JPS6153223A - Preparation of bonded compound of hemoglobin and polyalkylene glycol - Google Patents

Abstract

PURPOSE:To obtain the titled bonded compound useful as an oxygen carrier necessary in blood substitute, by reacting hemoglobin with a polyalkylene glycol having carboxyl group in the presence of an amino acid. CONSTITUTION:The objective hemoglobin-polyalkylene glycol bonded compound can be prepared by reacting (A) 2-30wt% (preferably 5-15%) hemoglobin (e.g. hemoglobin originated from human, cattle, pig, sheep, etc.) with (B) 1-100mol (preferably 5-20mol) of a polyalkylene glycol having carboxyl group (e.g. polyethylene glycol succinimidyl succinate) based on 1mol of hemoglobin, in the presence of (C) an amino acid (e.g. lysine, glycine, glutamic acid, etc.). The molecular weight of the bonded compound is 80,000-300,000.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for producing hemoglobin-polyalkylene glycol conjugates for use as necessary oxygen carriers in blood substitutes.

Conventional technology Hemoglobin as an oxygen carrier used in blood substitutes
Polyalkylene glycol conjugates are excellent in that they can significantly extend the lifespan in the bloodstream (Japanese Patent Laid-Open No. 56-12
No. 308, see No. 57-206622 BAIa8),
Its development is expected. However, if the molecular weight of this conjugate is too low, it will be excreted in the urine like unmodified hemoglobin, whereas if the molecular weight is too high (more than 300,000), it will give a highly viscous solution and become an obstacle when flowing through small blood vessels. . The preferred molecular weight of the hemoglobin-polyalkylene glycol conjugate when used as a blood substitute is 80,000 to 300,000 Daltons.

Problems to be Solved by the Invention The molecular weight of a hemoglobin-polyalkylene glycol conjugate is determined by the molar ratio and concentration of hemoglobin and polyalkylene glycol during their reaction;
In particular, in order to produce the conjugate having a molecular weight distribution within the above range, it is necessary to reduce the weight concentration of hemoglobin to 2% or less, and if the hemoglobin concentration is higher than that, a product with a molecular weight of 300,000 or more will be produced. It has been found that hemoglobin-4 realkylene glycoyl conjugates having a broad molecular weight distribution containing large amounts of t- are prepared.

On the other hand, the higher the hemoglobin concentration during the reaction, the more a large amount of hemoglopin-polyalkylene glycol conjugate can be produced at once, and as a result, the apparatus including the wide container can be made smaller.

It also facilitates concentration of the product.

To produce a hemoglobin-polyalkylene glycol conjugate having a molecular weight distribution within the range of 80,000 to 300,000 even if the hemoglobin concentration during the reaction is increased to produce a large amount of hemoglobin-polyalkylene glycol conjugate at one time. It is desired to develop a method that can provide a hemoglobin-realkylene glycol conjugate that is equivalent to or higher in quality than conventional methods.

As a result of intensive studies aimed at producing a hemoglobin-polyalkyle/glycol conjugate with a molecular weight distribution within the range of 80,000 to 300,000 even when the hemoglobin concentration during the reaction is high, the present inventor found that hemoglobin and cal have a xyl group. When the hemoglobin-polyalkylene glycol conjugate is reacted with polyalkylene glycol in the presence of an amino acid, the molecular weight distribution of the obtained hemoglobin-polyalkylene glycol conjugate is 8 to 30 even when the weight concentration of hemoglobin at the time of reaction exceeds 2. The inventors have found that the activity and physical properties are within the range of 1,000,000, and that the activity and physical properties are no inferior to those of conventional products, and based on this discovery, they have completed the present invention.

The hemoglobin used in the present invention is human, bovine, porcine,
It may be derived from animals that have hemoglobin, such as sheep, horses, dogs, monkeys, rabbits, and chickens. Hemoglobin as used herein refers to so-called abnormal hemoglobin (
K, Imai, A11osterlcEffect
s in Hasmoglobln, Cambrid
ge Un1v@ra1t7Pros♂, 1980) or pyridoxal-phosphate, e.g. pyridoxal-5'-phosphate, 2-true 2-formylpyridoxal-5'-phosphate, pyridoxal-sulfuric acid, e.g. pyridoxal-51-sulfate, glycerin Phosphoric acids, such as glycerin-2,3-syric acid, sugar phosphoric acids, such as glucose-6-phosphate, adenosine-5'-! J7
Hemoglobin derivatives such as [] may also be used.

