Polyoxazoline-conjugated albumin, an artificial plasma expander, and a resuscitation solution for hemorrhagic shock

Abstract

To provide: an albumin that is bound with a water-soluble polymer that has high biocompatibility and can be easily prepared (synthesized); and an artificial plasma expander and a resuscitation fluid for hemorrhagic shock each of which includes the water-soluble polymer-bound albumin.SOLUTION: The present invention discloses: a polyoxazoline-bound albumin 100 that has albumin 10 as a core and a polyoxazoline 20 as a shell covalently bound to the albumin 10 via a crosslinking agent; and an artificial plasma expander and a hemorrhagic shock resuscitation fluid each of which contains the polyoxazoline-bound albumin 100.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to polyoxazoline-conjugated albumin, an artificial plasma volume expander and a resuscitation fluid for hemorrhagic shock. [Background technology]

[0002] Serum albumin is a simple protein that accounts for approximately 60% of plasma proteins. Its role in the bloodstream is to maintain oncotic pressure and to store and transport various endogenous substances (e.g., metabolites and hormones) and exogenous substances (e.g., drugs). Albumin, separated and purified from donated blood (plasma fraction), is formulated and widely used clinically. The purpose of administering albumin preparations is to maintain oncotic pressure and ensure circulating blood volume (circulating plasma volume). Specifically, albumin preparations are administered when hypoalbuminemia occurs due to bleeding, increased capillary permeability, decreased hepatic albumin synthesis, excessive renal or intestinal excretion, increased metabolism, or dilution by intraoperative fluid infusion. There are two types of albumin preparations: isotonic and hypertonic. Isotonic albumin preparations are used in emergency hemorrhagic shock following trauma, sepsis, cardiac surgery using cardiopulmonary bypass, extracorporeal circulation during hemodynamic instability, severe burns, and pregnancy-induced hypertonicity. Hypertonic albumin preparations increase colloid oncotic pressure and draw fluid into the blood vessels, making them useful for hemorrhagic shock, as well as for conditions such as refractory ascites associated with liver cirrhosis, refractory edema, and nephrotic syndrome with pulmonary edema. Albumin preparations can be inactivated by heating, eliminating the risk of viral infection. However, Japan's self-sufficiency rate for albumin preparations is low at 64% (2018), and the "domestic self-sufficiency" principle of the Blood Act has not been achieved. With the declining birthrate and aging population, the number of elderly people requiring blood transfusions increases, and the number of blood donors (young people) decreases, raising concerns that the self-sufficiency rate could decline further.

[0003] As alternatives to albumin preparations, i.e., artificial plasma expanders and resuscitation fluids for hemorrhagic shock, preparations using polysaccharides have been developed since the 1970s, and currently, low-molecular-weight dextran preparations and hydroxyethyl starch (HES) preparations are in practical use (see, for example, Patent Documents 1 and 2). However, administration of HES preparations can cause side effects such as blood coagulation disorders, renal dysfunction, shock, and elevated amylase levels. Given this background, there is currently a desire to develop new artificial plasma expanders and resuscitation fluids for hemorrhagic shock.

[0004] Japan is a pet-loving nation, with over 18.13 million pet dogs and cats, far outnumbering the population of children under 15 (15.11 million). Furthermore, as pets continue to age, the demand for veterinary care continues to grow. However, the current situation is that transfusion therapy is not adequately supported due to the lack of veterinary blood banks. Naturally, there are no veterinary albumin preparations (e.g., canine serum albumin preparations separated and purified from canine blood or feline serum albumin preparations separated and purified from feline blood). While obtaining and transfusing the animal's own plasma is one way to treat animals with hypoalbuminemia, a stable supply is difficult to secure. While HES preparations are the only viable option, they suffer from the aforementioned side effects and short blood retention time. Given this background, there is currently great anticipation for the development of a safer, longer-lasting artificial plasma expander for animals.

[0005] Polyethylene glycol (PEG) is a water-soluble synthetic polymer with excellent biocompatibility. Coating the molecular surface of heterologous proteins with PEG confers immunological stealth properties. However, it has been reported that serum antibodies against PEG are produced in patients treated with PEG-conjugated asparaginase or PEG-conjugated uricase. The presence of anti-PEG antibodies results in rapid excretion of the administered PEG-conjugated formulation from the body (see, for example, Non-Patent Documents 1 and 2). Furthermore, it has been reported that over 25% of patients who have not received treatment with PEG-conjugated formulations have anti-PEG antibodies (see, for example, Non-Patent Documents 3 and 4). This is thought to be due to the use of PEG in various products, such as food and cosmetics, on the market. It has also been shown that repeated administration of PEG-conjugated hemoglobin induces cell vacuolation (see, for example, Non-Patent Document 5). Under these circumstances, the development of new biocompatible, water-soluble polymers to replace PEG is attracting attention. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 06-133791 [Patent Document 2] Special Publication No. 2007-525588 [Non-patent literature]

[0007] [Non-Patent Document 1] JK Armstrong et al., Cancer, 2007, 110, 103-111. [Non-patent document 2] MRSherman et al., Adv. Drug Delivery Rev., 2008, 6, 59-68. [Non-patent document 3] RM Leger et al., Transfusion, 2001, 41, 29S. [Non-patent document 4] C. Lubich et al., Pharm. Res., 2016, 33, 2239-2249. [Non-Patent Document 5] C. Conover et al., Artif. Cells, Blood Subs., Immob. Biotechnol., 1996, 24, 599-611. Summary of the Invention [Problem to be solved by the invention]

[0008] The safe use of readily available heterologous albumin preparations as artificial plasma expanders and resuscitation fluids for hemorrhagic shock in humans and animals would be a major contribution to both general and veterinary medicine. One approach to this is to confer immunological stealth properties by conjugating PEG to the surface of heterologous albumin, but this raises concerns about the production of PEG antibodies. Therefore, there is a strong need for the development of artificial plasma expanders and resuscitation fluids for hemorrhagic shock composed of albumin conjugated with a water-soluble polymer that is highly biocompatible (e.g., not excreted in the kidney and free of side effects such as renal dysfunction and antigen-antibody reactions) and easy to prepare (synthesize).

[0009] The present invention aims to provide albumin bound to a water-soluble polymer that is highly biocompatible and easy to prepare (synthesize), as well as an artificial plasma expander and a resuscitation fluid for hemorrhagic shock that contain the albumin as an active ingredient. [Means for solving the problem]

[0010] As a result of extensive research into the development of an artificial plasma expander and a resuscitation fluid for hemorrhagic shock that are highly safe and effective, the inventors have discovered that polyoxazoline-conjugated albumin, in which the water-soluble polymer polyoxazoline is covalently bound to the surface of albumin, acts as an immunologically inactive artificial plasma expander and a resuscitation fluid for hemorrhagic shock even when administered to different species of animals, thereby achieving the above-mentioned objectives.

[0011] Polyoxazoline is a nonionic (uncharged), water-soluble polymer with a pseudo-polypeptide structure that is highly biocompatible and non-immunogenic. It exhibits many of the favorable properties of PEG while avoiding some of its drawbacks. The notable features of polyoxazoline are as follows (disadvantages of PEG are listed in parentheses): (I) it can be easily synthesized by ring-opening polymerization of oxazoline, allowing for the preparation of various side-chain-substituted derivatives (PEG is difficult to polymerize and has no side chains); (II) it does not generate peroxides (PEG generates peroxides); (III) it has low viscosity (PEG has high viscosity at high concentrations); (IV) it is stable at room temperature (PEG is stable at low temperatures but unstable at room temperature); and (V) it is easily degraded and eliminated in the body (PEG may accumulate). The present inventors have discovered that polyoxazoline can be an alternative to PEG as a compound for modifying albumin.

