JP2999815B2 - Artificial blood - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a life-saving infusion having artificially adjusted oxygen-carrying capacity, which is used for life-saving treatment of a patient with massive bleeding in the medical field, that is, an artificial infusion. Regarding blood.

[Prior Art] It is known that plasma protein, which is a plasma colloid, plays an important role in maintaining blood volume in blood vessels of a living body. For this reason, a method of recovering a patient's hemorrhagic shock by supplementing a plasma expander having a colloid osmotic pressure equal to that of the blood of a living body has been conventionally used. However, if more than 30% of the circulating blood bleeds, there is insufficient oxygen supply to peripheral tissues,
In addition, it becomes necessary to administer an oxygen carrier.

Conventionally, as such an oxygen carrier, natural blood containing natural red blood cells or a red blood cell concentrate has been used. However, when using these natural red blood cells, the blood types of the donor and the recipient must be matched in order to avoid coagulation due to the antigen-antibody reaction, and therefore blood of various blood types is prepared. This necessitates excess stock and the cross-compatibility testing is tedious and time-consuming, making it difficult to respond in an emergency. Moreover, such a blood or red blood cell concentrate has a short effective storage period of three weeks (4 ° C.). Frozen blood, which can be stored for a long time by cryopreservation, is also used.However, it is expensive and red blood cells are vulnerable to osmotic shock during use due to long-term storage. There is a problem that it is easy. Furthermore, when natural blood or erythrocyte concentrate is used, there is a concern about the risk of infection with hepatitis, AIDS, and the like.

In order to solve such a problem, a blood substitute having modified polyethylene glycol, polypropylene glycol or ethylene glycol-propylene glycol copolymer by binding to hemoglobin to extend the residence time of hemoglobin in the circulating blood stream is also disclosed. (Japanese Patent Publication No. 2-6337). However, in this blood substitute, it has been pointed out that when the concentration of the modified hemoglobin is increased, the oncotic pressure becomes extremely excessive. That is, when the concentration of the modified hemoglobin is increased to the normal hemoglobin concentration of the living body of 14 to 15%, the oncotic permeability is increased.
It reaches up to 65-85mmHg. This is significantly higher than the normal oncotic pressure of natural blood, 25-35 mmHg,
When such artificial blood is administered to a living body, the influx of water into blood vessels becomes excessive, and extracellular cells may become dehydrated. There is a danger that it may rise, and it is dangerous. Also, when the hemoglobin concentration is increased, the viscosity of the blood substitute increases and becomes higher than that of natural blood. Therefore, artificial blood using this type of modified hemoglobin may be difficult to administer intravenously or may cause poor circulation in blood vessels when artificial blood is administered to a living body. Also, to make the oncotic pressure 25-35 mmHg, the normal value of natural blood,
The upper limit of the concentration of the modified hemoglobin is 6.0 to 6.5%, which is considerably lower than the hemoglobin concentration of natural blood of 14 to 15%. Therefore, it is difficult to say that the oxygen carrying capacity is sufficient.

[Problems to be Solved by the Invention] The present invention has been made in view of the above problems, and the hemoglobin concentration, the oncotic pressure and the viscosity are adjusted to the acceptable range of the living body, and the oxygen transport is sufficient. It is an object of the present invention to provide an artificial blood having both the function and the effect of increasing plasma.

[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 in which a liposome encapsulating hemoglobin and a colloid osmotic pressure regulator having an average molecular weight of 20,000 to 70,000 are suspended in a solvent, wherein the hemoglobin concentration in the artificial blood is 7 to 15%, and Pressure regulator concentration 0.3 ~ 4.0mo
Artificial blood characterized by l / l.

(2) Artificial blood in which a liposome encapsulating hemoglobin and a colloid osmotic pressure regulator are suspended in a solvent, wherein the concentration of hemoglobin in the solvent is 7 to 15% and the colloid osmotic pressure is
Artificial blood having a pressure of 25 to 35 mmHg.

(3) The artificial blood according to claim 1 or 2, wherein the viscosity of the artificial blood is 4.5 cp or less at a shear rate of 383 sec-1 and a temperature of 37 ° C.

(4) The artificial blood according to claim 1 or 2, wherein the oncotic agent is dextran or hydroxyethyl starch.

Hemoglobin-encapsulated liposome and a method for producing the same,
JP-A-52-151758, JP-A-58-183625, JP-A-61-37
No. 735, No. 62-178521, No. 63-209746, No. 63-21123
No. 0, 63-275522, 64-61426, 64-75418
And JP-A-1-180245.