The concentration of hemoglobin during reaction is 2-30% (Jt amount)
degree, preferably 5 to 15% (by weight).

The polyalkylene glycol used in the present invention is a highly water-soluble polymer such as polyethylene glycol, polypropylene glycol, or a copolymer of ethylene oxide and propylene oxide, and has a molecular weight of 300 to 2,000. From the viewpoint of the viscosity of the composite to be produced, it is preferably within the range of 750 to 10,000.

Furthermore, the polyalkylene glycol has at least two carboxyl groups, or is a so-called activated ester derivative obtained by dehydrating and condensing a carboxyl group with N-hydroxyconolimide, succinimide, imidazole, or the like.

The amount of polyalkylene glycol used is hemoglobin 1
About 1 to 100 moles, preferably 5 to 20 moles
It is on the order of moles.

As a method of imparting a carboxyl group to polyalkylene glycol, a carboxyl group of polycarzenic acid such as alkanedicarboxylic acid is used, and a carboxyl group is used to form an ester bond. A method of introducing a xyl group or a method of oxidizing the hydroxyl group of polyalkylene glycol to form a carboxyl group may be used. Examples of polycarboxylic acids include succinic acid, geltaric acid,
Dicarboxylic acids that do not have functional groups other than carboxyl groups such as adipic acid, malic acids, and those that have functional groups other than carboxyl groups such as asuno, laginic acid, and glutamic acid, and that the functional groups do not inhibit the desired reaction. protected,
Further, polycarboxylic acids such as nitrilotriacetic acid, tricarbaric97) or ethylenediaminetetraacetic acid may be used.

On the other hand, if the desired polyalkylene glycol containing xylyl groups is obtained by dehydrohalogenation with a hydroxyl group of polyalkylene glycol using, for example, a halogenated monocardic acid or an amine group-containing monocarboxylic acid, I can do that. Furthermore, the desired substance can also be obtained by reaction of polycarboxylic acid anhydride and polyalkylene glycol.

The amino acids present during the reaction may be, for example, naturally occurring amino acids, such as basic amino acids such as lysine, arginine, and histidine, neutral amino acids such as glycine and phenylalanine, and acidic amino acids such as glutamic acid and aspartic acid.

shown. On the other hand, one or more types of amino acids are used, and the amount of amino acids used is about 1 to 100 mol, preferably about 5 to 20 mol, per 1 mol of hemoglobin.

Other than reacting in the presence of the amino acid, known methods for bonding hemoglobin and polyalkylene glycol, such as 2,2'-dichlorobenzidine, P, P/-
Binders such as dichloro-m2, m'-dinitropheniclesulfone, 2,4-dichloronitrobenzene, etc. may be used, but in consideration of reaction selectivity, it is preferable to use the following method.

There is a method in which gv alkylene glycol is first made into a highly reactive derivative, and then this is reacted with hemoglobin.

Cal like this? When reacting polyalkylene glycol with xyl groups and hemoglobin, for example, N-
Hydroxysuccinimide, N-hydroxyphthalimide, p-nitrophenol, pentachlorophenol, etc. can be converted into active esters using carboxylic acid activators in the usual begtide synthesis, and can be reacted with hemoglobin to perform amidation exchange. Yes, you can use thionyl chloride etc.
It is also possible to make an acid halide of polyalkylene glycol by acting with a conversion agent, and to react this with hemoglobin.

At the time of the reaction for bonding hemoglobin and polyalkylene glycol, it is preferable to use oxygen absent or at a low oxygen concentration, and the oxygen partial pressure may be selected from about 0 to 30 wH1. Any reaction conditions other than the above oxygen concentration may be used as long as they do not involve denaturation of hemoglobin.

