JPS59104322A - Blood substitute - Google Patents

Abstract

PURPOSE:To provide a blood substitute having excellent oxygen-transporting capability, by reacting hemoglobin with inulin in the absence of oxygen or in an atmosphere having low oxygen concentration, and using the resultant hemoglobin-inulin adduct as an oxygen carrier. CONSTITUTION:In a blood substitute containing a hemoglobin-inulin adduct as an oxygen carrier, said adduct is prepared by the reaction of hemoglobin with inulin in the absence of oxygen or in an atmosphere having low oxygen content. The bonding is carried out through an amide bond formed by the dehydrative condensation of the carboxyl group of the carboxyl-modified inulin and the amino group of hemoglobin. An oxygen transporting substance having high oxygen supplying capability can be prepared under mild condition (the reaction temperature and pH) to keep the hemoglobin from denaturation, by substituting the air in the reactor with an inert gas such as nitrogen, argon, etc. to reduce the partial pressure of oxygen to 0-30mm.Hg.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel blood substitute containing a hemoglobin-inulin conjugate with extremely high oxygen supplying ability as an oxygen transport substance.

For example, among various proposals, using human hemoglobin itself as a blood substitute has been proposed because intravenously injected hemoglobin is quickly excreted in the urine, resulting in a short lifespan in the bloodstream and It has drawbacks such as causing complaints about administration.

Therefore, this problem had to be solved in developing a blood substitute with red blood cell and plasma volume expansion properties suitable for transfusion. Therefore, this point was improved by binding inulin to hemoglobin (see Japanese Patent Application Laid-open No. 16815/1983). However, the oxygen supplying ability of the -moglobin-inulin conjugate is lower than that of red blood cells, and there is a demand for the development of a compound with even higher oxygen supplying ability.

The oxygen supply capacity can be expressed as the ratio of oxygen captured by oxygen transport substances in the lungs to the bloodstream e and delivered to the terminal tissues, and this can be determined from the oxygen height 11 curve.

In addition, as an index representing the oxygen solution 1ii11 curve, it is convenient to use the oxygen partial pressure when oxygen transport substances transfer 50% of the oxygen captured in the lungs to tissues as I)Fllll, and to indicate the oxygen supplying capacity using this value. (K, Imai+Δ1losteri
cEffectsinHaemoglobinS+Ca
mbridgeUniversityPress+l9
82). That is, 1 n 1. . P, on the other hand, is high, which means that oxygen transfer is easily carried out. A low level of oxygen means that it is difficult for oxygen to reach the tissues. ? As stated earlier, the hemoglobin-inulin conjugate has a long life in the bloodstream and has excellent properties as a blood substitute, but it has been desired to further improve its oxygen supply ability.

The present inventors tried to improve this point by applying multiple coats of paint, and as a result,
When reacting hemoglobin and inulin, it is possible to obtain a -hemoglobin-inulin conjugate with excellent oxygen supply ability, that is, high PfiO, by using an oxygen-absent state or using low concentration oxygen. The people who discovered this and completed the present invention.

That is, the present invention provides a blood substitute containing a monomoglobin-inulin conjugate as an oxygen transport substance, in which the conjugate causes hemocropin and inulin to combine in the absence of oxygen or in the presence of low oxygen concentration. This is a blood substitute characterized in that it is prepared from this blood.

The hemoglobin used here may be derived from animals having hemoglobin, such as humans, orchids, pigs, pigs, horses, dogs, monkeys, rabbits, and nitrogen. As used herein, hemoglobin refers to modified hemoglobin, such as its pyridoxal-51-phosphate, pyridogyza-51-sulfate, 2-true 2-formylpyritoxy → no-ru 51-phosphate, etc. pyridoxal or glucose-6-phosphate ester,
These include sugar phosphate esters such as adenonone-51-phosphate ester, or phosphate ester derivatives such as chrycerin-2,3-silicate ester.

In the present invention, oxygen may be absent or at a low concentration during the reaction for binding hemocropin and inulin.

In the absence of oxygen or at low oxygen concentrations, is hemocurvin hepti-inulin? Other than the reaction, the reaction conditions and method for binding hemoglobin and inulin may be the same as those in 11. For example, cyanogen bromide, anuric acid chloride,
212-cyclohennsine, Φ, Φ1-dichlorom
, m'-dini(lonophenylsulfone, 2,4-dichloronitrobenzene) can be used as a simple bonding probe. Since the amide bond is superior in terms of properties and oxygen supply ability, we will explain the details of the reaction focusing on this bond.In this case, a carboxyl group is introduced into inulin, and a bond between this and the amine group of inulin is introduced. Is there a way to do dehydration condensation?