[0012] That is, the polyoxazoline-conjugated albumin of the present invention is characterized by having albumin as a core and polyoxazoline as a shell covalently bonded to the albumin via a crosslinking agent.

[0013] In the polyoxazoline-conjugated albumin of the present invention, the binding site of the albumin to the crosslinking agent is preferably lysine, a primary amine at the protein terminal, or cysteine.

[0014] In the polyoxazoline-conjugated albumin of the present invention, the binding site of the polyoxazoline with the crosslinking agent is preferably a terminal hydroxyl group or amino group of the polyoxazoline represented by the following general formula (1). [ka] [In general formula (1), R1 represents a hydrocarbon group having 1 to 8 carbon atoms, R2 represents a hydroxyl group, an amino group, or —NH—(CH2)2—OH, and n represents the number of repeating monomer units.]

[0015] In the polyoxazoline-conjugated albumin of the present invention, the covalent bond via the crosslinking agent preferably contains the following structure (1): [ka]

[0016] In the polyoxazoline-conjugated albumin of the present invention, it is desirable that the covalent bond via the crosslinking agent contains a structure derived from a maleimide group-introducing agent.

[0017] In the polyoxazoline-conjugated albumin of the present invention, it is desirable that the maleimide group-introducing agent contains at least one compound selected from the group consisting of compounds represented by the following general formula (2) or the following general formula (3). [ka] [ka] [In the general formula (2), R2 is a hydrogen atom or SO3 - Na + wherein R1 represents any one of the following general formula (4), general formula (5), chemical formula (1), and chemical formula (2): In addition, in general formula (3), R3 represents the following general formula (4), and R4 represents OH or Cl. [ka] [In the general formula (4), n represents an integer of 1 to 10.] [ka] [In general formula (5), n represents an integer of 2, 4, 6, 8, 10, or 12.] [ka] [ka]

[0018] In the polyoxazoline-conjugated albumin of the present invention, the covalent bond via the crosslinking agent preferably contains the following structure (2): [ka] [In the structure (2), R1 represents any one of the following general formula (4), the following general formula (5), the following chemical formula (1), and the following chemical formula (2)] [ka] [In the general formula (4), n represents an integer of 1 to 10.] [ka] [In general formula (5), n represents an integer of 2, 4, 6, 8, 10, or 12.] [ka] [ka]

[0019] In the polyoxazoline-conjugated albumin of the present invention, it is desirable that the covalent bond via the crosslinking agent further contains a structure derived from a thiol group introducing agent, and that the thiol group introducing agent is at least one compound selected from the group consisting of compounds represented by the following chemical formula (3), the following general formula (6), or the following general formula (7). [ka] [ka] [In the general formula (6), n represents an integer of 1 to 10.] [ka] [In the general formula (7), R1 represents OH or Cl, and n and m represent integers of 1 to 10.]

[0020] In the polyoxazoline-conjugated albumin of the present invention, the covalent bond via the crosslinking agent preferably contains the following structure (3) or structure (4). [ka] [ka] [In structures (3) and (4), m represents an integer of 1 to 10.]

[0021] In the polyoxazoline-conjugated albumin of the present invention, the polyoxazoline preferably has a weight-average molecular weight of 500 to 100,000 daltons.

[0022] The artificial plasma expander of the present invention is characterized by containing the polyoxazoline-bound albumin.

[0023] The resuscitation fluid for hemorrhagic shock of the present invention is characterized by containing the polyoxazoline-conjugated albumin. [Effects of the Invention]

[0024] According to the present invention, it is possible to provide a novel polyoxazoline-conjugated albumin, an artificial plasma volume expander, and a resuscitation fluid for hemorrhagic shock, which are highly biocompatible and easy to prepare (synthesize). [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows an example of the polyoxazoline-conjugated albumin of the present invention. [Figure 2] 1 is a graph showing the production of anti-PSA IgM antibodies in the porcine albumin (PSA) administration group, the polyethylene glycol-conjugated porcine albumin (PEG(5k)-eSM-PSA) administration group, and the polyoxazoline-conjugated porcine albumin (POx(5k)-eSM-PSA) administration group. [Figure 3]3(A) shows the number of red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs) in blood mixed with polyoxazoline-conjugated porcine albumin (POx(5k)-eSM-PSA). [Figure 4] 4A and 4B are graphs showing mean arterial blood pressure (MAP) (FIG. 4(A)) and heart rate (HR) (FIG. 4(B)) in the polyoxazoline-conjugated porcine albumin (POx(5k)-eSM-PSA) administration group and the hydroxyethyl starch (HES, Volven infusion) administration group after 50% blood removal. DETAILED DESCRIPTION OF THE INVENTION

[0026] Hereinafter, embodiments of the present invention will be specifically illustrated and described with reference to the drawings as necessary.

[0027] <Polyoxazoline-bound albumin> The polyoxazoline-conjugated albumin of this embodiment has at least albumin as a core and polyoxazoline as a shell, and may further have other moieties as necessary. The albumin and the polyoxazoline are covalently bonded via a crosslinking agent.

[0028] For example, as shown in Figure 1, the polyoxazoline-conjugated albumin 100 of this embodiment may have albumin 10 as a core and six polyoxazolines 20 as shells. In Figure 1, the albumin 10 and the polyoxazolines 20 are covalently bonded via a crosslinker.

[0029] [albumin] Albumin is a simple protein with a molecular weight of approximately 66,500 daltons. The polyoxazoline-conjugated albumin of this embodiment may be any albumin in which polyoxazoline is conjugated to albumin via a crosslinking agent.

[0030] The albumin is not particularly limited and can be appropriately selected depending on the purpose, and albumin purified from serum derived from vertebrates, including humans, can be used. The albumin is preferably at least one selected from the group consisting of human albumin, porcine albumin, bovine albumin, equine albumin, canine albumin, feline albumin, and recombinant albumin. These may be used alone or in combination. Among these, porcine albumin and bovine albumin are preferred in terms of securing raw materials. Furthermore, albumin derived from SPF (specific pathogen-free) porcine is particularly preferred from the viewpoint of safety. Furthermore, the recombinant albumin can be easily produced by protein synthesis (culture).

[0031] -Human albumin- The human albumin may be one purified from human serum, and is not particularly limited and can be appropriately selected depending on the purpose.

[0032] -Pig albumin- The porcine albumin may be purified from serum derived from pigs, and is not particularly limited and may be appropriately selected depending on the purpose. Furthermore, the albumin derived from SPF pigs may be purified from serum derived from SPF pigs.

[0033] -Bovine albumin- The bovine albumin may be one purified from bovine serum, and is not particularly limited and can be appropriately selected depending on the purpose.

[0034] -Equine albumin- The equine albumin may be one purified from serum derived from horses, and is not particularly limited and can be appropriately selected depending on the purpose.

[0035] -Canine albumin- The canine albumin may be one purified from serum derived from dogs, and is not particularly limited and can be appropriately selected depending on the purpose.

