JPS59104323A - Blood substitute - Google Patents

Abstract

PURPOSE:To provide a blood substitute having excellent oxygen-transporting capability, by reacting hemoglobin with a polyalkylene glycol in the absence of oxygen or in an atmosphere having low oxygen concentration, and using the resultant adduct as an oxygen carrier. CONSTITUTION:In a blood substitute containing a hemoglobin-polyalkylene glycol adduct as an oxygen carrier, said adduct is prepared by the reaction of hemoglobin with a polyalkylene glycol (e.g. polyethylene glycol) in the absence of oxygen or in an atmosphere having low oxygen concentration. The bonding is carried out through an amide bond formed by the dehydrative condensation of the amino group of hemoglobin and the carboxyl group of polyalkylene glycol imparted with carboxyl group. An oxygen transporting substance having high oxygen supplying capability can be prepared 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.

The conditions that should be met by a blood substitute with oxygen supply ability are (
1) The substance must be safe for animals.

(2) have a suitably long bloodstream lifetime, and (:
It is necessary to have a high oxygen supply capacity. From a safety point of view, it would be better to use moglohin in animals, but hemoglobin itself has a short lifespan in the bloodstream of about 2 hours and rapidly disappears from the +rn flow through urine or blood vessel walls. It has the following drawback. This drawback has been demonstrated, for example, by covalently binding polyalkylene glycols to hemoglobin, which was found to have a half-life in the bloodstream of 12 to 24 hours in 80% of exchange transfusions (particularly See JP-A-56-12308.

). However, as the oxygen supply capacity decreases, the reaction with polyalkylene glycol tends to decrease.

Generally, the oxygen enzyme 11 curve under the same measurement conditions as the physiological conditions is widely used for evaluating the oxygen supply capacity of blood. P2 is the oxygen partial pressure that is explained by P2.
It is defined as O and is widely used as an indicator of the oxygen carrying capacity of blood. That is, l],.

1. Thick blood transfers oxygen to tissues, P5o
High blood flow means that it easily transfers oxygen.

By the way, animal whole blood has a pH of 7,4,25C and PIit+
is approximately 14++nnHg.

However, the pio of many hemoglobin/-polyalkylene glycol conjugates is ]OfflllHg. It was desired to obtain an oxygen-carrying substance with a high

As a result of extensive research in order to develop an excellent blood substitute, the present inventors discovered a hemoglobin-polyalkylene glycol hemoglobin obtained by reacting polyalkylene glycol hemoglobin in the absence of oxygen or under oxygen-low conditions. The present invention was completed by discovering that the kyrenoglyfur conjugate has extremely high PIiO and is highly practical as an oxygen transport substance for blood substitutes.

The polyalkylene glycol used in the present invention is a polymer with high water solubility, such as polyethylene glycol, polypropylene/glycol, or a copolymer of ethylene oxide and propylene oxide.
Its molecular weight is 300-20. From the viewpoint of OOO1 viscosity, etc. (If it is within the range of 750 to IO,000).

Furthermore, one of the hydroxyl terminals of the polyalkylene glycol used in this conjugate may be protected. For example, C1-C16 realkyl ethers such as monomethyl ether, monoethyl ether, monoacetyl ester, monophlohilnisder', 5° carbon A
M 2-18 fat III IMI”, -ry.

The methyl or hydroxyl group may be substituted with 1 to 18 (7) carbon atoms.

- way, hemogrohy/ha, human, education, futa, pino 7,
Any animal derived from hemocropoid species such as horses, cows, monkeys, rabbits, chickens, etc. may be used.

This specification 111 (・U-・Moglobin and (yo, so-called abnormal hemoglobin (K, 1mai + A11osle
ric Effectsin Haemoglobin
＋Cambridge University Pr
ess+1980) or pyridoquinol-51-phosphate, 2
-Tru-2-formylpyridoxal-5'-9noic acid,
Bilitoxal-sulfate, e.g. pyryl-xal-5'
- sulfuric acid, chrycerin phosphates, e.g. chrycerin-2,
It may also be a hemocropino derivative such as 3-nophosphoric acid, sugar phosphoric acid, glucose-6-phosphate, adeno/'/-5'-phosphoric acid, etc. Except for the use of oxygen-free or low-oxygen concentration conditions (4), the bonding method of hemoglobin and polyalkylene/glycol is known, such as +f-2,
2゛-Dichloroben7nono, l', P'-/riI' l
j −n1m”; z = l o Phoenic Lepon,
A binder such as 2,4-dichlorni-1-lobenzene may be used, but in consideration of reaction selectivity, it is preferable to use the following method.

First, add polyalkylene glycol to a high 1. - There is a method of making a reactive derivative and then reacting it with -hemocrohin. As a method for activating such polyalkylene glycol, the following method may be used.

