JPH029564B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel oxygen transport agent for artificial blood. The present inventor has discovered that a hemoglobin-inulin conjugate has the following properties and is excellent as an oxygen carrier for artificial blood, and has completed the present invention. Long residence time in blood vessels. It has the same oxygen carrying capacity as hemoglobin. Not bioaccumulative in humans. It is harmless. The hemoglobin used in the production of the hemoglobin-inulin conjugate used in the present invention is human, bovine, porcine, ovine, equine, canine, monkey, rabbit,
Any animal that has hemoglobin, such as chicken, may be used. In this specification, when referring to hemoglobin, it refers to hemoglobin derivatives, such as
Hemoglobin and pyridoxal phosphate, pyridoxal sulfate, 2,3-diphosphorylated glyceric acid,
It includes covalent bonds with inositol phosphate, sugar phosphate esters, phosphoenolpyruvate, covalent bonds with monosaccharides or polysaccharides such as glucose, and covalent bonds with amino sugars. Among these, derivatives modified with pyridoxal derivatives such as pyridoxal phosphate and pyridoxal sulfate are preferred in that they provide target conjugates with particularly excellent oxygen transport ability. On the other hand, inulin is not particularly limited, but has a molecular weight of
The number is preferably 3,000 to 6,000, and it may also have branches. Any method may be used to bond hemoglobin and inulin, but it is preferable to use a chemical bonding agent to form a covalent bond. For example, using a chemical binder such as cyanogen bromide,
By bonding between the amino group of hemoglobin and the hydroxyl group of inulin, cyanuric chloride, 2,2'-dichlorobenzidine, p,p'-difluoro-m,m'-
There is a method of bonding using a chemical binder such as dinitrodiphenyl sulfone or 2,4-dichloronitrobenzene. In particular, carboxylic acids are good as chemical binders;
For example, halogenated monocarboxylic acids, monocarboxylic acids such as amino group-containing monocarboxylic acids, succinic acid,
Dicarboxylic acids that do not have functional groups other than carboxyl groups such as glutaric acid and adipic acid; dicarboxylic acids that have functional groups other than carboxyl groups such as malic acid, aspartic acid, and glutamic acid; tricarboxylic acids such as nitriacetic acid and tricarbalyllic acid; acid, ethylenediaminetetraacetic acid and other polycarboxylic acids. For example, when using halogenated monocarboxylic acids such as iodoacetic acid, chloroacetic acid, bromoacetic acid, etc.
It is preferable to carry out dehydrohalogenation with the hydroxyl group of inulin to produce an inulin derivative having a carboxyl group, and then bind this to hemoglobin. Furthermore, when using a polycarboxylic acid, it is preferable to condense the acid anhydride with the hydroxyl group of inulin to similarly produce an inulin derivative having a carboxyl group, and bind this to hemoglobin. When using polycarboxylic acids having functional groups other than carboxyl groups, such as hydroxydicarboxylic acid and aminodicarboxylic acid, protect the functional groups in advance with a protective reagent that is commonly used to protect each functional group. For example, it is desirable to protect a hydroxyl group or an amino group with carbobenzyloxychloride or the like before carrying out the above reaction.
Of course, the protecting group can be removed by a commonly used deprotection method such as hydrogenation. Next, as a method for forming an amide bond between an inulin derivative into which a carboxyl group has been introduced and hemoglobin, N-hydroxysuccinimide,
Active esters using carboxyl group activators in peptide synthesis such as p-nitrophenol, pentachlorophenol, succinimide, N-hydroxyphthalimide, carbodiimidazole, imidazole, etc., and reacting with hemoglobin to form amide bonds. You can force it.
Alternatively, for example, the carboxyl group can be reacted with an acid halogenating agent such as thionyl chloride or phosphorus oxychloride to form an acid halide, which can then be reacted with hemoglobin. The hemoglobin-inulin conjugates used in the present invention are those in which about 1 to 15 inulins are bound to hemoglobin per hemoglobin subunit, those in which about 1 to 10 hemoglobins are bound to inulin, and those in which inulin and hemoglobin are cross-linked with each other. or a mixture thereof can be used. The oxygen transport agent for artificial blood of the present invention includes one consisting essentially of a hemoglobin-inulin conjugate, a mixture of this and other substances having oxygen transport ability, or a plasma expander, glucose, salts, etc. All oxygen carriers for artificial blood containing a hemoglobin-inulin conjugate, such as a mixture, are included. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 10g (0.002 mol) of inulin (Tokyo Kasei Co., Ltd.) was added to water.
Dissolve in 100ml, cool on ice, add 1g of cyanogen bromide (0.01
mol) dissolved in 10 ml of dioxane was added dropwise. At this time, the pH was maintained at 9 to 10 with a 2N caustic soda aqueous solution. It was added dropwise over about 30 minutes, and then stirred for 20 minutes. Add this reaction solution to ice-cold acetone.
The activated inulin prepared above was precipitated. After standing still for a while, the supernatant was removed, and residual cyanogen bromide was washed away with 300 ml of ice-cold acetone.
After completely removing acetone, add an aqueous solution (50 ml) containing 5.2 g (0.08 mmol) of hemoglobin under ice cooling.