[0036] -Feline albumin- The feline albumin may be one purified from serum derived from a cat, and is not particularly limited and can be appropriately selected depending on the purpose.

[0037] -Recombinant Albumin- The recombinant albumin is not particularly limited as long as it is produced by ordinary gene recombination procedures, culture procedures, etc., and can be appropriately selected depending on the purpose.

[0038] [Crosslinking agent] Examples of the crosslinking agent include a crosslinking agent containing a maleimide group introducing agent, a crosslinking agent containing a thiol group introducing agent, etc. Specific examples include a maleimide group introducing agent, a thiol group introducing agent, etc. The crosslinking agent may be used alone or in combination of two or more types.

[0039] (Maleimide group introduction agent) The maleimide group-introducing agent is not particularly limited as long as it is a reagent capable of introducing a maleimide group into albumin or polyoxazoline, and can be appropriately selected depending on the purpose. A compound having a maleimide group is preferred, and from the viewpoint of reaction efficiency, at least one compound selected from the group consisting of bifunctional compounds having a succinimidyl group at one end and a maleimide group at the other end, such as those represented by the following general formula (2), and compounds represented by the following general formula (3) is preferred.

[0040] [ka] [ka]

[0041] In the general formula (2), R2 is a hydrogen atom or SO3 - Na + and R1 represents any one of the following general formula (4), the following general formula (5), the following chemical formula (1), and the following chemical formula (2). In addition, in the general formula (3), R3 represents the following general formula (4), and R4 represents OH or Cl.

[0042] [ka] In the general formula (4), n represents an integer of 1 to 10.

[0043] [ka] In the general formula (5), n represents an integer of 2, 4, 6, 8, 10, or 12.

[0044] [ka] [ka]

[0045] When a maleimide group is introduced using a compound represented by the above general formula (3) in which R4 is OH, it is preferable to use it together with a condensing agent to introduce the maleimide group into the NH2 terminal or OH terminal of albumin or polyoxazoline. Examples of preferred condensing agents include N,N'-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Furthermore, the compound represented by the above general formula (3) in which R4 is OH may be reacted with a compound such as thionyl chloride or oxalyl chloride to convert the OH in R4 to Cl before use.

[0046] As the maleimide group-introducing agent, compounds represented by the above general formula (2) or (3) are preferred for introducing maleimide groups into the NH2 group of lysine residues in albumin, the NH2 group of primary amines at the protein terminal, and the terminal NH2 group of polyoxazoline, and compounds represented by the above general formula (3) are preferred for introducing maleimide groups into the terminal OH group of polyoxazoline. These may be used alone or in combination of two or more.

[0047] For example, by reacting the succinimidyl group in the maleimide group-introducing agent with the amino group (NH2 group) of a lysine residue in albumin or the amino group (NH2 group) at the protein terminus, a maleimide group can be introduced into the amino group (-NH2) of the lysine residue or the amino group (NH2 group) at the protein terminus.

[0048] Examples of methods for introducing the maleimide group include stirring albumin and a maleimide group-introducing agent at 0°C to 30°C for 0.5 to 10 hours.

[0049] (Thiol group introducing agent) The thiol group-introducing agent is not particularly limited as long as it is a reagent capable of introducing a thiol group into albumin or polyoxazoline, and can be appropriately selected depending on the purpose. From the viewpoint of reaction efficiency, at least one compound selected from the group consisting of compounds represented by the following chemical formula (3) (2-iminothiolane hydrochloride), the following general formula (6), or the following general formula (7) is preferred.

[0050] [ka]

[0051] [ka] In the general formula (6), n represents an integer of 1 to 10, and from the viewpoint of ease of crosslinking reaction, n is preferably 2 to 5, more preferably 2 to 3, and particularly preferably n=2.

[0052] [ka] In general formula (7), R1 represents OH or Cl, and n and m represent integers of 1 to 10. Each R1 may be the same or different. R1 is preferably OH, and both R1 are more preferably OH. n and m may be the same or different. From the viewpoint of ease of crosslinking reaction, n and m are preferably 2 to 5, more preferably 2 to 3, and particularly preferably n=m=2.

[0053] When a thiol group is introduced using a compound represented by the above general formula (7) in which R1 is OH, it is preferable to use it together with a condensing agent to introduce the thiol group into the NH2 terminal or OH terminal of albumin or polyoxazoline. Examples of preferred condensing agents include N,N-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Furthermore, the compound represented by the above general formula (7) in which R1 is OH may be reacted with a compound such as thionyl chloride or oxalyl chloride to convert the OH in R1 to Cl before use.

[0054] As the thiol group-introducing agent, for introducing thiol groups into the NH2 group of lysine residues of albumin, the NH2 group of primary amines at the protein terminal, and the terminal NH2 group of polyoxazoline, the compound represented by the above chemical formula (3), the above general formula (6), or the above general formula (7) is preferred, and for introducing thiol groups into the terminal OH group of polyoxazoline, the compound represented by the above general formula (7) is preferred. These may be used alone or in combination of two or more.

[0055] [Polyoxazoline] Polyoxazolines are nonionic, water-soluble polymers with a pseudo-polypeptide structure that are highly biocompatible and non-immunogenic. They can be synthesized in large quantities, various functional groups can be introduced, and they are safe for use in animals. Polyoxazolines exhibit many of the excellent properties of PEG while avoiding some of its drawbacks. Furthermore, polyoxazolines are excreted by the kidney without accumulating in tissues. The polyoxazolines may be used alone or in combination of two or more.

[0056] Examples of the polyoxazoline include compounds represented by the following general formula (1). [ka] In general formula (1), R1 represents a hydrocarbon group having 1 to 8 carbon atoms, preferably a methyl group, an ethyl group, a propyl group, or an isopropyl group. R2 represents a hydroxyl group, an amino group, or -NH-(CH2)2-OH. n represents the number of repeating monomer units. Here, the bonding site of the polyoxazoline with the crosslinking agent is preferably a terminal hydroxyl group or amino group of the polyoxazoline represented by the general formula (1) above. The polyoxazoline may be a homopolymer or a heteropolymer.

[0057] The weight average molecular weight of the polyoxazoline is preferably 500 to 100,000 daltons.

[0058] <Method of producing polyoxazoline-bound albumin> Examples of the method for producing polyoxazoline-conjugated albumin of this embodiment include a method of reacting at least an albumin derivative derived from albumin and a polyoxazoline derivative derived from polyoxazoline.

[0059] (albumin derivative) The albumin derivative is preferably at least one selected from the group consisting of maleimide-group-introduced albumin, thiol-group-introduced albumin, and unmodified albumin.

[0060] In particular, when a polyoxazoline having a terminal thiol group is used as the polyoxazoline derivative, it is desirable to use a maleimide group-introduced albumin as the albumin derivative. The maleimide-introduced albumin is preferably a maleimide-introduced albumin obtained by binding the maleimide-introducing agent (e.g., the compound represented by the general formula (2)) to a lysine residue (NH2 group) of albumin or an amino group (NH2 group) at the protein terminal. The maleimide-introduced albumin can be obtained, for example, by stirring albumin and the maleimide-introducing agent at 0°C to 30°C for 0.5 to 10 hours.