1) Convert at least one terminal hydroxyl group of the polyalkylene/glycol to a carhokyfur group, and further dehydrate and condense the carhokyfur group with N-hydroxycono-phosphoric acid imide, cono-phosphoric acid imide, imitalyl, etc. to obtain the so-called active Method for producing chemical ester 1. 2) A method in which the terminal hydroxyl J of polyalkylene glycol is reacted with at least one of the following with bromide/ammonium.

3) A method in which a polyalkylene glycol is chlorinated/an-I or reacted with a derivative thereof and used as a polyalkylene gelcol, for example, 4,6-7chloro-5-1-lyazine.

4) A method of deriving at least one terminal hydroxyl group of a polyalkylene glycol into an aldehyde or an acid salt compound.

Next, which Pl' is preferable from the viewpoint of oxygen supply ability and safety of this substance? The activated ester method 1) with t will be explained in detail below.

Polyalkylene glycol P has a calhokyfur group τj
As a sipping method, the carhokyfur group of a polycarphonic acid such as alkali/dinorphonic acid may be used to oxidize the hydroxyl group of the polyalkylene/glycol to form a carhokyfur group.

Examples of polycarphonic acids include succinic acid, curcuric acid, nocarphonic acid having no functional group other than a carboquinol group such as anopine, carboquinol group U such as apple alcohol, aspartic acid, glutamic acid, etc. having a functional group other than carboquinol group, It may also be a polycarboxylic acid such as one in which the functional group is protected so as not to inhibit the desired reaction, or diI-lilotriacetic acid, tricarno-lic acid, or ethylenediaminetetraacetic acid.

On the other hand, for example, chlorogenated monocarboxylic acid, monocarboxylic acid containing amine 7 groups, and dehydrogenation with the hydroxyl group of polyalkylene/glycol.
In this way, the desired polyalkylene glycol containing a carboxy/kyl group can be obtained. The desired substance can also be obtained by reacting polycarboxylic acid anhydride with polyalkylene glycol.

This J, huh? In this reaction of polyalkylene glycol hemoglobin with a carboxylic group, for example, N-hyperoxylic acid imide, N-hydroxyfluoric acid imide, p-nitrophenol , Peck chlorophenol and other carbonic acid activators used in normal peptide synthesis can be used to form active esters, which can be reacted with hemoglobin to perform amidation exchange.
Thionyl chloride, etc. It is also possible to react with a roquefating agent to form an acid halide of polyalkylene glycol, and to react this with hemoglopino.

In the present invention, during the reaction of combining -moglopine and polyalkylene glycol, oxygen is absent or the oxygen concentration is low, and the oxygen partial pressure is 0 to 30.
Approximately mmHg may be selected. The reaction conditions other than the oxygen concentration mentioned in Section 2 may be any conditions that do not cause denaturation of hemoglobin.

Low concentrations of oxygen, nitrogen, argon, -
The air in the reaction vessel is replaced with a reducing agent (Na
BH4, Na2520. Known methods can be employed, such as a method of reducing and removing the gas using a pump or the like, and a method of removing the gas using a pump or the like and replacing it with an inert gas such as argon.

Also, the oxygen solution curve 1 of the hemoglobin-polyogiaethylene conjugate, P. Measurement +i Now this method (KIma
l + l'l MOrllnOlO+ M, KOl
anl+H, Watarl+H, WakaandM
, Kusoda, Biochim, Biophys.

AC+, a 200. 189-196 (1970)
According to

1.1, below, the present invention will be described in detail from Example 1 onwards.

Example 1 4 ml of 10.9% hemoglobin solution! (0,00
millimoles) in 0.122 M Tris buffer (p[
16,8) I 8ml 1f dissolved solution 1 volume 25
m, l! Place in a three-ringed flask and add 1 cup of argon.
Time ventilation L ta.

Subsequently, 6.6 m9 (0
, 025 mmol) and then (after aerating the albon gas for 1 hour, 5.0 #J (0.13 mmol) of borohydride,
The tube was then vented with an argon gaff for an additional hour. Add monomer/polyethylene glycol mono(zaku/
imidyl→)゛ri/un-l・)(average molecular weight 5000)
After adding 657 mg (0,124 F, remole) and stirring overnight at 4C for 1°, ultrafiltration was repeated using an ultrafiltration membrane with a molecular weight rejection of 30,000 to remove unreacted active esters and other decomposition products. Modification of section 4.5 from 1 by removing -
8.2 ml of moglobin solution was obtained. Molecular weight 97, OO
Degree of O1 substitution 6.2゜'5+1 (lt', (011vl phosphate buffer, p
H7,40,25'C) 11.0 ++lmHg 0
In addition, the reaction of the same stroke as described in -1 is carried out under normal pressure air (l).
It was confirmed that an improvement in oxygen transport ability was observed.

