JPH0359883B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a novel blood substitute, more specifically, a blood substitute containing a hemoglobin-polyalkylene glycol conjugate as an oxygen carrying substance,
The present invention relates to a blood substitute characterized in that the hemoglobin and polyalkylene glycol of the conjugate are bonded through an amide bond. It is known that a hemoglobin-polyalkylene glycol conjugate is useful as an oxygen carrying substance for blood substitutes (see Japanese Patent Application Laid-open No. 12308/1983). As a result of extensive research into the above-mentioned substances in order to further improve their oxygen supply ability, the present inventor found that a compound in which hemoglobin and polyalkylene glycol are bonded through an amide bond is even more superior in terms of its oxygen supply ability. Among these, a conjugate in which hemoglobin is bound to both ends of a polyalkylene glycol is particularly superior in terms of colloid osmotic pressure and has the industrial advantage of being able to halve raw material costs, and is practically effective as an oxygen transport substance. We have discovered something, and based on this discovery, we have completed the present invention. Examples of the polyalkylene glycol used in the conjugate include polyethylene glycol, polypropylene glycol, and ethylene oxide-propylene oxide copolymer. The hemoglobin may be derived from animals having hemoglobin such as humans, cows, pigs, sheep, horses, dogs, monkeys, rabbits, and chickens. In this specification, hemoglobin refers to modified hemoglobin, such as pyridoxal derivatives (pyridoxal-5'-phosphate, pyridoxal-5'-sulfate, 2-nor-2-formylpyridoxal-5'-phosphate). etc),
Sugar phosphate derivatives (glucose-6-phosphate, adenosine monophosphate, etc.) are also included. The molecular weight of the polyalkylene glycol is 300
It is preferably about 750 to 10,000 in terms of oxygen supply ability and viscosity. In order to bond polyalkylene glycol to hemoglobin through an amide bond, for example, a carboxyl group may be added to the polyalkylene glycol, and a condensation reaction may be performed between this and the amino group of hemoglobin to form an amide bond. In order to impart carboxyl groups to polyalkylene glycol, for example, known methods such as the US Pat.
4179337 specification and US Patent No. 3941710, a method of reacting at least one carboxyl group of a polycarboxylic acid such as an alkanedicarboxylic acid with a terminal hydroxyl group of a polyalkylene glycol to form an ester of a polycarboxylic acid. should be adopted. When reacting such an alkylene glycol with a carboxyl group with hemoglobin,
For example, N-hydroxysuccinimide, N-hydroxyphthalimide, P-nitrophenol,
It is also possible to form an active ester using a carboxylic acid activator in ordinary peptide synthesis such as pentachlorophenol, and then react with hemoglobin to perform amidation exchange, or to form an active ester with an acid halogenating agent such as thionyl chloride to form a carboxylic acid ester. It is also possible to prepare an acid halide of polyalkylene glycol and react it with hemoglobin. Examples of the polycarboxylic acids include malonic acid, succinic acid, glutaric acid, adipic acid, malic acid, and citric acid; however, the oxygen supplying ability of the present oxygen transport substance varies depending on the type of these polycarboxylic acids. It doesn't change. Some hemoglobin-polyalkylene glycol conjugates include those in which one molecule of polyalkylene glycol is bound to one molecule of hemoglobin through an amide bond, those in which two or more molecules of polyalkylene glycol are bound to one molecule of hemoglobin through an amide bond, and those in which one molecule of polyalkylene glycol is bound to one molecule of hemoglobin through an amide bond. These include those in which hemoglobin is bound to both ends, and various other copolymers of hemoglobin and polyalkylene glycol. Among these, in particular, a conjugate in which one molecule of hemoglobin is bound to each end of one molecule of polyalkylene glycol, or a copolymer mainly composed of this, brings colloid osmotic pressure and oxygen co-supply ability close to those of human blood. It has been found to be favorable in this sense. In the hemoglobin-polyalkylene glycol conjugate, polyalkylene glycol having a terminal hydroxyl group may be protected, such as etherification with an alcohol having 1 to 16 carbon atoms, esterification with a fatty acid having 2 to 18 carbon atoms, Alternatively, a protection method by imination with an amine having 1 to 18 carbon atoms may be employed. The number of polyalkylene glycol bonds per subunit and the molecular weight per subunit in hemoglobin with polyalkylene glycol bound through amide bonds are determined by the method of Ajisaka et al. (K. Ajisaka and Y. Iwashita, Biochem.
Biophys.Res.Commun. 97 , 1076-1081 (1980). Polymer, such as polyether carboxylic acid, has 1 molecule per molecule of hemoglobin subunit.
~10 molecules are bound. The hemoglobin to which a polymer is bonded via an amide bond, which is used in the present invention, can be prepared, for example, as follows. (1) Polyether carboxylic acid and 1 to 10 times the mole, preferably 2 times the mole of N-hydroxysuccinimide, in N,N-dimethylformamide,
The mixture is stirred at room temperature or under heating for 3 to 20 hours, preferably 8 to 14 hours, in the presence of 1 to 10 times the mole, preferably 2 times the mole of dicyclohexylcarbodiimide.
