JPH03215499A - Hemoglobin complex material - Google Patents

Abstract

PURPOSE:To obtain the subject complex material effective as a medicine having a long intravascular preservation time, a high oxygen-transportability and harmless without any cumulative property in human body by bonding hemoglobin and a styrene-maleic anhydride copolymer. CONSTITUTION:(A) Hemoglobin is bonded with (B) a styrene-maleic anhydride copolymer to afford the object complex material. Besides, for instance, human hemoglobin, etc., is preferably used as the component A and a copolymer containing about 50% styrene and having about 100-3000 molecular weight is preferably used as the component B.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a composite of hemoglobin and styrene anhydride maleic a copolymer, which is useful as an oxygen carrier for artificial blood.

[Prior Art/Problems to be Solved by the Invention] Blood transfusion plays a very important role in surgical operations, and currently holds the key to the success or failure of the operation.

However, today, a number of problems related to blood transfusions have become serious, especially the supply issue, as well as the AIDS virus, n.
The problem of contamination with on-A and non-B viruses is also serious, and the development of artificial blood is extremely significant in terms of solving these problems.

By the way, various attempts have been made to develop artificial blood. Under these circumstances, many studies have been conducted to use hemoglobin as a substitute for red blood cells.

Hemoglobin is a complex protein compound containing iron molecules that constitutes the major part of vertebrate red blood cells.

Using hemoglobin solution instead of blood also solves the problem of blood type. Hemoglobin can be isolated from blood (animal or human) and can be lyophilized for longer storage than blood.

However, hemoglobin is quickly excreted from the kidneys into the urine. Therefore, hemoglobin solutions often have to be injected in large quantities, and in addition to having a high rate of excretion, they also have the disadvantage of being potentially dangerous for patients with kidney disease.

To solve this problem, hemoglobin was mutually bonded with glutaraldehyde (Special Publication No. 62-16932).
Publication No.), hemoglobin was converted into dextran (Special Publication No. 1983-
No. 21124) or those bound to hydroxyethyl starch (West German Publication No. 2616086), those bound to polyalkylene glycol (Japanese Patent Publication No. 1983-
23084) etc. were proposed. However, these products do not always give satisfactory results in terms of oxygen carrying capacity or antigenicity.

On the other hand, liposomalization of hemoglobin (Japanese Patent Application Laid-open No. 61377
No. 35), microencapsulation (Japanese Unexamined Patent Publication No. 56152)
424) have been attempted, but it is currently difficult to put them into practical use due to the problem of phagocytosis by the reticuloendothelial system.

As is clear from the above, in order to eliminate the drawbacks of the conventional technology, it is necessary to use hemoglobin that has a long retention time in blood vessels (in other words, hemoglobin with low clearance), has an oxygen carrying capacity comparable to that of hemoglobin, and has a There is a need for an oxygen carrier for artificial blood that has neutral and non-hazardous properties.

(Means for Solving the Problems) In order to solve the problems, the present inventors have made various studies and have combined hemoglobin with a styrene-maleic anhydride copolymer that has strong protein binding properties with human serum albumin. By allowing the styrene-maleic anhydride copolymer to function as an anchor for hemoglobin, the glomerular filtration of hemoglobin from the kidneys is prevented, and satisfactory results can be obtained as an oxygen carrier for artificial blood. I found out that it can be done.

The present invention was completed based on the above findings, and its gist relates to a hemoglobin complex in which hemoglobin and a styrene-maleic anhydride copolymer are combined.

The hemoglobin used in the present invention may be derived from animals having hemoglobin such as humans, cows, sheep, horses, dogs, monkeys, rabbits, chickens, etc., but preferably,
Human hemoglobin is good. Hemoglobin as used herein refers to so-called abnormal hemoglobin (K. Imai,
AIlsteric Effects inHaemo
globin, Cambridge Universe
ity Press, 1980) or pyridoxal-5'-phosphoric acid, pyridoxal-5'-phosphoric acid, 2-nor-2-formylpyridoxal-5'-phosphoric acid, pyridoxal-sulfuric acid, e.g. pyridoxal-5
"monosulfuric acid, glycerin phosphates, e.g. glycerin-2
．． 3-diphosphoric acid, sugar phosphoric acid, e.g. glucose-6-
Hemoglobin derivatives such as phosphoric acid and adenosine-5'-phosphoric acid may also be used.