Example 2 20.49 potassium dihydrogen phosphate was added to 1325 ml of distilled water for injection, and the pH was adjusted with IN aqueous caustic soda solution.
Adjusted by 6.81. Add 76 ml (0.47 E!I mol) of human hemoglobin solution M+ (17.l candy) and aerate the argon gas in the flask for 3 hours.
493 mg (1,93 mmol) of 1-phosphoric acid ester was added, and after further heating for 1 hour, borohydride was added.
2 mg (8.8 mmol) were added. After stirring for an additional 1 hour and adding polyethylene glycol bis(Zaku/Imifurusaku/F-+・) (average molecular weight 4000) while aerating the argon gas, ultrafiltration was repeated through an ultrafiltration membrane with a molecular weight blocking capacity of 10,000. 6.5 Section repair ff
1li hemogrohi/solution 380ml (!) was obtained.

This solution is N + ICI 2.17 If', Na
1lCO(1,+4?CaC1,2o,I I
IF, KCI O, +1 2
, MgCl2.6H,,00,057r, then 0. Adjust the pH to 7.4 with IN NaOH and reduce to 0.
A blood substitute solution was obtained by filtration through a 22μ membrane.

Average published % I ] 0.000, P5o: I O,
4mmHg (0.1M phosphate buffer, pH 7, 4, 25
C) In addition, the same reaction as above is carried out with the presence of normal pressure air 1τ and tj
The hemocrohine-polyethylene glycol conjugate obtained was P. -6.5 mmHg.

Example 3 Polyethylene glycol monooleyl ether (average molecule @ 4.000) 5 f (1.3 mmol) was added to 4
00 mi of Hensen V was dissolved and reacted with Nork 52 carbonate and Affl chloride 2.75 f (15 mmol) overnight under water cooling to obtain petroleum ether insoluble 2-0-oleyl polyethylene glycol-4,6-7. Kuroro S-h
Rhianon (activated polyethylene glycol oleyl ether) 6.2 y obtained.

Cow hemokurohi/(USA/manufactured by Boots Inc.) 2 y (0,
03 mmol) to 200 m1! Boric acid buffer (pH 9,
After dissolving in 2), high-purity nitrogen was aerated, and then hydrosulfide sulfur and lithium (Na25204) was added.
0.52 was added to remove oxygen.

The reaction was carried out under water cooling, 4.09% active polyethylene glycol monooleyl ether in a stream of hydrogen gas,
After the reaction was completed for 3 hours, unreacted hemoglobin and activated polyethylene glycol monooleyl ether were removed using an ultrafiltration membrane with a molecular weight of 10,000.

The amount of hemoglobin-polyethylene glycol monooleyl ether obtained was 482. That P5 (, is l)
H7,4,25+1: 4 in 0.1 molar phosphate buffer
．． lmmHg and Pl of the reaction product in the presence of air
,. -19 Hg compared to +41! An oxygen supply capacity of -4 was observed.

Example 4 Polyethylene glycol (average molecular weight 750) o, s
y (] mmol) was dissolved in 15 m (!) of water, and 0.42 (4 mmol) dissolved in 2 ml of dioxane was added dropwise under water cooling. After stirring for an hour, IN
The pH was adjusted to 7 with HC:l. The product was concentrated through an ultrafiltration membrane with a molecular weight of 500.
200 ml of phosphate buffer with a pH of 7.5.
+++(!) was added and concentrated.

The Jz Me fm polyethylene glycol solution was deoxygenated with an alkonous solution, and this was added to 20 mL of a water-cooled and deoxygenated phthalemoglobin IO% solution.

CM-Sephadex 2 balanced to p+-16,0 (
After purifying the product using an ultrafiltration membrane with a molecular weight of 50,000, it was desalted and concentrated using a 0.4511 Millipore membrane (USA, MIJ). Collection of 1° produced hemocrohine-polyethylene/glycol conjugate that was sterilized using Boa Co., Ltd. 6
212.1) H7,4 (phosphate buffer 0.1 M)
, 25C, Pho = 3.2 mmHg. In addition, the reaction product P in the presence of oxygen. Under the same conditions 1, 12 mL and 11 g were used.

Example 5 Polypropylene/Glicol (average molecular ft 4.000
) 12S' (3 mmol) was first reacted with succinic anhydride in pyrinone, the pyrinone was removed under reduced pressure, and then dehydrated with N-hypoquine succinimide and zinclohequinol sulfozoimide in methylformamide [. Toki: Sasae, polypropylene glycol bis(→]-kunoimidirusaku/ne+-)+2.5? I got it. This one B
The method of unn et al. (M, J, McDonald+M,
Bleichman+ H,F, Bunn and
R,W, Robert+J, Biol, Che
Human-\mocrohin curcose-6-phosphate 52 prepared by J. M. 254, 702-707 (1979) in 10% phosphate buffer 1&.