[0061] When a polyoxazoline having a terminal maleimide group is used as the polyoxazoline derivative, it is desirable to use a thiol group-introduced albumin or a non-modified albumin as the albumin derivative. The thiol group-introduced albumin is preferably obtained by binding the thiol group-introducing agent (e.g., 2-iminothiolane hydrochloride) to the amino group (NH2 group) of a lysine residue of albumin or the amino group (NH2 group) at the protein terminal. The thiol group-introduced albumin can be obtained, for example, by stirring albumin and a thiol group-introducing agent such as 2-iminothiolane hydrochloride at 0°C to 30°C for 0.5 to 10 hours. As the unmodified albumin, albumin treated with a reducing agent may be used.

[0062] (Polyoxazoline derivatives) The polyoxazoline derivative is preferably at least one selected from the group consisting of thiol-terminated polyoxazolines and maleimide-terminated polyoxazolines, and more preferably at least one selected from the compounds represented by the following general formulas (8) to (13): Among these, when the albumin derivative is a maleimide-introduced albumin, a terminal thiol group polyoxazoline is preferred, and when the albumin derivative is a thiol-introduced albumin or an unmodified albumin, a terminal maleimide group polyoxazoline is preferred. The polyoxazoline derivative can be used, for example, as a crosslinking agent when introducing polyoxazoline into a compound (for example, a compound having a thiol group at the end, a compound having a maleimide group at the end, etc.).

[0063] -Thiol-terminated polyoxazoline- The thiol-terminated polyoxazoline is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include compounds represented by the following general formulas (8) to (10). These may be used alone or in combination of two or more. The weight-average molecular weight of the thiol-terminated polyoxazoline is preferably 500 to 100,000 daltons.

[0064] [ka] In the general formula (8), n represents the number of repeating monomer units, and R1 represents a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0065] [ka] In the general formula (9), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R1 represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0066] [ka] In the general formula (10), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R1 represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0067] The structures of the repeating units in the thiol-terminated polyoxazoline may be the same or different.

[0068] The thiol-terminated polyoxazoline can be used as a crosslinking agent for adding polyoxazoline to a compound having a maleimide group at the end (for example, a protein modified with a maleimide group).

[0069] The thiol-terminated polyoxazoline can be obtained, for example, by reacting polyoxazoline with a thiol group-introducing agent such as the compound of general formula (7) above (e.g., by stirring at 25°C for 1 to 96 hours). The mixing ratio may be 1 mole of polyoxazoline to 2 to 20 moles of the thiol group-introducing agent such as the compound of general formula (7) above. After the reaction, the product may be treated with a reducing agent or purified by centrifugation, filter filtration, gel filtration, or the like. The resulting thiol-terminated polyoxazoline is 1 The structure can be analyzed by H-NMR or the like.

[0070] -Maleimide-terminated polyoxazoline- The maleimide-terminated polyoxazoline is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include compounds represented by the following general formulas (11) to (13). These may be used alone or in combination of two or more. The weight-average molecular weight of the maleimide-terminated polyoxazoline is preferably 500 to 100,000 daltons.

[0071] [ka] In the general formula (11), n ​​represents the number of repeating monomer units, and m represents an integer of 1 to 10. R1 represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0072] [ka] In the general formula (12), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R1 represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0073] [ka] In the general formula (13), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R1 represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0074] The structures of the repeating units in the maleimide-terminated polyoxazoline may be the same or different.

[0075] The maleimide-terminated polyoxazoline can be used as a crosslinking agent for adding polyoxazoline to a compound having an SH group at its terminal (for example, a cysteine ​​residue in a protein, a protein modified with a thiol group, etc.).

[0076] The maleimide-terminated polyoxazoline can be obtained, for example, by reacting polyoxazoline with a maleimide-group-introducing agent such as the compound of general formula (3) above (for example, if R4 is Cl, the reaction is carried out as is, or if R4 is OH, a condensing agent is added and the reaction is stirred at 25°C for 1 hour to 96 hours). The mixing ratio may be 2 to 20 moles of the maleimide-group-introducing agent such as the compound of general formula (3) above per mole of polyoxazoline. After the reaction, the product may be purified by centrifugation, filter filtration, gel filtration, or the like. The resulting maleimide-terminated polyoxazoline is 1 The structure can be analyzed by H-NMR or the like.

[0077] In the general formulas (8) to (13), those in which R1 is a methyl group tend to exhibit higher water solubility than PEG, while those in which R1 is a propyl group tend to exhibit lower water solubility upon heating. Therefore, those in which R1 is an ethyl group are preferred in that they have a good balance between hydrophilicity and hydrophobicity.

[0078] In all of the general formulas (8) to (13), m is preferably the minimum value of 2, which has high chemical stability.

[0079] More specifically, examples of methods for producing the polyoxazoline-conjugated albumin include the following methods (a) to (c).

[0080] -Production Example of Polyoxazoline-Conjugated Albumin (a)- By reacting the thiol-terminated polyoxazoline with the maleimide-group-introduced albumin, the thiol group in the thiol-terminated polyoxazoline forms a covalent bond with the maleimide group in the maleimide-group-introduced albumin. Examples of methods for introducing the polyoxazoline include stirring maleimide group-introduced albumin and thiol-terminated polyoxazoline at 0° C. to 30° C. for 1 hour to 72 hours.

[0081] -Production Example of Polyoxazoline-Conjugated Albumin (b)- By reacting the maleimide-terminated polyoxazoline with the thiol group-introduced albumin, the maleimide group in the maleimide-terminated polyoxazoline forms a covalent bond with the thiol group of the thiol group-introduced albumin. Examples of methods for introducing the polyoxazoline include stirring the thiol group-introduced albumin and the terminal maleimide group polyoxazoline at 0°C to 30°C for 1 hour to 72 hours.

[0082] -Production example of polyoxazoline-bound albumin (c)- By reacting the maleimide-terminated polyoxazoline with the unmodified albumin, the maleimide group in the maleimide-terminated polyoxazoline forms a covalent bond with a cysteine ​​in the unmodified albumin. Examples of methods for introducing the maleimide-terminated polyoxazoline include stirring unmodified albumin and the maleimide-terminated polyoxazoline at 0° C. to 30° C. for 1 hour to 72 hours.

[0083] <Characteristics of polyoxazoline-bound albumin> In the polyoxazoline-conjugated albumin of this embodiment, the binding site of the albumin to the crosslinking agent is preferably lysine, a primary amine at the protein terminal, or cysteine. In the polyoxazoline-conjugated albumin of this embodiment, the binding site of the polyoxazoline with the crosslinking agent is preferably the terminal hydroxyl group or amino group of the polyoxazoline represented by the general formula (1) above. The binding site of the maleimide group-introduced albumin with the terminal thiol group-containing polyoxazoline is preferably an introduced maleimide group. The binding site of the thiol group-introduced albumin with the terminal maleimide group-containing polyoxazoline is preferably an introduced thiol group. The binding site of the unmodified albumin with the terminal maleimide group-containing polyoxazoline is preferably a cysteine ​​residue.