In order to introduce a carboxyl group into inulin, a method such as activating one carboxyl group of polycarphonic acid and performing transesterification, or...l) Saponified carbonic acid,
Alternatively, inulin may be reacted with a dicarboxylic acid anhydride or the like.

When reacting P-carboxyl group-introduced inuri/ with haemocrine, for example, carboxyl/acid in conventional peptide synthesis such as N-hydroquine/succinimide, N-hydroquine phthalimide, etc. It is also possible to react with hemoglobin as an active ester using an activator to perform amidation exchange.

Seven methods of inulin activation can be made by selecting the following reactions, taking into account the physical properties of the reactants and the ease of purification.

1) One or more of the dog J7 hydroxyl groups are
1 to 10 times the molar amount of N.
- Activated ester θ, typified by ester obtained by dehydration condensation of hytroquine succinimide hepta; 2) A method of reacting inulin with hydroxyl of inulin and ammonium bromide.

3) A method of reacting inulin with chloride/anul or its derivatives to form inulin-4,6-dichloro-8-tria7no: 4) Deriving inulin to an aldehyde or acid halide by oxidation or other methods. Method.

As described above, the reaction in the absence or low concentration of oxygen in the present invention is carried out in the step of binding inulin (activated inulin is used if necessary) and hemoglobin.

In terms of oxygen concentration, zero oxygen partial pressure is 30 mHg.
It is good if it is below. When the oxygen partial pressure is 30 mmHg or less, an oxygen transport substance with a high P50 can be obtained under reaction conditions (reaction temperature, pH) that do not cause denaturation of hemoglobin.

To obtain a reaction environment with zero or low oxygen concentration, i) a method of blowing an inert gas such as nitrogen, argon helium, etc. into the reaction liquid to expel the reaction liquid and air from the container;
11) A method of reducing and removing oxygen with a reducing agent (NaBH4, Na2S2O5, etc.) that does not interfere with the reaction between activated inulin and hemoglobin, 111) A solid oxygen absorber, for example, 1--Rusj manufactured by Mitsubishi Gas Chemical ■. (2) Or a method of deoxidizing by coexisting a liquid oxygen absorber, such as pirocalol liquid, in the container. (IV) After deaerating the frozen moglobin solution using a tube or the like, it is redissolved and activated. This can be carried out by a method such as reacting with inulin or a combination of these methods.

The measurement of the oxygen decomposition Ht and curve of the hemoglobin-inulin conjugate was performed using our method (K, Imai+H, Morimo
to+M, Kotani+H, Watag++H, Wa
kaandKusoda+Biochim, Bioph
ys, Acta20yu, 189~+96(+970))
According to

Hereinafter, the present invention will be described in detail from specific examples.

Example 1 4 ml (0,006 mmol) of 98% human hemoglobin solution was added to O, 12 M Tris buffer (pH 6,8) 20
After dissolving the solution in a volume of 1.5 ml, the mixture was placed in a three-necked flask, and argon was bubbled through the flask for 2 hours. Sodium borohydride (NaBH) is
4) After adding 3.5 mg (0,006 mmol), argon gas was further bubbled through for 1 hour. In this container, 160ml of inulin, which had been activated in advance with 24mg of succinic anhydride and 33mg of N-hydroquinsquenoimide, was added.
1/ was added and reacted for 8 hours using a 4CF trowel under the same argon stream.

The reaction solution was repeatedly subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight blocking of 30,000 to remove low molecular substances such as unreacted active esters, and 9ml of a 38% hemoglobin-inulin binding solution was obtained.
I got it.

The P50 of this product was determined using Method P described above.

Product of Example 1 of the invention: 47 mmHg (25C10
, IM/acid buffer) was obtained by reacting in the presence of air (2sc, o, +M phosphate buffer), indicating that the oxygen supply ability was improved by the reaction in the absence of oxygen. Ta.

Example 2 200ml of 10% human hemoglobin solution was added to 1000ml of pyrogen-free potassium phosphate buffer at pH 6.8.
ml (0.31 mmol) was added and argon gas was bubbled in for 3 hours.

For this, Bocker's method (A, Pocker+J, Osg
.

Chem, 38 (25) 4295-4299 (197
3) See. 510 mg (2.0 mmol) of 2-true 2-pormyl-pyridoxal-5-phosphoric acid prepared in detail were added, and after 1 hour, 84380 ml of NaB was added.