After stirring for 4 hours, dialysis and concentration were performed using an "XM-100" membrane manufactured by Amicon. The product was converted into water-based gel “G-” manufactured by Toyo Soda Co., Ltd.
3000SW" column, and if necessary, molecular weight fractionation was performed by gel filtration using Pharmacia's "Sephadex G-100", and two types of fractions (hereinafter referred to as
They are called "inulin-Hb1" and "inulin-Hb2." ) was obtained. Example 2 To a solution of 5 g (0.001 mol) of inulin dissolved in 100 ml of water, 1 g (0.005 mol) of cyanuric chloride was added.
A solution dissolved in a mixed solvent of 10 ml of water and 20 ml of acetone was added dropwise. At this time, the pH value was maintained at 10 to 11 with a 2N caustic soda aqueous solution. 5 after completion of dripping
After stirring for a minute, the pH value was brought to 3 with 2N hydrochloric acid. 500 ml of acetone was added to this, and the resulting precipitate was filtered off and further washed thoroughly with acetone. After removing the acetone well, add 0.2M borate buffer 100
ml, then add 15 ml of 5.6% membrane component-free hemoglobin solution (stroma-free hemoglobin).
was added and stirred for 3 hours under ice cooling. The subsequent treatments were performed in the same manner as in Example 1. Molecular weight fractionation was performed by gel filtration, and two types of fractions (hereinafter referred to as "inulin-Hb3") were
and “inulin-Hb4”. ) was obtained. Example 3 In Example 2, instead of hemoglobin
6.3% pyridoxal phosphorylated hemoglobin 13.3
ml was used to carry out the same reaction as in Example 2 to obtain an inulin-hemoglobin-pyridoxal phosphate conjugate (referred to as "inulin-Hb5"). Example 4 Inulin 5.0g (0.001 mol) and succinic anhydride 3
g (0.03 mol) was dissolved in 50 ml of pyridine and heated under reflux for 2 hours. After cooling, add 200ml of hexane,
The resulting precipitate was filtered. 2.8 g of this precipitate, 2.4 g (0.021 mol) of N-hydroxysuccinimide and 4.2 g (0.021 mol) of dicyclocarbodiimide were added.
It was dissolved in 60 ml of dimethylformamide (DMF) and stirred for 1 hour. After filtering off the formed precipitate, 300 ml of hexane was added to the filtrate to obtain 3.5 g of inulin succimidyl succinate crystals. 2.1 g (0.0004 mol) of these crystals were added to a 1% hemoglobin solution (0.2 M, borate buffer, PH 8.5) with stirring. After stirring at 4° C. for 16 hours, unreacted inulin and salts were removed by ultraviolet 3 filtration using Amicon's "PM-30" membrane, and concentrated to obtain an inulin-hemoglobin conjugate (referred to as "inulin-Hb6"). ) was obtained. Example 5 In Example 4, a reaction was carried out in the same manner as in Example 4 using pyridoxalic acid-bound hemoglobin instead of hemoglobin, and an inulin-hemoglobin-pyridoxal phosphate conjugate ("inulin-
Hb7”. ) was obtained. Example 6 Inulin 10g (0.002 mol) and succinic anhydride 6.2
g (0.062 mol) in a mixed solvent of 40 ml of dimethylformamide and 4 ml of pyridine to give 110
Stirring was carried out at ℃ for 2 hours. After cooling, acetone was added and the resulting crystals were filtered to obtain 8 g of inulin succinate crystals. 8g (0.0013mol) of this crystal
and N-hydroxysuccinimide 1.76g (0.015
Dissolve mol) in 60ml of DMF and add 3.16g to it.
(0.015 mol) of dicyclohexylcarbodiimide was added and stirred overnight at room temperature. After filtering the precipitate,
When 120 ml of ethyl ether was added to the filtrate, 5.2 g of inulin succimidyl succinate was obtained. Add this to 30ml (0.00006 mol) of 13% hemoglobin solution.
Dissolve in 200ml of 0.1M borate buffer (PH8.5) and add the above inulin succimidyl succinate 3 to this.
g (0.0006 mol) and stirred at 4°C for 1 hour. After the reaction solution was dialyzed against distilled water, it was desalted and concentrated by ultrafiltration using a membrane with a molecular weight of 30,000 (PM30, manufactured by Amicon), and then
Sterile filtration is performed using Millipore's "GS" membrane.
Inulin-hemoglobin conjugate (inulin-hemoglobin conjugate)
Hb8”. ) was obtained. Example 7 5.1 g of inulin succinate was obtained in the same manner as in Example 6 using 10 g (0.002 mol) of inulin and 4 g (0.04 mol) of succinic anhydride.
Mol) from inulin succimidyl succinate
4.8g was obtained. Using this inulin succimidyl succinate, an inulin-hemoglobin conjugate ("inulin-Hb9") was prepared by the same reaction as in Example 6.