[0084] In the polyoxazoline-conjugated albumin of the present embodiment, the bond via the crosslinking agent preferably includes a structure derived from a maleimide group-introducing agent and / or a thiol group-introducing agent, and more preferably a structure derived only from a maleimide group-introducing agent and a thiol group-introducing agent. The bond via the crosslinker is formed by the following structure (1): [ka] and preferably comprises the following structure (2), structure (3) or structure (4): [ka] [In the structure (2), R1 represents either the general formula (4), the general formula (5), or the chemical formula (1), or the chemical formula (2).] [ka] [ka] [In structures (3) and (4), m represents an integer of 1 to 10.] More preferably, the compound has the following structure (5) or (6): [ka] [ka] [In Structure (5) and Structure (6), R1 represents either the general formula (4), general formula (5) or the chemical formula (1) or chemical formula (2), and m represents an integer of 1 to 10.] It is more preferable that the compound has the structure:

[0085] The polyoxazoline-conjugated albumin of this embodiment has a clear three-dimensional structure despite being easy to synthesize, etc. The average particle size of the polyoxazoline-conjugated albumin is preferably 8 to 30 nm, more preferably 10 to 20 nm.

[0086] The number of polyoxazolines bound to the core albumin in the polyoxazoline-bound albumin of this embodiment is preferably 1 to 10. In the polyoxazoline-conjugated albumin of this embodiment, the number of polyoxazolines bound to core albumin can be measured by measuring the dry weight of the polyoxazoline-conjugated albumin.

[0087] The polyoxazoline-conjugated albumin of this embodiment has a higher colloid osmotic pressure than unmodified albumin, and when administered to a living body, is more effective in maintaining circulating blood volume than unmodified albumin at the same concentration.

[0088] The polyoxazoline-conjugated albumin of this embodiment does not exhibit immunogenicity even when administered to animals of different species, since the core albumin is surrounded by polyoxazoline.

[0089] The polyoxazoline-conjugated albumin of this embodiment does not cause precipitation or aggregation even when mixed with blood, and is highly compatible with blood.

[0090] When administered to a living body, the polyoxazoline-conjugated albumin of the present embodiment does not undergo renal excretion or leakage from vascular endothelial cells, and therefore has a longer blood retention time than unmodified albumin. Furthermore, polyoxazoline is highly water-soluble and easily metabolized.

[0091] When the polyoxazoline-conjugated albumin of this embodiment is administered to a living body in a state of hemorrhagic shock, it restores circulating blood volume and improves blood pressure.

[0092] From the above, the polyoxazoline-conjugated albumin of the present invention can function as an unprecedented artificial plasma expander and resuscitation fluid for hemorrhagic shock, which is both biocompatible (safe) and effective.

[0093] <Artificial plasma expander> The artificial plasma expander of this embodiment contains the polyoxazoline-conjugated albumin of the above embodiment. The artificial plasma expander is a substance having colloid osmotic pressure, and when administered to a living body, functions as a substitute for the animal's own albumin. The artificial plasma expander can be used as a substitute for albumin in vertebrates such as humans, pigs, cows, horses, dogs, cats, monkeys, and rabbits.

[0094] <Hemorrhagic Shock Resuscitation Fluid> The resuscitation solution for hemorrhagic shock of this embodiment contains the polyoxazoline-conjugated albumin of the above embodiment. The resuscitation solution for hemorrhagic shock is a substance having colloid osmotic pressure, and when administered to a living body, functions as a substitute for the animal's own albumin. The resuscitation fluid for hemorrhagic shock can be used as a substitute for albumin in vertebrates such as humans, pigs, cows, horses, dogs, cats, monkeys, and rabbits. [Example]

[0095] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

[0096] Example 1 -Preparation Example 1: Preparation of maleimide group-introduced porcine albumin (PSA-M)- To introduce a maleimide group into porcine albumin (PSA), the following procedure was carried out. 119.8 mg of N-succinimidyl 3-maleimidopropionate (SMP, Fujifilm Wako Pure Chemical Industries, Ltd.) was placed in an 8 mL sample bottle and dissolved in 3 mL of dimethyl sulfoxide to prepare a 0.15 M SMP solution. Next, 30 mL of porcine albumin (1 mM) was placed in a 100 mL single-necked flask, and 3 mL of SMP solution (N-succinimidyl 3-maleimidopropionate / albumin (SMP / PSA) = 15 (mol / mol)) was added and stirred at 25 °C for 1 hour. After filtering through a filter (Merck Millipore, Millex-GP, 0.22 μm, PES), the solution was applied to a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with phosphate-buffered saline (PBS, pH 7.4) to remove excess N-succinimidyl 3-maleimidopropionate. PBS was added to the resulting solution to make the total volume 90 mL.

[0097] Preparation Example 2: Preparation of thiol-terminated polyoxazoline (weight average molecular weight 5,000 Da, POx(5k)-eSH) The following procedure was carried out to synthesize thiol-terminated polyoxazoline (weight average molecular weight 5,000 Da, POx(5k)-eSH). [ka] In chemical reaction formula (1), n ​​represents the number of repeating monomer units. A three-necked recovery flask (300 mL capacity) was charged with 5 g of hydroxyl-terminated poly(2-ethyl-2-oxazoline) (weight-average molecular weight 5,000 Da, POx(5k)-OH, Sigma-Aldrich), 1.26 g of 3,3'-dithiodipropionic acid (DTDPA, Tokyo Chemical Industry Co., Ltd.), 1.26 g of N,N'-dicyclohexylcarbodiimide (DCC, Tokyo Chemical Industry Co., Ltd.), and 160 mg of 4-dimethylaminopyridine (DMAP, Tokyo Chemical Industry Co., Ltd.). After aerating with nitrogen, 60 mL of tetrahydrofuran (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and the mixture was stirred at 25°C for 72 hours. The solvent in the reaction solution was removed using a rotary evaporator (EYELA), and the white solid was dried using a vacuum pump. 50 mL of pure water was added, and the precipitate was removed by centrifugation. The pH was adjusted to 7 using a 5 M NaOH solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and then 1.85 g of dithiothreitol (DTT, Fujifilm Wako Pure Chemical Industries, Ltd.) was added. Nitrogen was bubbled through the solution for 5 minutes, and the mixture was stirred at 25°C for 2 hours (dithiothreitol / polyoxazoline (DTT / POx(5k)- O H) = 12 (mol / mol). The solution was filtered through a filter (Merck Millipore, Millex-GP, 0.22 μm, PES) and dialyzed using a dialysis membrane (molecular weight cutoff 3500 Da, Spectrum Laboratories) to remove unreacted substances. The resulting aqueous solution was frozen with liquid nitrogen and then freeze-dried under vacuum. 1 The structure was identified by H NMR. The thiol concentration in the resulting polyoxazoline solution was quantified using the exchange reaction of thiol groups and disulfide bonds. 4,4'-Dithiopyridine (4,4'-DTP) reacts with free thiol (SH) groups to produce 4-thiopyridinone (4-TP), which allows the introduction of thiol groups. PolyoxazolineThe amount of thiol groups can be quantified by adding 4,4'-dithiopyridine (4,4'-DTP) to the solution and measuring the amount of 4-thiopyridinone (4-TP) produced. The introduction rate of thiol groups in the thiol-terminated polyoxazoline (POx(5k)-eSH) was calculated from the obtained concentration and found to be approximately 96%.