After stirring for 1 hour, 18 g of succimidyl succinate inulin (average molecular weight: about 5,000) was added while aerating Alcon, and further deoxidation was performed using 20 g of Na, S, and O5R, and the reaction was allowed to proceed for 2 hours.

Relatively low-molecular substances such as unreacted hemoglobin were removed from the reaction solution using an ultrafiltration membrane with a molecular weight restriction of 100,000 to obtain about 400 ml of a 6% hemoglobin-inulin conjugate solution.

Add to this solution NaC1°CaC, which corresponds to the salt concentration of plasma.
After adding 1,,1<C1 and MgCl2, the pH was adjusted to 7.4 with caustic soda, and sterile filtration was performed to obtain a blood substitute. Average molecular weight: 13.53, P, n=24+m11
g (25C, 0,1M phosphate buffer pl(7,4).

On the other hand, the P2O value of the reactor in which oxygen was removed was 4 mm and 11 g.

Example 3 500 ml (0.15 mmol) of 2% human hemoglobin solution and 0.2 g O.75 of pyridoxal-51-phosphate
millimoles under a nitrogen stream by Bcnes method (R, Bcnes
ch, S, Yung+T, 5uzuki+C, Baue
r.

R, 13enesch, Proc, Na order, Acud,
SciUSA Engineering 1 (9), 2595-2599 (+97
3) See. ), followed by 5 times molar NaB
Pyridoxal-51- of hemoglobin is reduced with H4.
One ton of phosphoric acid derivative solution (0.2M phosphate buffer) was obtained. After aerating high-purity woodpecker at 4C for 20 minutes,
Inulin succimidyl sacinate (see Example 1 for the preparation method of this material) was added in an amount of 5 times the molar amount relative to hemoglobin, and the pH was adjusted to 8,5,4C with vigorous stirring.
Allowed time to react.

During this reaction, the oxygen partial pressure was maintained at 5 mm by the nitrogen gas flow.
Maintained below Hg. The reaction product was purified using an ultrafiltration membrane "PM-30J [F]" manufactured by Amicon Corporation in the United States to remove unreacted inulin and salts, and then concentrated and sterile filtered, and inorganic substances were added by the method of Example 1. A blood substitute was obtained.

P of this example. Value +2. :3mm12r of the sample prepared in the presence of 1g air, :l:4.4mmHg However, P
,. The measurement conditions are the same as in Example 1.

Example 4 Four drops of a solution of inulin IOS' (2 mmol) dissolved in 100 mef of water were added with a solution of chloride/anul 2f' (IO mmol) dissolved in a mixed solvent of 10 ne of water and 401 ne of acetone. During this time, the pH value was maintained at 10-11 with a 2N aqueous caustic soda solution. After the dropwise addition was completed, the pH value was adjusted to 3 with 2N hydrochloric acid.

The produced inulin-2,4-dichlorotrianone was purified by precipitation by adding acetone. The purified activated inulin was dissolved in 100 ml of 0.2M phosphate buffer, frozen, degassed using an oil pump, and then dissolved repeatedly to remove oxygen. To this, 15 ml of a 56% hemoglobin solution deoxygenated in the same manner was added under an argon atmosphere, and the mixture was allowed to react for 3 hours under water cooling.

The hemoglobin-inulin conjugate bound with cyanuric chloride was purified by the method of Example 3, and salts were added to prepare Blood Substitute Trial 1.

Obtained molecular weight: 204,000 (as a tetramer of subunits) P50 (25°C, 0.1M phosphate buffer) Hemoglobin-inulin conjugate prepared under anoxic conditions (cyanuric chloride method): 3.50 mmHg air Hemocrobin-inulin conjugate prepared in the presence (chloride/anul method): 1.46 mm
Hg Example 5 5 g (approximately 1 mmol) of inulin was dissolved in 150 ml of water, and 42 cyanogen bromide (40 mmol) dissolved in 20 ml of dioxane was slowly added dropwise under water cooling.
The reaction solution was stirred for 2 hours while maintaining the pH at 9 to LO with 2N Kasinoke aqueous solution, and then adjusted to pH 7.5 with IN hydrochloric acid.
Adjusted to. The product was concentrated to 75 ml using an ultrafiltration membrane with a molecular weight of 1000, and 500 ml of 0.2 M phosphate buffer at pH 7.5 was added to this, and 50 ml was concentrated using the same method.
It was concentrated for 1 hour.

The above activated inulin solution was deoxygenated with argon gas, added to 180 ml of a 10% horse hemoglobin solution deoxygenated with argon gas in a water-cooled downward V-V, and reacted at 4C under a small argon atmosphere.