To reduce the concentration of oxygen, replace the air in the reaction vessel with an inert gas such as nitrogen, argon, helium, etc.
Known methods can be employed, such as reducing and removing oxygen in the container using a reducing agent (NaBH4, Na2S2O5, etc.), degassing with a pump, etc., and replacing the oxygen with an inert gas such as argon.

Example Hereinafter, the present invention will be explained in detail by way of examples.

Example 1 50 rnl of red blood cells from an expired transfusion blood cell were washed 4 times with 50 d of 0.9% saline using a centrifuge at 4°C.

This was hemolyzed with the same volume of water, 0.4 volume of toluene was added, and the mixture was stirred for 1 hour. After that sooo.

Membrane components and hemoglobin were separated by centrifugation on rotation for 1 hour. The obtained hemoglobin solution was diluted to 0.
The membrane components were removed using a 22 μm cellulose acetate membrane (manufactured by Millipore, USA) to obtain a hemoglobin solution (15% weight concentration).

This hemoglobin solution was mixed with 0.1M phosphate buffer (pH
= 7-4') and then dialyzed against the same 0.1
A 6% hemoglobin solution was obtained by diluting the solution with M's phosphoric acid solution (pH=7.4).

Glycine 10 in this 6% hemoglobin solution iooag
After adding 4.7 μl (15 times equivalent to hemoglobin), 3.53 Il of polyethylene glycol succinimidyl succinate (molecular weight 3800) (10 times equivalent to hemoglobin) was added.

After 30 minutes, the - was adjusted to 7.8 with IN sodium hydroxide solution, and the reaction was continued for another 30 minutes.

Note that polyethylene glycol succinimidyl succinate was prepared as follows. First, polyethylene glycol (molecular weight 3400) and succinic anhydride were reacted without a solvent at 100°C for 3 hours to synthesize a succinic acid ester of polyethylene glycol. Polyethylene glycol succinimidyl succinate was synthesized by reacting this polyethylene glycol edge/minister with N-hydroxysuccinimide in the presence of dicyclohexylcargodiimide in a dimethylformamide solvent.

The 400 MHz 'HNMR spectrum of this polyethylene glycol succinimidyl succinate was measured in dichloroform and the chemical shift of the methylene proton signal of cono-phosphoric acid bound to polyethylene glycol was 2.
．． They were triplets at 97 ppm and 2.78 ppm, respectively. In addition, the chemical shift of the methylene proton signal of N-hydroxysuccinimide is 2.85 pp.
O hemoglobint that was a singlet in m? After the reaction of lyethylene glycol succinimidyl succinate was completed, the molecular weight distribution of the product was measured by high performance liquid chromatography using dull filtration. The column used was Toyo Soda RA-3000SW J, and the eluent was 0. IM phospho-a buffer (P! (=6.8) was used. The flow rate of the eluent was 1.9 ml 7 minutes.

A high performance liquid chromatogram of the obtained hemoglobin-polyethylene glycol conjugate is shown in FIG. As shown in FIG. 1, the resulting conjugate had a small proportion of components with molecular weights of 300,000 or more and 80,000 or less.

Example 2 A 0.1 M') acid buffer solution of the hemoglobin obtained in Example 1 (hemoglobin weight: ii concentration 6% volume = 7.4
) After adding 163.2 da of L-lysine (12 times the mole relative to hemoglobin) to 100 d of polyethylene glycol succinimidyl succinate, 4.24 d of polyethylene glycol succinimidyl succinate was added. li' (12 times molar relative to belly button globin) was added.

Thereafter, the molecular weight distribution of the product was measured in the same manner as in Example 1. A high performance liquid chromatogram of the product is shown in FIG.

As in Example 1, the ratio of components with molecular weights of 300,000 or more and 100,000 or less was small.

Comparative Example 1 A 0.1M phosphate buffer solution of the hemoglobin obtained in Example 1 (hemoglobin weight concentration 6% P[(=7.4
) Polyethylene glycol 1.24II (3.5 equivalents) without addition of amino acids to 100d, 1.
411I (4.0 equivalents), 1.59.9 (4.5 equivalents)
added.