After the reaction, dicyclohexyl urea produced is removed by filtration, and then ether or the like is added to obtain a polyether carboxylic acid activated ester. The obtained substance was adjusted to pH7
~9, preferably PH7~9, preferably 8~
8.5 in an aqueous solution or a buffer solution with 1 to 1/100 times, preferably 1/5 to 1/30 times, hemoglobin. In the above reaction, N-hydroxyphthalimide is substituted for N-hydroxysuccinimide,
It can be similarly prepared using p-nitrophenol or pentachlorophenol. (2) N,N-
Dissolve in dimethylformamide and add 1 to 10
Two times the mole, preferably five times the mole of dicyclohexylcarbodiimide is added, and the mixture is reacted under heating and reflux for 5 to 20 hours, preferably 10 hours. After the reaction is cooled, the resulting dicyclohexyl urea is removed by filtration, and ether or the like is added to the filtrate to obtain polyether carboxylic acid amide. This polyether carboxylic acid amide has a pH of 7 to 9, preferably 8 to 9.
8.5 in aqueous solution or buffer solution 1x to 1/100x,
Preferably, the reaction is performed with about 1/5 to 1/30 times as much hemoglobin. In the above reaction, succinimide or phthalimide may be used in place of imidazole. In addition, as a method for preparing carboxylic acid amide, polyether carboxylic acid and 1 to 10 times the mole, preferably 2 times the mole of carbonyldiimidazole are mixed with N,
The reaction may also be carried out in N-dimethylformamide. (3) Polyether carboxylic acid and a large excess of thionyl chloride are mixed at 60 to 90°C, preferably 75 to 80°C.
After reacting for ~5 hours, preferably 1.5 to 2 hours, thionyl chloride is distilled off under reduced pressure, and the resulting polyether carboxylic acid chloride is about 1/10 to 1/100 times, preferably 1/30 to 1/50. About twice as much hemoglobin
React in an aqueous solution or buffer solution with a pH of 8.5 to 9.5. In the above reaction, an acid halogenating agent such as phosphorus oxychloride or phosphorus pentachloride may be used in place of thionyl chloride. (4) Polyether dicarboxylic acid and 1 to 10 times the mole of N-hydroxysuccinimide, preferably 3 times the mole, in N,N-dimethylformamide.
The mixture is stirred at room temperature or under heating for 3 to 20 hours, preferably 8 to 14 hours, in the presence of ~10 times the mole, preferably 3 times the mole of dicyclohexylcarbodiimide. After the dicyclohexylurea produced after the reaction is removed by filtration, ether or the like is added to obtain a polyether dicarboxylic acid activated diester. The reaction after that was
React with hemoglobin in the same manner as in (1). Note that in (1) to (4), hemoglobin naturally includes modified hemoglobin as described above. The present invention will be explained in detail below using Examples and various tests. Example 1 Monomethoxypolyoxyethylene succinic acid monoester (average molecular weight 5000) 5 g (0.001 mol),
N-hydroxysuccinimide 0.23g (0.002mol), dicyclohexylcarbodiimide 0.42g
(0.002 mol) was dissolved in 300 ml of N,N-dimethylformamide and stirred at room temperature overnight. The precipitated dicyclohexyl urea was filtered, 600 ml of ethyl ether was added to the filtrate, and the formed monomethoxypolyethylene glycol mono(succiimidyl succinate) crystals were filtered, thoroughly washed with ether and dried to obtain 4.6 g of white crystals. Ta. Pyridoxal phosphate-bound hemoglobin 0.5g
(0.0077 mmol) in 100 ml borate buffer (PH
8.5) Add 0.5 of the above active ester under ice cooling to the solution dissolved in
g (0.1 mmol) was added. After stirring for 4 hours under ice-cooling, ultrafiltration was repeated using an ultrafiltration membrane with a molecular weight blocking of 30,000 to remove unreacted active ester or its decomposition products, thereby obtaining a modified hemoglobin solution. This product showed a single peak in high-performance liquid chromatography using a TSK G3000SW column (manufactured by Toyo Soda Co., Ltd.). This product was freeze-dried to obtain 0.58 g of modified hemoglobin. Degree of substitution 6.0, molecular weight 95000.
However, the degree of substitution and molecular weight correspond to one molecule of hemoglobin (α ₂ β ₂ type). Example 2 Polyoxyethylene adipate monoester (average molecular weight 2000) 2 g (1 mmol), pentachlorophenol 0.27 g (1 mmol), dicyclohexylcarbodiimide 0.25 g (1.2 mmol)
was reacted in 30 ml of N,N-dimethylformamide at room temperature overnight, and the dicyclohexylurea formed was filtered off. Add ethyl ether to the filtrate, filter the precipitated crystals, and recrystallize from toluene to give 1.5 g.
crystals were obtained. 0.05 g (0.1 mmol) of the above active ester
% carbon monoxide hemoglobin aqueous solution 20ml (0.016
mmol) in small portions. At that time, the pH was maintained at 8.5 with a 0.2N caustic soda aqueous solution. The reaction solution was repeatedly ultrafiltered through a membrane with a molecular weight blocking of 30,000 to obtain 2 ml of a 0.4% modified hemoglobin solution. The substance showed a single peak in high performance liquid chromatography. Degree of substitution
3.5, molecular weight 72000. Example 3 8.5 g (0.01 mol) of polyethylene glycol monomalonic acid ester (average molecular weight 850), 2.07 g (0.015 mol) of paranitrophenol, and 2.3 g (0.015 mol) of dicyclohexylcarbodiimide were dissolved in 300 ml of N,N-dimethylformamide. The mixture was reacted overnight at room temperature. After the reaction is complete, the dicyclohexyl urea produced is filtered off, and the filtrate is mixed with 600% ethyl ether.