The product was desalted four times using an ultrafiltration membrane with a molecular blocking capacity of 100,000, and was further subjected to sterile filtration using a 0.22μ membrane.

P5 measured using 25C,0,IM phosphate buffer
0 = 3.4 mmHg, and an improvement in oxygen supply ability was observed compared to the equine hemoglobin-inulin conjugate P50 = 0, 9 mmHg prepared by the same method in the presence of air.

In addition, the hemoglobin-inulin conjugate in the anoxic, hypoxic, or hypoxic 7s degrees Fahrenheit shown in the example and the hemoglobin-inulin conjugate obtained by the same method in the presence of air are reduced by half in the bloodstream. The period was measured using rats. The half-life in the bloodstream was measured using the method of Ajisaka et al.
andY, Iwashi+a+Biocl+em, Bi
ophys, Res, Commun,, 97.

1076~+081 (1980) 80% (7) from Ic
I got it after an exchange transfusion. As a result, it was revealed that the half-life of these hemoglobino-inulin conjugates in the bloodstream depends mainly on the molecular weight and is 9 to 24 hours regardless of the presence or absence of oxygen.

As is clear from the above, the present invention provides a hemoglobin-inulin complex whose oxygen carrying capacity has been completely improved without adversely affecting the half-life in the bloodstream, and therefore it can be expected to be useful as a blood substitute.

Patent applicant Ajinomoto Co., Ltd. 1'<4+n+1iif,'j! 1 (j for 0 11j1) 81 November Toru 11 1, ll'l expression 11j (sum!, 1'7'l'1.'+: 1'liη1 1st nl'3!'+f32 C;2, Name of the invention (;II Substitute 1111 liquid 3, N+1i Correct spelling right ijl'l Which relationship 'L'li)'l 3 out of 11 applicants
+! fil 2 medium heat 1 zo Lヱ1i---+I-1! No. 1 (
Man',? IIil, ', 1wJ-, riincrease invention'), none 7, f'F in i1ijt (1) clearlytillr'H7i:il,I:(Jl-1
MI7)r-i6aill]5fAl;z,,rI
J'1i111&, lnii'i'J correct.

(2) Akii 111. ! ith [;11.4(, ill to h
Self-pleasure [inulin]'Mino J, tj]l\1ri11
Hishi's)) Mino' 1! Corrected to 1.

(3) Akira1tlllF! 1u'1, l-/11t-,
>: IIiIIIlj description (7) rKllsodr+J7
．． ! 1Kurod+1] revised i[Ruru,.

(/'l) Akira1tlli', ith! lj'4.8・!
1 fjtl 1 賎 1 小柚水;);ノ[-1 嗆sim (Na13114) 3. ! +++r! l(0,00(
3 mmol) 1 in January - November - reel-j)゛-phosphoric acid fi, 4111jl ((1,024 mmol)'j)
Jbi boronated water book [~lium ((car) mouth 114) 4.
! im! J (0.12 milliliter)" (corrected), 1
(5) Ming 4' River i'ii'r! ! ')j'H1I;1-
il, IMi self-loaded [N-human 1j-1 cisc 1 neuimi 1: to [N-hi)]-] 11-cis succinoimi 1-
Old city Noru.

(6) Specifications, +1, page 13, 10 (-f)

(7) 1 old year 111: 1st () (“1, l'1irrl
ni; 1l book of [he[gurohi? ? 1 eyebrow 4 [l\] - 11 bin so 1 i stage 1 (Kosha II Ujru.

]

Claims (1)

[Scope of Claims] 1) A blood substitute containing a hemoglobin-inulin conjugate as an oxygen-carrying substance, in which the conjugate is prepared by reacting hemoglobin and inulin in the absence or low concentration of oxygen. A blood substitute characterized by: 2) The blood substitute according to claim 1, wherein the inulin is a modified inulin provided with at least one functional group selected from a carboxyl group, a formyl group, and a triazinyl group. 3) Hemoglobin is pyridoxal-51-phosphate, 2
-nor-2-formylpyridoxal-51-phosphoric acid,
The blood substitute according to claim 1, which is modified with a phosphoric acid derivative selected from the group consisting of: and glucose-6-phosphate. 4) The bond between inulin and hemoglobin in the hemoglobin-inulin conjugate is a dehydration condensation P of the carboxyl group of the carboxyl group-added modified product of inulin and the amine group of hemoglobin.
The blood substitute according to claim 1, which has an amide bond.