[0098] Preparation Example 3: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-eSM-PSA) To prepare polyoxazoline-bound albumin (POx(5k)-eSM-PSA) bound to polyoxazoline (weight-average molecular weight 5,000 Da), the following procedure was carried out. In a single-neck flask (300 mL capacity) was placed 90 mL of the maleimide group-introduced porcine albumin solution (PSA-M, 333 μM) obtained in Preparation Example 1, and 60 mL of a PBS solution (3.75 mM) of the thiol-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eSH) obtained in Preparation Example 2 was added, followed by stirring at 25°C for 24 hours (thiol-terminated polyoxazoline / maleimide group-introduced albumin (POx(5k)-eSH / PSA-M) = 7.5 (mol / mol)). The reaction mixture was subjected to circulating ultrafiltration (Merck, Pelicon XL casette, ultramolecular weight 100 kDa) to remove unreacted polyoxazoline. The dry weight of polyoxazoline-bound albumin was measured, and the number of polyoxazoline bound to core albumin was calculated to be approximately 6 per PSA.

[0099] Example 2 Preparation Example 1: Preparation of thiol-terminated polyoxazoline (weight average molecular weight 5,000 Da, POx(5k)-aSH) To synthesize thiol-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-aSH), the following procedure was carried out. [ka] In chemical reaction formula (2), n represents the number of repeating monomer units. A two-necked recovery flask (100 mL capacity) was charged with 200 mg of amino-terminated poly(2-ethyl-2-oxazoline) (weight-average molecular weight 5,000 Da, POx(5k)-NH2, Sigma-Aldrich) and 124 mg of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Tokyo Chemical Industry Co., Ltd.), and nitrogen was purged (N-succinimidyl 3-(2-pyridyldithio)propionate / polyoxazoline (SPDP / POx(5k)-NH2) = 10 mol / mol). 20 mL of dichloromethane (Fujifilm Wako Pure Chemicals) was added and the mixture was stirred at 25°C for 15 hours. The solvent was removed from the reaction solution using a rotary evaporator (EYELA). 12 mL of purified water and 124 mg of dithiothreitol (DTT, Fujifilm Wako Pure Chemicals) were added and the mixture was stirred at 25°C for 2 hours (dithiothreitol / polyoxazoline (DTT / POx(5k)-NH2) = 20 (mol / mol)). After removing the precipitate by centrifugation, the supernatant was filtered through a filter (Merck Millipore, Millex-GP, 0.22 μm, PES) and applied to a gel filtration column (GE Healthcare Japan, PD-10) equilibrated with purified water to remove unreacted material. The resulting aqueous solution was frozen with liquid nitrogen and then freeze-dried under vacuum. 1 The structure was identified by H NMR. The thiol concentration in the resulting polyoxazoline solution was quantified using the exchange reaction of thiol groups and disulfide bonds, and the introduction rate of thiol groups in the terminal thiol polyoxazoline (POx(5k)-aSH) was calculated to be approximately 95%.

[0100] Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-aSM-PSA) To prepare polyoxazoline-bound albumin (POx(5k)-aSM-PSA) bound to polyoxazoline (weight-average molecular weight 5,000 Da), the following procedure was carried out. A single-neck flask (30 mL capacity) was charged with 9.0 mL of the maleimide group-introduced albumin solution (PSA-M, 333 μM) obtained in Preparation Example 1 of Example 1, and 6.0 mL of a PBS solution (3.75 mM) of the thiol-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-aSH) obtained in Preparation Example 1 of Example 2 was added thereto, followed by stirring at 25° C. for 24 hours (thiol-terminated polyoxazoline / maleimide group-introduced albumin (POx(5k)-aSH / PSA-M)=7.5 (mol / mol)). The reaction mixture was subjected to circulating ultrafiltration (Merck, Pelicon XL casette, ultramolecular weight 100 kDa) to remove unreacted polyoxazoline. The dry weight of polyoxazoline-bound albumin was measured, and the number of polyoxazoline bound to core albumin was calculated to be approximately 6 per PSA.

[0101] Example 3 Preparation Example 1: Preparation of thiol-terminated polyoxazoline (weight average molecular weight 10,000 Da, POx(10k)-eSH) A thiol-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eSH) was prepared in the same manner as in Preparation Example 2 in Example 1, except that a hydroxy-terminated poly(2-ethyl-2-oxazoline) (weight-average molecular weight 10,000 Da, POx(10k)-OH) was used instead of a hydroxy-terminated poly(2-ethyl-2-oxazoline) (weight-average molecular weight 5,000 Da, POx(5k)-OH) in Preparation Example 2 in Example 1. The thiol concentration in the resulting polyoxazoline solution was quantified using an exchange reaction between the thiol group and a disulfide bond, and the introduction rate of the thiol group in the thiol-terminated polyoxazoline (POx(10k)-eSH) was calculated to be approximately 95%.

[0102] Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 10,000 Da)-bound albumin (POx(10k)-eSM-PSA) Polyoxazoline-conjugated albumin (POx(10k)-eSM-PSA) was prepared in the same manner as in Preparation Example 3 in Example 1, except that the thiol-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eSH) obtained in Preparation Example 1 in Example 3 was used instead of the thiol-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eSH) in Preparation Example 3 in Example 1. The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazolines bound to core albumin, which was approximately 6 bonds / PSA.

[0103] Example 4 -Preparation Example 1: Preparation of maleimide group-introduced human albumin (HSA-M)- Maleimide group-introduced human albumin (HSA-M) was prepared in the same manner as in Preparation Example 1 in Example 1, except that human albumin was used instead of porcine albumin.

[0104] Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-eSM-HSA) Polyoxazoline-conjugated albumin (POx(5k)-eSM-HSA) was prepared in the same manner as in Preparation Example 3 in Example 1, except that the maleimide-conjugated human albumin (HSA-M) obtained in Preparation Example 1 in Example 4 was used instead of the maleimide-conjugated porcine albumin (PSA-M) in Preparation Example 3 in Example 1. The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazolines conjugated to core albumin, which was approximately 6 per HSA.

[0105] Example 5 Preparation Example 1: Preparation of polyoxazoline (weight-average molecular weight 10,000 Da)-bound albumin (POx(10k)-eSM-HSA) Polyoxazoline-conjugated albumin (POx(10k)-eSM-HSA) was prepared in the same manner as in Preparation Example 3 in Example 1, except that the thiol-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eSH) obtained in Preparation Example 1 in Example 3 was used instead of the thiol-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eSH) in Preparation Example 3 in Example 1, and the maleimide-conjugated human albumin (HSA-M) obtained in Preparation Example 1 in Example 4 was used instead of the maleimide-conjugated porcine albumin (PSA-M). The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazoline bonds relative to core albumin, which was approximately 6 bonds / HSA.

[0106] Example 6 -Preparation Example 1: Preparation of maleimide group-introduced bovine albumin (BSA-M)- Maleimide group-introduced bovine albumin (BSA-M) was prepared in the same manner as in Preparation Example 1 in Example 1, except that bovine albumin was used instead of porcine albumin.

[0107] Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-eSM-BSA) Polyoxazoline-conjugated albumin (POx(5k)-eSM-BSA) was prepared in the same manner as in Preparation Example 3 in Example 1, except that the maleimide-conjugated bovine albumin (BSA-M) obtained in Preparation Example 1 in Example 6 was used instead of the maleimide-conjugated porcine albumin (PSA-M) in Preparation Example 3 in Example 1. The number of polyoxazoline bonds relative to core albumin was calculated by measuring the dry weight of the polyoxazoline-conjugated albumin, and was found to be approximately 6 bonds / BSA.