That's what it means. ) was obtained. Example 8 2 g (0.02 mol) of succinic anhydride and 0.73 g of N-hydroxysuccinimide as in Example 6
(0.0063 mol), 4.4 g of inulin succimidyl succinate was obtained. Further, using this product, an inulin-hemoglobin conjugate (referred to as "inulin-Hb10") was obtained by the same reaction as in Example 6. Example 9 Same as Example 6, 1 g (0.01 mol) of succinic anhydride and 0.58 g of N-hydroxysuccinimide.
(0.005 mol), 4.1 g of inulin succinimidisuccinate was obtained. Furthermore, using this, in Example 6, a reaction similar to that of Example 6 was carried out using pyridoxal phosphorylated hemoglobin (8%, 49 ml) instead of hemoglobin, thereby producing an inulin-hemoglobin conjugate (referred to as "inulin-Hb11"). ) was obtained. Example 10 Add potassium dihydrogen phosphate to 225 ml of distilled water for injection.
1.7g and 26.6ml of 13.2% hemoglobin solution (0.000054
After adding mol), the pH was adjusted to 6.8 with a 2N aqueous solution of caustic soda. In this, pyridoxal
Add 51 mg (0.0002 mol) of 5'-phosphoric acid and stir at 4°C.
After stirring for an hour, add sodium borohydride to this
36 mg (0.00097 mol) was added. After further stirring for 1 hour, PH was added with 2N caustic soda aqueous solution.
8.5, 6 g (0.0011 mol) of inulin succimidyl succinate prepared by the method described in Example 4 was added under ice cooling. After stirring this at 4°C overnight, it was ultrafiltered and concentrated using a "Pallicon" concentrator manufactured by Millipore, resulting in 6.5% of the inulin-hemoglobin complex (referred to as "inulin-Hb12"). A solution was obtained. Example 11 Regarding the inulin-hemoglobin conjugate obtained above, the number of inulin bound per hemoglobin subunit was determined, and the molecular weight per subunit was determined. In addition, the intravascular residence time and 50% oxygen dissociation pressure were determined for the same sample. [Molecular weight measurement method] Cyanmethemoglobin method (D. Drabkin, Am.
Take a certain amount (Vo) of the inulin-hemoglobin conjugate solution (concentration Co) whose concentration was determined according to J.Med.Sci., vol217, 710-711 (1949)), freeze-dry it,
The weight was measured. When the average molecular weight of the inulin used is M _I and the molecular weight of hemoglobin is M _H , the number n of inulin bonds can be determined by the following formula. n=(mo-CoVo)/M _I /CoVo/M _H However, the molecular weight is the average value per subunit. From this, the molecular weight M of the inulin-hemoglobin conjugate was determined using the following formula. M=M _H +nM _I [Intravascular residence time] Two rats were used for each sample, and after intravenously injecting 5 ml/Kg of the 4-6% sample based on the rat's body weight, 5 min.
0.5 ml of blood was collected after 30, 60, 90, and 120 minutes, and the concentration of the sample in the centrifuged supernatant was determined by the cyanmethemoglobin method. From the graph, the half-life of the injected sample in plasma was calculated. [50% oxygen dissociation pressure] For the same sample solution, the method of Imai et al.
H.Morimoto, M.Kotani, H.Watari, H.
Waka, and M. Kuroda, Biochim. Biophys.
Acta, 200 , 189-196 (1970)), an oxygen equilibrium curve was drawn and the 50% oxygen dissociation pressure (P ₅₀ ) was determined from it. The results are shown in Table 1.

[Table] Example 12 Inulin-Hb12 was added to renal reflux fluid (NaCl570mg/
dl, NaHCO ₃ 48.8mg/dl, KCl4.9mg/dl,
After preparing a 5.7% solution containing 4.6 mg/dl of CaCl ₂ , 2.4 mg/dl of MgCl ₂ .6H ₂ O, and 1.8 g/dl of glucose, an exchange transfusion experiment was conducted in rats. For replacement, insert a polyethylene tube (outer diameter 1 1/3
mm) and inserted a cannula. After removing 1 ml of blood over 1 minute, 1 ml of blood was removed from the same crab urea.
The procedure of injecting 1 ml of the sample solution over several minutes and then resting for 1 minute was repeated 20 to 30 times to achieve 85% exchange. Dextran 70 was used as a control at the same exchange rate.
In the solution, the animals died within 5 minutes, but in this sample solution, they survived for more than 2 hours. From the above results, the hemoglobin-inulin conjugate has a disappearance rate (half-life) from the blood that is twice as long as that of hemoglobin, and maintains a value similar to that of hemoglobin in terms of oxygen affinity. It is understood that it is suitable as an oxygen carrier for artificial blood.

Claims (1)

[Scope of Claims] 1. An oxygen transport agent for artificial blood containing a hemoglobin-inulin conjugate using a carboxylic acid as a binding agent. 2 The oxygen-carrying active ingredient is substantially hemoglobin.
The oxygen transport agent according to claim 1, which comprises an inulin conjugate. 3. The oxygen transport agent according to claim 1, wherein the hemoglobin is modified with a pyridoxal derivative. 4. The oxygen transport agent according to claim 1, wherein the carboxylic acid is a carboxylic acid selected from the group consisting of monohalogenated carboxylic acids and dicarboxylic acids.