ml was added to obtain 6.2 g of crystals. The active ester was reacted with 10 ml of a 1% pyridoxal sulfate-bound hemoglobin solution in the same manner as in Example 1 to obtain 10.5 ml of a 0.8% modified hemoglobin solution. Degree of substitution 6.2, molecular weight 70000. Example 4 Monomethoxypolyoxyethylene succinic acid monoester (average molecular weight 2000) 4 g (0.002 mol),
0.7 g (0.004 mol) of N,N-carbodiimidazole was stirred in 100 ml of N,N-dimethylformamide at room temperature overnight. Add 200 ml of ethyl ether to the reaction solution, filter the precipitate, and wash well with ethyl ether to obtain 2.2 g of imidazole monomethyl polyoxyethylene succinic acid monoimide.
I got it. 6% pyridoxal phosphate-conjugated hemoglobin 6
ml (0.0055 mmol) of 0.1 M Tris buffer (PH
8.0) Dissolve in 60 ml and add 1 g (0.5
mmol) was added. After reacting for 4 hours under ice cooling, ultrafiltration was repeated using an ultrafiltration membrane with a molecular weight blocking of 30,000 to obtain 3.6 ml of an 8.3% modified hemoglobin aqueous solution.
Degree of substitution 10.4, molecular weight 86000. Example 5 5 g (0.001 mol) of monomethoxypolyoxyethylene succinate (average molecular weight 5000), 0.5 g (0.005 mol) of succinimide, and 1 g (0.005 mol) of dicyclohexylcarbodiimide were mixed with N,N-
It was dissolved in 50 ml of dimethylformamide and heated under reflux for 12 hours. After filtering the produced dicyclohexyl urea, 150 ml of ethyl ether was added and the resulting precipitate was filtered. The precipitate was thoroughly washed with ethyl ether and dried to obtain 3.2 g of crystals. The above imide was reacted with 7 ml of 3.2% pyridoxal phosphate-conjugated hemoglobin in the same manner as in Example 4 to obtain a 4.8% modified hemoglobin solution.
Got ml. Degree of substitution 11.2, molecular weight 120000. Example 6 5.2 g (0.001 mol) of polyethylene glycol malonic acid ester (average molecular weight 5200) was dissolved in 20 ml of thionyl chloride and heated to 75-80°C for 1.5 hours.
Thionyl chloride was distilled off under reduced pressure, and the resulting acid chloride crystals were thoroughly dried. 20ml of 1% pyridoxal phosphate-bound carbon monoxide hemoglobin
Dissolve in 0.7M borate buffer (PH10.0) and cool on ice.
5 g of the above acid chloride was added little by little. After stirring for 3 hours under ice-cooling, ultrafiltration was repeated through a membrane with a molecular weight inhibition of 30,000 to obtain 4.6 ml of a 4.1% modified hemoglobin solution. Degree of substitution 9.3, molecular weight
114000. Example 7 Pharmacopoeia polyethylene glycol (average molecular weight
4000) 100g (0.025mol), 6.3g succinic anhydride
(0.063 mol) in N,N-dimethylformamide
The mixture was dissolved in 100 ml and stirred at 100°C for 3 hours. After cooling, 400 ml of ethyl ether was added and the resulting precipitate was filtered. The precipitate was thoroughly washed with ethyl ether and dried to obtain 97.5 g of polyethylene glycol diester succinate crystals. 97.5 g (0.024 mol) of the above crystals, 6.3 g (0.054 mol) of N-hydroxysuccinimide, and 11.4 g (0.054 mol) of dicyclohexylcarbodiimide were added to 100
After heating and dissolving in ml of N,N-dimethylformamide, the mixture was stirred at 30°C overnight. The produced dicyclohexyl urea was filtered, 300 ml of ethyl ether was added to the filtrate, and the produced polyethylene glycol di(succimidyl succinate) crystals were filtered, thoroughly washed with ether and dried to obtain 95 g of white crystals. 10 ml of 16.4% hemoglobin solution (0.025 mmol)
was dissolved in 35 ml of 0.2M borate buffer (PH8.5), and 4.3 g (1.0 mmol) of polyethylene glycol di(succiimidyl succinate) crystals were added thereto. After stirring overnight at 4°C, ultrafiltration was repeated using an ultrafiltration membrane with a molecular weight restriction of 100,000 to obtain 51 ml of a 3.2% modified hemoglobin aqueous solution. Degree of substitution 11.2, molecular weight 122000. Example 8 14.3 ml (0.1 mmol) of a 15% pyridoxal phosphate-bound hemoglobin aqueous solution was dissolved in 270 ml of phosphate buffer (PH7.0), and the polyethylene glycol di(saccharide) prepared in Example 7 was heated at 4°C. 4.43 g (1.1 mmol) of crystalline imidyl succinate were added in small portions. After stirring at 4° C. for 4 hours, ultrafiltration was repeated through an ultrafiltration membrane with a molecular weight inhibition of 100,000 to obtain 11 ml of a modified hemoglobin solution with a concentration of 11.6%. Degree of substitution 6.6, molecular weight
93000. Test 1 The method of Imai et al. (K. Imai, H.
Morimoto, M.Kotani, H.Watari.H.Waka,
and M.Kuroda, Biochim.Biophys.Acta, 200 ,
189-196 (1970)) to measure the oxygen equilibrium curve,
The 50% oxygen dissociation pressure was then determined. The results are shown in Table 1. Furthermore, the residence time in the bloodstream of the same modified hemoglobin was measured. The measurement method is to use two rats (average weight 200 g) per sample, inject 5 ml/Kg of a 4-6% modified hemoglobin solution based on the rat's body weight through the tail vein, and then inject 5 minutes, 10 minutes, and 30 minutes after injection.
After 0.2 ml of blood was collected at the lapse of 1, 60, 90, and 120 minutes and centrifuged, the concentration of modified hemoglobin in the plasma was determined colorimetrically by the cyanmethemoglobin method. The half-life of the reduction of modified hemoglobin from plasma was determined from the graph of concentration changes over time. The results are shown in Table 1.