The solution was dissolved in 50 ml (pH 8, 9), deoxygenated, and purified by the method of Example 2. The yield estimated from the extinction coefficient of ultraviolet absorption of the reaction product, hemocrohin-polypropylene glycol conjugate mW, was 122%.

1) H7,4 (phosphate buffer) P at 25C! it1
”=9.2+nmHg.

In addition, P of the same hemoglobin-polypropylene glycol conjugate as above, which was reacted in the presence of air. -42 Taki H
It was g.

Example 6 9.8% human hemoglobin solution 10 m/' (0,0
15 mmol) total 0.2M 1-1) buffered Kf, 5
This 1.6% solution of -\Mokurohino was blown into a reaction tube of 200 + + + (!) with argon gas for 3 hours.
POCker+ J, OsgChem, 38 (25
) 4295-4299 (+973)) was added, and 14 mg (o, o 54 mmol) of 2-true 2-formylpyridoxal-51-phosphate prepared by 4295-4299 (+973)) was added, and alkonol was heated for 1 hour at +11 atmospheres, and then N
a B H i Add 97n9 and create an alcono atmosphere 1.
+++ and allowed to react for 5 minutes.

This solution V is the average molecule of ethylene glycol (saku/noi/rurik/r, -1) (;l-ly-1-lene glycol/r, -1) (;5000) 1.8?
(0.3 mmol) was added and reacted at 4C for 12 hours under an argon stream. The reaction mixture is filtered four times through an ultrafiltration membrane with a molecular weight of 50,000 to remove unreacted active Nisdel, O, and non-ions.
, a solution with a hemoglobin concentration of 7.1% +0.5
I got mJ. The average molecular weight was measured by gel filtration chroma 1-craphy method] 02. There was OOO.

P. (Phosphate buffer 0.1MlpH7,4,25C
) was 26 mmHg. In addition, the combined product "F+fl" obtained by performing the reaction part of 2-true 2-formylpyridoxal-5+-phosphate-hemoglobin and activated polyethylene glycol in normal pressure air was measured at 13 mmHg under the same measurement conditions. there were.

In addition, the product of Example 1.2.3.4.5 was obtained by the method already published by Ajisaka et al.
ka and Y, Iwashita+
Biochem.

B i 01) h y s , I g e s ,
Co+nmun, 9 units, +1076~+0
81 (1!l l(o) ) +, = , an exchange transfusion of 80% of Rano I's blood lfk was carried out, and the half-life in the bloodstream was measured, but no effects that appeared to be due to the low oxygen concentration t were observed. I couldn't.

As is clear from the following, in the absence of oxygen or with low oxygen
A. The reaction of hemoglobin with polyethylene glycols in the seat is effective in extending the lifespan of hemoglobin in the bloodstream without impairing its original oxygen carrying ability.

Claims (1)

[Claims] I) In a blood substitute containing a hemoglobin-polyalkylene glycol conjugate as an oxygen transport substance, +
'+ij combination is also low in the absence of oxygen'/R
1. A blood substitute characterized in that it is a substance prepared by drying hemochlorhyde and polyalkylene/glycol. 2) The blood substitute according to claim 1, which is a polymer selected from the group consisting of polyalkylene glycol, polyethylene glycol, polypropylene glycol, and copolymers of ethylene oxy-noi and fluoro-copylene oxy-noi. . 3) Hemoglohine-polyalkylene/glycol conjugate e
Here, the terminal hydroxyl group of polyalkylene glycol is
A blood substitute according to claim 1, wherein the blood substitute is substituted with a compound selected from the group consisting of alcohols, fatty acids and O-amines. 4) Hemocrobin-polyalkylene/glycol conjugate 1
The blood substitute according to claim 1, wherein the blood substitute is all modified with a compound selected from the group consisting of hemoglobin, bilitoxaluric acid, pyridogyzarulic sulfuric acid, and υ sugar phosphoric acid. . 5) The blood substitute according to claim 1, wherein in the hemoclohy/-polyalkylene glycol conjugate 1, hemoclohy/ and polyalkylene glycol are bonded via an amide bond. 6) The blood substitute according to claim 85, wherein the amide bond is formed by dehydration condensation between the amine group of hemoglobin and the carboxyl group of polyalkylene/glycol containing a carfoquinol group. 7) The molecular weight of polyalkylene glycol is 300-20
．． The blood substitute according to claim 1, having an ip range within the OOO range. ,