Figure 3 shows the high performance liquid chromatograms of each after the completion of the reaction.
, 4 and 5. In both cases, it can be seen that the ratio of components with molecular weights of 300,000 or more and 100,000 or less is greater than that of the conjugates obtained in Examples 1 and 2.

Therefore, it is impossible to produce a hemoglobin-4-reproduced tylene glycol conjugate with a molecular weight distribution of 80,000 to 300,000 as obtained in Examples 1 and 2 without adding amino acids in a 6% hemoglobin solution. It was possible.

Example 3 As in Example 1, 100 m of hemoglobin in 0.1M phosphate buffer (hemoglobin weight concentration 12% pH = 7.4) was prepared.
A! is prepared, L-alanine 82.9rn9 (10 times the mole relative to hemoglobin) is added, and then polyethylene glycol succinimidyl succinate a, 5ay (10 times the mole relative to navel globin) is added. Thereafter, the molecular weight distribution of the product was measured in the same manner as in Example 1. A high performance liquid chromatogram of the product is shown in FIG. As shown in this figure, even if the hemoglobin weight concentration becomes 12 or more, 6
As in the case of H, the ratio of components with molecular weights of 300,000 or more and 100,000 or less was small.

Example 4 As in Example 3, a 0.1M phosphate buffer solution of hemoglobin (hemoglobin weight concentration 12% - = 7.4) 10
Glycine 1as, 7m9 (20 times the mole relative to hemoglobin) was added to 01d, and then polyethylene glycol succinimidyl 5.30.9 (15 times the mole relative to hemoglobin) was added.

Thereafter, the molecular weight distribution of the product was measured in the same manner as in Example 1. A high performance liquid chromatogram of the product is shown in FIG.

As in Example 3, the ratio of components with molecular weights of 300,000 or more and 100,000 or less was small.

Comparative Example 2 As in Example 3, hemoglobin was added in 0.1 M phosphate buffer (hemoglobin weight concentration 12% - = 7.4) 100
nA! 0.707 g (2 times mole relative to hemoglobin) or 1.06 g (3 times mole relative to hemoglobin) of polyethylene glycol succinimidyl succinate was added to the solution without adding any amino acids. High performance liquid chromatograms after the completion of the reaction are shown in Figures 8 and 9.

Similar to Comparative Example 1, when no amino acid is added, the molecular weight is 30.
It can be seen that the proportion of components with a value of 10,000 or more and 100,000 or less is large.

Therefore, even when the weight concentration of hemoglobin was 12%, it was impossible to prepare a hemoglobin-polyethylene glycol conjugate with a molecular weight range of 100,000 to 300,000 without adding amino acids.

As is clear from the above, according to the present invention, by performing the reaction between navel globin and polyethylene glycol in the presence of amino acids, a hemoglobin-polyethylene glycol conjugate with a narrow molecular weight distribution can be obtained even when high alkali hemoglobin is used. It became possible to manufacture.

[Brief explanation of the drawing]

Figures 1 to 2 and 6 to 7 show high performance liquid chromatograms of the conjugates obtained in Examples 1 to 4, respectively, Figures 3 to 5 show the results obtained in Comparative Example 1, and Figures 8 to 9 show the results obtained in Comparative Example 1. The results obtained in Comparative Example 2 are shown.

Claims (1)

[Claims] 1. A method for producing a hemoglobin-polyalkylene glycol conjugate by reacting hemoglobin with a polyalkylene glycol having a carboxyl group,
1. A method for producing a hemoglobin-polyalkylene glycol conjugate, which comprises reacting in the presence of an amino acid. 2. The method according to claim 1, wherein the amino acid contains at least one naturally occurring amino acid. 3. Amino acid has a molar ratio of 1 to 1 to hemoglobin
00. The method according to claim 1, wherein the method is such that the number is 00. 4. The method according to claim 1, wherein the concentration of hemoglobin is 2 to 30% (by weight). 5. A hemoglobin-polyalkylene glycol conjugate that can be obtained by reacting hemoglobin with a polyalkylene glycol having a carboxyl group in the presence of an amino acid.