 hydroxyethyl starch in a 6% physiological saline solution (Salin Hess, manufactured by Kyorin Pharmaceutical Co., Ltd.). The colloid osmotic pressure of this artificial blood is 28.6 mmHg.

Crystalloid osmotic pressure was 290 mosm.

The colloid osmotic pressure and quality osmotic pressure were measured using a colloid osmometer 4400 (manufactured by Wesco, USA), respectively.
The measurement was carried out using an osmometer Model 13C2 (manufactured by Advance Co., Ltd.).

In addition, the viscosity of the artificial blood was measured using an E-type viscometer ELD.
When measured with a mold (manufactured by Tokyo Keiki Co., Ltd.), it was 4.0 (shear rate 3835 ec-1, temperature 37°C).

[Example 2; Example 2 except that the precipitated liposomes were suspended in an 8% saline solution of hydro-17 ethyl starch with a hemoglobin concentration of 10%, with a bi-average molecular weight of 30,000 to 40,000. Artificial blood (Example 2) was prepared in the same manner as in 1.
) was obtained. The colloid osmotic pressure of this artificial blood is 33,1
mmHg, quality osmotic pressure is 290IIIO5111.
·Ta. Well, the viscosity of artificial blood is 3°0cp (shear rate 3835ec-1, temperature 37°C).

Comparative Example 11 Same as Example 1 except that the precipitated liposomes were suspended in a 3% physiological saline solution of hydroquinoethyl starch having an average molecular weight of 30,000 to 40,000 so that the hemoglobin concentration was 12%. Similarly, artificial blood (Comparative Example 1)
I got it. The colloid penetration length L of this artificial blood is 15.1 mm.
) Ig, quality osmolality was 290 mosm. In addition, the viscosity of artificial blood is 3°2 cp (shear rate 3835
ec-1, temperature 37°C).

[Comparative Example 2j Precipitated liposomes were mixed with hydrochloride having an average molecular weight of 30.000 to 40.000 so that the hemoglobin concentration was 3%.
Artificial blood (Comparative Example 2) was obtained in the same manner as in Example 1, except that ethyl starch was suspended in a 6% physiological saline solution. The colloid osmotic pressure of this artificial blood is 28.6 mmHg,
Crystalloid osmotic pressure is 290! It was Osm. The viscosity of artificial blood was 1.4 cp (shear rate 3835 ec-1, temperature 37°C).

[Comparative Example 3j Monometho 4-7 polyoxyethylene cono\phosphoric acid monoester (+average molecular weight 5000) 5g, N-hydroxysuccinimide 10.23g, dicyclohequinyluponimide 0.42g, 300mQ of N,N - Dissolved in dimethylpolamide and stirred overnight at room temperature.

The generated 7-chlorohequinyl urea is filtered, and the filtrate contains 6
0 Onn of ethyl ether was added, and the formed monomethquine polyethylene glycol mono (succimidyl sane/nate) crystals were filtered, washed with ether and dried with IQ to obtain 4.6 g of white crystals.

Pyridoxal phosphate-bound hemoglobin 0.59 to 10
0.5 g of the above active ester was added to a solution of 0rrlQ in a boric acid grade buffer solution (pH 8.5) while cooling with water.

After stirring for 4 hours under water cooling, ultrafiltration was repeated using an ultrafiltration membrane with a molecular weight blocking of 30,000 to remove unreacted active esters or their decomposition products. I got it. (42 cases 3
) The colloid osmotic pressure of the obtained modified hepglobin solution is 8Q m
m11g, quality osmotic pressure is 290 mos+n, viscosity is 5
゜2cp (shear rate 3835ec-1, temperature 37℃)
Met.

<Blood Exchange Experiment> Furthermore, an advanced blood exchange experiment was conducted in domestic rabbits using the artificial blood of Examples and Comparative Examples. In other words, the big NWr of the rabbit
25 mJ/ from a catheter inserted into the abdominal aorta from the pulse.
kg, 20 mul kg of blood was removed, and the same amount of artificial blood was administered each time. Finally, 20 m Ql
K was exsanguinated and 40 mQ/kg of artificial blood was administered.
5 to 6 or more blood was replaced with artificial blood.

After 2.1 hours, the condition of the rabbits was observed, and after another 24 hours, the rabbits were placed in a sacrificial line and their tissues were observed.

The results are shown in Table 1.

(Left below) Table 1 [Efficacy of invention! J-] As mentioned above, the artificial blood of the present invention has the colloid osmotic pressure and hemoglobin concentration adjusted to values acceptable to the living body, so it has sufficient oxygen carrying capacity and appropriate plasma volume expansion capacity. able to demonstrate. Furthermore, the viscosity is equal to or lower than that of natural blood, so it has sufficient fluidity and can be administered intravenously smoothly, and there is no risk of poor circulation, so it is extremely safe. There is.

Claims (2)

[Claims] (1) Artificial blood made by suspending liposomes containing hemoglobin in a solvent, characterized in that the hemoglobin concentration in the solvent is adjusted to 7 to 15%, and the colloid osmotic pressure is adjusted to 25 to 35 mmHg. Artificial blood. (2) The viscosity of the artificial blood is such that the shear rate is 383 sec.
The artificial blood according to claim 1, which has a concentration of 4.5 cp or less at a temperature of 37°C.