The styrene maleic anhydride copolymer used as an anchor for hemoglobin has molecules 1 of about 100 to 3000,
The composition ratio of styrene and maleic anhydride is exemplified as about 40:60 to 80:20.

Specifically, one made of 50χ styrene with a molecular weight of 1600 (Cosmo Bio General Catalog, No. 6, pp665,
(See product number 3106).

As a method for preparing a hemoglobin-styrene-maleic anhydride copolymer complex, about 1 to 10 moles of a styrene-maleic anhydride copolymer are bonded to 1 mole of hemoglobin by an amide reaction according to a conventional method. That is, the NHz of hemoglobin and the CO of styrene maleic anhydride copolymer
This causes OH to react.

The properties of the hemoglobin-styrene maleic anhydride copolymer complex thus prepared include a molecular weight of 50,000 to 10
10,000, bonding ratio: hemoglobin to 1 liter of styrene maleic anhydride copolymer to 1 to 10 (molecular ratio), and in the present invention, hemoglobin and albumin are bonded using a styrene maleic anhydride copolymer as an anchor. Complexes are also included in this scope.

The albumin used in the present invention is not limited to albumin derived from plasma, but may also be albumin obtained by cell culture or by recombinant genetic engineering techniques. In view of antigenicity, albumin of human origin is preferred, and there are no particular limitations as long as it is purified for medical use. The purity is preferably 80% or more albumin when analyzed by electrophoresis. As a method for obtaining human-derived albumin, the ethanol fractionation method (Japanese Patent Publication No. 47-2869,
Japanese Patent Publication No. 35-5297), heating method in the presence of an organic acid (
Examples include Japanese Patent Publication No. 43-1604 and Japanese Patent Publication No. 51-40132). Particularly preferably, albumin is used which has been heat-treated (preferably at 60° C. for about 10 hours) to inactivate hepatitis viruses and the like.

These complexes may be administered to the patient, e.g., as an aqueous solution (which may be an isotonic buffered salt solution) in admixture with a pharmaceutically acceptable excipient, diluent or carrier. good. Generally, these complexes will be administered using various types of formulations, packaging, and dosage forms conventionally used for the administration of plasma expanders. These complexes may also be lyophilized, optionally with cryoprotectants, and reconstituted for later use.

The amount of conjugate administered will vary depending on the patient's weight, symptoms, or desired treatment. However, in severe cases, substantially all of the patient's blood may be replaced by a composition containing the conjugate of the invention.

〔Effect of the invention〕

The complex obtained according to the present invention has a long intravascular residence time, has an oxygen carrying capacity comparable to that of hemoglobin, does not accumulate in the human body, is harmless, and has strong affinity with human serum albumin (ISA) in vivo. Because it binds to proteins, it has properties such as preventing glomerular filtration of Hb in the kidney and allowing it to remain in the body for a long time. for tissues and organs to be preserved when it is impossible or impossible to determine blood type and compatibility within a given time, and when the patient is unsafe or refuses regular blood transfusions. It can be used for the purpose of donating oxygen, for priming the extracorporeal circulatory system, or for other conditions in which red blood cells are normally used, allowing new developments in the medical industry and clinically as well. .

〔Example〕

EXAMPLES Examples and experimental examples are given to explain the present invention in more detail, but the present invention is not limited thereto.

Example 100 mg of human hemoglobin was dissolved in 10 ml of a 0.2 M sodium bicarbonate aqueous solution (pH 8) at 4° C. with stirring. After dissolving, styrene maleic anhydride copolymer (50χ styrene, molecule it 1600
, Cosmo Bio General Catalog, No. 6, pp6
65, product number 3106) was added, and 1.
The reaction was allowed to proceed for 5 hours.

The obtained reaction solution was treated with Sephadex (S6phadex■
) G-50 (manufactured by Pharmacia) and gel filtration. Distilled water was used as an eluent, and the effluent was detected by absorbance at 280 nm, and the desired fraction was collected and freeze-dried to obtain a hemoglobin complex.

Claims (1)

[Claims] A complex consisting of hemoglobin and styrene maleic anhydride copolymer.