In the artificial blood of the present invention, since the hemoglobin is encapsulated in the liposome, the oncotic pressure is substantially 0 mmHg unless the oncolytic agent is added. However, if the oncotic pressure is extremely low compared to the normal oncotic pressure of living blood, the ability to maintain circulating blood volume will be insufficient, and if it is too high, extravascular cells will be dehydrated. There is a possibility that the state may be brought about or the blood pressure may rise extremely. Therefore, the artificial blood of the present invention
It is desired that the osmotic pressure is adjusted by adding a colloid osmotic pressure adjusting agent, specifically, the osmotic pressure is adjusted to 25 to 35 mmHg.

Such colloid osmotic agents include albumin,
Examples include natural plasma-derived proteins such as globulin, and artificial water-soluble polymer compounds such as acacia gum, modified gelatin, polynivirpyrrolidone, dextran, polyethylene glycol, carboxymethylcellulose, and hydroxyethyl starch.

The average molecular weight of such oncotic agents is 20,000
7070,000 is preferable, and 30,000 to 40,000 is more preferable. If the average molecular weight is too large, the viscosity of the artificial blood becomes too high to allow smooth intravenous injection, which is not preferable.

Further, the concentration of such a colloid osmotic pressure adjusting agent in artificial blood is 0.3 to 4.0 mol / μm in the above-mentioned molecular weight.
The degree is preferred. If it exceeds this range, the viscosity of the artificial blood will increase and it will not be possible to smoothly inject intravenously.
Not preferred. In addition, when the amount falls below this range, it becomes difficult to substantially adjust the oncotic pressure to a range acceptable for the living body.

The liposome-forming lipid in the present invention is not particularly limited, and natural or synthetic lipids can be used as long as they form liposomes. In particular, phospholipids are preferably used.For example, in order to obtain the artificial blood of the present invention, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, phosphatidylcholine, and other phospholipids represented by yolk, soybean, and other natural materials Or those obtained by organic chemical synthesis means can be used alone or in combination as the main component. Further, sterols such as cholesterol and cholestanol may be added as a film stabilizer, and phosphatidic acid, dicetyl phosphate, higher fatty acids and the like may be added as a charging substance.

In the artificial blood of the present invention, the hemoglobin concentration is adjusted to 7 to 15%. As described above, in the artificial blood of the present invention, since the hemoglobin is encapsulated in the liposome, the oncotic pressure is not affected by the hemoglobin concentration. Therefore, a hemoglobin concentration of 7
It can be increased to 1515%, preferably to 14-15%, which is preferably the same concentration as natural plasma. In addition, there is an advantage that the rate of disappearance in the bloodstream can be effectively suppressed by the encapsulation. The hemoglobin concentration can be adjusted at the stage of dissolving and dispersing the hemoglobin-encapsulated liposome suspension in an aqueous solution of a colloid osmotic pressure regulator.

The concentration and viscosity of the hemoglobin solution taken into the liposome are not particularly limited, but the viscosity is 10 to 3,000 cp (4 ° C.), and the hemoglobin concentration is 30 to 60%.
(W / w) is desirable.

In the present invention, the ratio (L / H) of the weight (L) of the lipid forming the liposome membrane to the weight (H) of the solute of the aqueous solution taken into the liposome is determined by the encapsulation efficiency and the effect on the living body. From the viewpoint of safety, it is desired to be 0.40 to 1.67. If this value is greater than 0.40, the lipid membrane has a sufficient thickness without risk of solute leakage, while 1.67
When the ratio is smaller, the lipid dose is reduced, and the viscosity of the hemoglobin-encapsulated liposome is also reduced, which is advantageous for a circulating body to which artificial blood is administered.

As for the viscosity of such artificial blood, the shear rate is 383 s
It is desirable that the viscosity be adjusted to ec ^-1 and a temperature of 37 ° C. to be equal to or less than that of natural blood, that is, 4.5 cp or less.

Further, in the artificial blood of the present invention, if the crystalline osmotic pressure is too high or too low compared to the normal crystalline osmotic pressure of a living body, there is a possibility that natural red blood cells of the living body may be lysed. Therefore, the crystal osmotic pressure 250-300
Preferably, it is adjusted to m0sm. The electrolyte composition is also preferably adjusted to be substantially equal to that of natural blood, Ringer's solution or Ringer's lactate.