【table】

[Table] From the above results, the half-life of the reduction in rat plasma of the hemoglobin-polyalkylene glycol conjugate of the present invention is considerably longer than that of hemoglobin itself or the conjugate in the conventional method. Therefore, it can be seen that the substance of the present invention is significantly superior in its ability to supply oxygen to tissues within the body. Test 2 Bohr effect was measured. E.Bucci and C.Fronticelli, Method in
The experiment was conducted according to the method of Enzymology, 76 , 523-533, 1981), and the results are shown in Table 2.

[Table] From the above results, it is understood that the Bohr effect of the material of the present invention is approximately three times larger than that of the conventional product. This result means that the substance of the present invention is superior to conventional products in its ability to transport carbon dioxide from body tissues to the lungs. Test 3 The colloid osmotic pressure of the hemoglobin conjugate blood substitute was measured. The results are shown in Table 3. Hemoglobin equivalent concentration: 6%.

【table】

[Table] Manufactured items
blood 31.0

Claims (1)

[Scope of Claims] 1. A blood substitute containing a hemoglobin-polyalkylene glycol conjugate as an oxygen transport substance, wherein the conjugate has hemoglobin bound to both ends of the polyalkylene glycol via an amide bond. 2. The blood substitute according to claim 1, wherein the polyalkylene glycol is a polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, and ethylene oxide-propylene oxide copolymer. 3. The substitute according to claim 1, wherein the hemoglobin is modified with a compound selected from the group consisting of pyridoxal-5'-phosphate, glucose-6-phosphate, and pyridoxal-5'-sulfuric acid. blood. 4 The molecular weight of polyalkylene glycol is 300~
Claim 1 falling within the scope of 20,000
Blood substitutes as described in section.