[0108] Example 7 Preparation Example 1: Preparation of polyoxazoline (weight average molecular weight 10,000 Da)-bound albumin (POx(10k)-eSM-BSA) Polyoxazoline-conjugated albumin (POx(10k)-eSM-BSA) was prepared in the same manner as in Preparation Example 3 in Example 1, except that the thiol-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eSH) obtained in Preparation Example 1 in Example 3 was used instead of the thiol-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eSH) in Preparation Example 3 in Example 1, and the maleimide-conjugated bovine albumin (BSA-M) obtained in Preparation Example 1 in Example 6 was used instead of the maleimide-conjugated porcine albumin (PSA-M). The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazoline bonds relative to core albumin, which was approximately 6 bonds per BSA.

[0109] Example 8 -Preparation Example 1: Preparation of maleimide group-introduced porcine albumin (PSA-MC)- Maleimide group-introduced porcine albumin (PSA-MC) was prepared in the same manner as in Preparation Example 1 in Example 1, except that N-succinimidyl 4-(N-Maleimidomethyl)cyclohexanecarboxylate (SMCC, Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead of N-succinimidyl 3-maleimidopropionate (SMP, Fujifilm Wako Pure Chemical Industries, Ltd.).

[0110] Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-eSMC-PSA) Polyoxazoline-conjugated albumin (POx(5k)-eSMC-PSA) was prepared in the same manner as in Preparation Example 3 in Example 1, except that the maleimide-conjugated porcine albumin (PSA-MC) obtained in Preparation Example 1 in Example 8 was used instead of the maleimide-conjugated porcine albumin (PSA-M) in Preparation Example 3 in Example 1. The number of polyoxazoline bonds relative to core albumin was calculated by measuring the dry weight of the polyoxazoline-conjugated albumin, and was found to be approximately 6 bonds / PSA.

[0111] Example 9 Preparation Example 1: Preparation of polyoxazoline (weight average molecular weight 10,000 Da)-bound albumin (POx(10k)-eSMC-PSA) A polyoxazoline-conjugated albumin (POx(10k)-eSMC-PSA) was prepared in the same manner as in Preparation Example 3 in Example 1, except that the thiol-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eSH) obtained in Preparation Example 1 in Example 3 was used instead of the thiol-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eSH) in Preparation Example 3 in Example 1, and the maleimide-conjugated porcine albumin (PSA-MC) obtained in Preparation Example 1 in Example 8 was used instead of the maleimide-conjugated porcine albumin (PSA-M). The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazoline bonds relative to core albumin, which was approximately 6 bonds / PSA.

[0112] Example 10 -Preparation Example 1: Preparation of thiol group-introduced porcine albumin (PSA-SH)- To introduce thiol groups into porcine albumin, the following procedure was carried out. A 0.1 M 2-iminothiolane solution was prepared by adding 13.8 mg of 2-iminothiolane hydrochloride (2-IT, Fujifilm Wako Pure Chemical Industries, Ltd.) to a 1.5 mL microtube and diluting with 1 mL of phosphate-buffered saline (PBS, pH 7.4). Next, 1 mL of 1 mM porcine albumin was added to a 10 mL single-neck flask, and 400 μL of the 2-iminothiolane solution (2-iminothiolane / albumin (2-IT / PSA) = 40 (mol / mol)) was added. The mixture was stirred at 25 °C for 3 hours. The resulting solution was applied to a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with phosphate-buffered saline (PBS, pH 7.4) to remove excess 2-iminothiolane (2IT). 20 mL of the resulting solution was placed in a centrifugal concentrator (Merck, Amicon Ultra-15, ultramolecular weight 10 kDa) and centrifuged to concentrate it to 2.5 mL (0.4 mM).

[0113] -Preparation Example 2: Preparation of maleimide-terminated polyoxazoline (weight average molecular weight 5,000 Da, POx(5k)-eM)- The following procedure was carried out to synthesize maleimide-terminated polyoxazoline (weight average molecular weight 5,000 Da, POx(5k)-eM). [ka] In chemical reaction formula (3), n represents the number of repeating monomer units. A two-necked recovery flask (50 mL capacity) was charged with 120 mg of hydroxyl-terminated poly(2-ethyl-2-oxazoline) (weight-average molecular weight 5,000 Da, POx(5k)-OH, Sigma-Aldrich) and 45 mg of 3-maleimidopropanoyl chloride (MPC) prepared by reacting 3-maleimidopropionic acid with thionyl chloride, and nitrogen was purged (3-maleimidopropanoyl chloride / polyoxazoline (MPC / POx(5k)-OH) = 10 (mol / mol)). 5 mL of dichloromethane (Fujifilm Wako Pure Chemical Industries, Ltd.) and 66 μL of triethylamine (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the mixture, which was then stirred for 18 hours at 25°C. The solvent was removed from the reaction solution using a rotary evaporator (EYELA Corporation), 3 mL of purified water was added, and the mixture was stirred thoroughly. After centrifugation to remove the precipitate, the supernatant was filtered through a filter (Merck Millipore, Millex-GP, 0.22 μm, PES) and purified using a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with purified water. The resulting aqueous solution was frozen with liquid nitrogen and then freeze-dried under vacuum. 1 The structure was identified by 1 H NMR.

[0114] -Preparation Example 3: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-eMS-PSA)- To prepare polyoxazoline-conjugated albumin (POx(5k)-eMS-PSA) bound to polyoxazoline (weight-average molecular weight 5,000 Da), the following procedure was carried out. A single-neck flask (5 mL capacity) was charged with 625 μL of the thiol group-introduced albumin solution (PSA-SH, 0.4 mM) obtained in Preparation Example 1, and 12.5 mg of the maleimide-terminated polyoxazoline obtained in Preparation Example 2 (weight-average molecular weight 5,000 Da, POx(5k)-eM) was added thereto, followed by stirring at 25° C. for 14 hours (maleimide-terminated polyoxazoline / thiol group-introduced albumin (POx(5k)-eM / PSA-SH)=10 (mol / mol)). The reaction mixture was applied to a gel filtration column (GE Healthcare Japan, Superdex 200 pg) equilibrated with phosphate-buffered saline (PBS, pH 7.4) to remove unreacted polyoxazoline. The dry weight of polyoxazoline-conjugated albumin was measured, and the number of polyoxazoline conjugated to core albumin was calculated to be approximately 6 conjugates per PSA.

[0115] Example 11 -Preparation Example 1: Preparation of maleimide-terminated polyoxazoline (weight average molecular weight 5,000 Da, POx(5k)-aM)- To synthesize maleimide-terminated polyoxazoline (weight average molecular weight 5,000 Da, POx(5k)-aM), the following procedure was carried out. [ka] In chemical reaction formula (4), n represents the number of repeating monomer units. 50 mg of amino-terminated poly(2-ethyl-2-oxazoline) (weight-average molecular weight 5,000 Da, POx(5k)-NH2, Sigma-Aldrich) and 26.5 mg of N-succinimidyl 3-maleimidopropionate (SMP, Fujifilm Wako Pure Chemical Industries) were placed in a two-necked recovery flask (50 mL capacity), and nitrogen was purged (N-succinimidyl 3-maleimidopropionate / polyoxazoline (SMP / POx(5k)-NH2) = 10 (mol / mol)). Five mL of dichloromethane (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the mixture, which was then stirred for 18 hours at 25°C. The solvent was removed from the reaction solution using a rotary evaporator (EYELA), and 2 mL of purified water was added. The mixture was thoroughly stirred and centrifuged to remove the precipitate. The supernatant was then filtered through a filter (Merck Millipore, Millex-GP, 0.22 μm, PES) and applied to a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with purified water to remove unreacted N-succinimidyl 3-maleimidopropionate (SMP). The resulting aqueous solution was frozen with liquid nitrogen and then freeze-dried under vacuum. 1 The structure was identified by 1 H NMR.