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

Known methods can be used to produce hemoglobin-encapsulated liposomes, but preferably, distilled water is added to a homogenous mixed powder obtained by freeze-drying liposome membrane-forming lipids,
Heat the lipid to 8080 ° C. to hydrate the lipid, add an aqueous hemoglobin solution to the hydrate, and use a high-speed stirrer at 5-10 ° C. so that the average particle size of the suspended particles in the mixture is 180-300 nm. It is achieved by stirring until it is.

Mixing of liposome membrane-forming lipid and hemoglobin aqueous solution
Stirring is performed by stirring using a stirring cell disrupter (Waring blender) until the outer diameter of the suspended particles becomes 180 to 300 nm. Stirring is 10,000 to 20,000 rpm
It is desirable to carry out at 00 rpm for 6 to 10 minutes.

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

The washed and concentrated liposome suspension is added and mixed with a colloid osmotic pressure adjusting agent so that the colloid osmotic pressure is 25 to 35 mmHg and the hemoglobin concentration is 7 to 15%, and is used as the artificial blood of the present invention. . The oncotic pressure regulator may be added and dissolved in an aqueous suspension of liposomes in the form of a raw material powder.

Next, the present invention will be described more specifically with reference to examples and comparative examples.

[Example 1] A homogeneous mixture of freeze-dried powder of purified soybean phosphatidylcholine, cholesterol, myristic acid, and vitamin E [presome, manufactured by Nippon Seika Co., Ltd.] having a hydrogenation rate of 90% was added to 108 g of an equal amount of distilled water at 60 ° C and 60 ° C. Hydration treatment was performed.

The hydrated lipid thus obtained has a hemoglobin concentration of 50%
(W / w) of a red blood cell membrane-removed hemoglobin solution (60 ml),
While cooling to 5 ° C., a stirring treatment was performed at 10,000 rpm for 60 minutes using a high-speed stirrer and an Ajihomomixer (Tokiki Kagaku Co., Ltd.). The average particle size of the suspended particles was measured using a light scattering particle size analyzer [DLS-700, manufactured by Otsuka Electronics Co., Ltd.].
220 nm.

The obtained liposome suspension was diluted 10-fold with physiological saline, and sterilized by filtration with a circulating filtration device using a 0.45 μ filter, and the particle size was controlled. The resulting filtrate was treated with a plasma separator to remove hemoglobin and microparticles that were not incorporated into the liposome. Physiological saline was added to the filtrate until no hemoglobin was detected, and the circulation washing was repeated, and concentrated until the hemoglobin concentration finally reached 5%. The volume of the obtained liposome suspension is 1200 m
l, the recovery of hemoglobin was 20%, and the average particle size of the liposome was 210 nm. Hemoglobin concentration in suspension: H
The ratio L / H of (mg / ml) to the liposome membrane-forming lipid concentration: L (mg / ml) was 0.70.

The liposome suspension is centrifuged (17,000 rpm, 30
The precipitated liposomes are suspended in a 6% physiological saline solution (Sarinhes, manufactured by Kyorin Pharmaceutical Co., Ltd.) of hydroxyethyl starch having an average molecular weight of 30,000 to 40,000 so that the hemoglobin concentration becomes 15%. Example) was obtained.
The oncotic pressure of this artificial blood is 28.6 mmHg and the crystalline osmotic pressure is 29
It was 0m0sm.

The colloid osmotic pressure and the crystalline osmotic pressure were measured using a colloid osmometer 4400 (manufactured by Wesco, USA) and an osmometer Model3C2 (manufactured by Advance), respectively.

Further, the viscosity of the obtained artificial blood was measured with an E-type viscometer ELD type (manufactured by Tokyo Keiki Co., Ltd.) and found to be 4.0 (shear speed 383 sec ^-1 , temperature 37 ° C.).

[Example 2] Artificial blood was prepared in the same manner as in Example 1 except that the precipitated liposome was suspended in an 8% physiological saline solution of hydroxyethyl starch having an average molecular weight of 30,000 to 40,000 so that the hemoglobin concentration became 10%. (Example 2) was obtained. The colloid osmotic pressure of this artificial blood is 33.1 mmHg, and the crystalline osmotic pressure is 290 mOs
m. The viscosity of artificial blood is 3.0 cp (shear speed
383 sec ⁻¹ , temperature 37 ° C.).

Comparative Example 1 An artificial liposome was artificially prepared in the same manner as in Example 1 except that the precipitated liposome was suspended in a 3% physiological saline solution of hydroxyethyl starch having an average molecular weight of 30,000 to 40,000 so that the hemoglobin concentration became 12%. Blood (Comparative Example 1) was obtained. The colloid osmotic pressure of this artificial blood is 15.1 mmHg, and the crystalline osmotic pressure is 290 m
It was 0sm. The viscosity of the artificial blood was 3.2 cp (shear speed 383 sec ⁻¹ , temperature 37 ° C.).

Comparative Example 2 Artificial blood was prepared in the same manner as in Example 1, except that the precipitated liposome was suspended in a 6% physiological saline solution of hydroxyethyl starch having an average molecular weight of 30,000 to 40,000 so that the hemoglobin concentration became 3%. (Comparative Example 2) was obtained. The colloid osmotic pressure of this artificial blood is 28.6mmHg, and the crystalline osmotic pressure is 290m0s
m. The viscosity of artificial blood is 1.4 cp (shear speed
383 sec ⁻¹ , temperature 37 ° C.).

Comparative Example 3 5 g of monomethoxypolyoxyethylene succinic acid monoester (average molecular weight 5000), 0.23 g of N-hydroxysuccinimide, and 0.42 g of dicyclohexylcarbodiimide were added to 30
It was dissolved in 0 ml of N, N-dimethylformamide and stirred at room temperature overnight.

The resulting dicyclohexylurea was filtered, and the filtrate was 600 m
l of ethyl ether was added, and the resulting monomethoxy polyethylene glycol mono (succinimidyl succinate) crystals were filtered, washed with ether and dried, and 4.6 g
Was obtained as white crystals. 0.5 g of the above-mentioned active ester was added to a solution of 0.5 g of molybbin in pyridoxal phosphate linkage dissolved in 100 ml of borate buffer (pH 8.5) under ice-cooling. After stirring under ice-cooling for 4 hours, ultrafiltration was repeated with an ultrafiltration membrane having a molecular weight inhibition of 30,000 to remove unreacted active ester or its degradation product, thereby obtaining 5 ml of a 15% modified hemoglobin solution (artificial blood). I got (Comparative Example 3) The oncotic pressure of the obtained modified hemoglobin solution was 80 mmH
g, crystalline osmotic pressure is 290m0sm, viscosity is 5.2cp (shear speed 383se)
c ⁻¹ , temperature 37 ° C.).

<Blood exchange experiment> Further, an advanced blood exchange experiment was performed on rabbits using the artificial blood of Examples and Comparative Examples. That is, 25 ml / kg, 2 from the catheter inserted into the abdominal aorta by the femoral artery of the rabbit.
Blood was removed at 0 ml / kg, and an equal volume of artificial blood was administered each time. Finally, blood was similarly removed at 20 ml / kg, and artificial blood at 40 ml / kg was administered to replace 85% or more of the blood with artificial blood. After that, the condition of the rabbit was observed for 24 hours, and after 24 hours, the rabbit was sacrificed and the tissue was observed.

 Table 1 shows the results.

[Effects of the Invention] As described above in detail, the artificial blood of the present invention has a sufficient oxygen-carrying ability and an adequate plasma volume-enhancing ability because the oncotic pressure and the hemoglobin concentration are adjusted to values acceptable to the living body. Can be demonstrated. In addition, since the viscosity is equal to or less than that of natural blood, it has sufficient fluidity, can be administered smoothly intravenously, and has no danger of causing poor circulation. I have.

Claims (4)

(57) [Claims] 1. An artificial blood in which a liposome encapsulating hemoglobin and a colloid osmotic agent having an average molecular weight of 20,000 to 70,000 are suspended in a solvent, wherein the hemoglobin concentration in the artificial blood is 7 to 15%. Colloidal osmotic pressure regulator concentration of 0.3
Artificial blood characterized by being ~ 4.0 mol / l. 2. An artificial blood in which a hemoglobin-encapsulated liposome and a colloid osmotic pressure regulator are suspended in a solvent, wherein the hemoglobin concentration in the solvent is 7 to 15% and the colloid osmotic pressure is 25 to 35 mmHg. Artificial blood characterized by the fact that: 3. The artificial blood has a viscosity of 383 sec.
3. The artificial blood according to claim 1, which has a temperature of 4.5 cp or less at a temperature of 37 ° C. 4. The artificial blood according to claim 1, wherein said oncotic agent is dextran or hydroxyethyl starch.