[0116] -Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-aMS-PSA)- Polyoxazoline (weight-average molecular weight 5,000 Da)-conjugated albumin (POx(5k)-aMS-PSA) was prepared in the same manner as in Preparation Example 3 in Example 10, except that the maleimide-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eM) obtained in Preparation Example 1 in Example 11 was used instead. The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazoline bonds relative to core albumin, which was approximately 6 bonds / PSA.

[0117] Example 12 Preparation Example 1: Preparation of maleimide-terminated polyoxazoline (weight average molecular weight 10,000 Da, POx(10k)-eM) A maleimide-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eM) was prepared in the same manner as in Preparation Example 2 in Example 10, except that poly(2-ethyl-2-oxazoline) (weight-average molecular weight 10,000 Da, POx(10k)-OH) having a hydroxy group at its terminal was used instead of poly(2-ethyl-2-oxazoline) (weight-average molecular weight 5,000 Da, POx(5k)-OH) having a hydroxy group at its terminal in Preparation Example 2 in Example 10.

[0118] -Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 10,000 Da)-bound albumin (POx(10k)-eMS-PSA)- Polyoxazoline-conjugated albumin (POx(10k)-eMS-PSA) was prepared in the same manner as in Preparation Example 3 in Example 10, except that the maleimide-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eM) obtained in Preparation Example 1 in Example 12 was used instead of the maleimide-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eM) in Preparation Example 3 in Example 10. The number of polyoxazoline bonds relative to core albumin was calculated by measuring the dry weight of the polyoxazoline-conjugated albumin, and was found to be approximately 6 bonds / PSA.

[0119] Example 13 -Preparation Example 1: Preparation of thiol group-introduced human albumin (HSA-M)- Thiol group-introduced human albumin (HSA-SH) was prepared in the same manner as in Preparation Example 1 in Example 10, except that human albumin was used instead of porcine albumin.

[0120] -Preparation Example 2: Preparation of polyoxazoline (weight average molecular weight 5,000 Da)-bound albumin (POx(5k)-eMS-HSA)- Polyoxazoline-conjugated albumin (POx(5k)-eMS-HSA) was prepared in the same manner as in Preparation Example 3 in Example 10, except that the thiol group-introduced human albumin (HSA-SH) obtained in Preparation Example 1 in Example 13 was used instead of the thiol group-introduced porcine albumin (PSA-SH) in Preparation Example 3 in Example 10. The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazolines bound to core albumin, which was approximately 6 per HSA.

[0121] Example 14 -Preparation Example 1: Preparation of polyoxazoline (weight average molecular weight 10,000 Da)-bound albumin (POx(10k)-eMS-HSA)- Polyoxazoline-conjugated albumin (POx(10k)-eMS-HSA) was prepared in the same manner as in Preparation Example 3 in Example 10, except that the maleimide-terminated polyoxazoline (weight-average molecular weight 10,000 Da, POx(10k)-eM) obtained in Preparation Example 1 in Example 12 was used instead of the maleimide-terminated polyoxazoline (weight-average molecular weight 5,000 Da, POx(5k)-eM) in Preparation Example 3 in Example 10, and the thiol group-introduced human albumin (HSA-SH) obtained in Preparation Example 1 in Example 13 was used instead of the thiol group-introduced porcine albumin (PSA-SH). The dry weight of the polyoxazoline-conjugated albumin was measured to calculate the number of polyoxazoline bonds relative to core albumin, which was approximately 6 bonds / HSA.

[0122] The summary of Examples 1 to 14 is shown in Table 1. [Table 1]

[0123] As shown in Table 1, it is possible to prepare polyoxazoline-conjugated albumin from various albumin derivatives and polyoxazoline derivatives.

[0124] Example 15 -Dynamic Light Scattering (DLS) Measurement- Dynamic light scattering (DLS) measurements of a solution of polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) obtained in Preparation Example 3 in Example 1 in phosphate-buffered saline (PBS, pH 7.4) were performed using a zeta potential, particle size, and molecular weight measurement system (Otsuka Electronics Co., Ltd., ELSZ-2000). Unmodified porcine albumin (PSA) was also tested in the same manner. The average particle size of unmodified porcine albumin (PSA) was 8 nm. The average particle size of polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) was 13 nm. It was found that binding of polyoxazoline to albumin increased the molecular size.

[0125] Example 16 -Collagen osmotic pressure measurement- The colloid osmotic pressure of a solution of polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) obtained in Preparation Example 3 in Example 1 in phosphate buffered saline (PBS, pH 7.4, [PSA] = 5 g / dL) was measured using a colloid osmometer (OSMOMAT 050, Gomotec). Similarly, polyoxazoline-conjugated albumin (POx(10k)-eSM-PSA) obtained in Preparation Example 2 in Example 3 and unmodified porcine albumin (PSA) were also tested. The oncotic pressure of unmodified porcine albumin (PSA, 5 g / dL) was 18 mmHg. The oncotic pressure of polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) was 36 mmHg. The oncotic pressure of polyoxazoline-conjugated albumin (POx(10k)-eSM-PSA) was 62 mmHg. It was found that the oncotic pressure increased when polyoxazoline was bound to albumin.

[0126] Example 17 Preparation Example 1: Preparation of polyethylene glycol (weight average molecular weight 5,000 Da)-bound albumin (PEG(5k)-eSM-PSA) To prepare polyethylene glycol-conjugated albumin (PEG(5k)-eSM-PSA) bound with polyethylene glycol (weight-average molecular weight 5,000 Da), the following procedure was carried out. A single-neck flask (5 mL capacity) was charged with 1.7 mL of the maleimide group-introduced albumin solution (PSA-M, 333 μM) obtained in Preparation Example 1 of Example 1, and 28 mg of polyethylene glycol (weight-average molecular weight 5,000 Da, PEG(5k)-SH, NOF Corporation) with a terminal thiol group was added thereto, followed by stirring at 25° C. for 24 hours (thiol group-introduced polyethylene glycol / maleimide group-introduced albumin (PEG(5k)-SH / PSA-M)=10 (mol / mol)). The reaction mixture was subjected to circulating ultrafiltration (Merck, Pelicon XL casette, ultramolecular weight 100 kDa) to remove unreacted polyethylene glycol. The dry weight of polyethylene glycol-conjugated albumin was measured to calculate the number of polyethylene glycol bonds to core albumin, which was approximately 8 bonds / PSA.

[0127] -Immunogenicity test using rats- A solution of albumin (PSA) in phosphate-buffered saline (PBS, pH 7.4, [PSA] = 5 g / dL), a solution of polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) obtained in Preparation Example 3 of Example 1 in phosphate-buffered saline (PBS, pH 7.4, [PSA] = 5 g / dL), and a solution of polyethylene glycol-conjugated albumin (PEG(5k)-eSM-PSA) obtained in Preparation Example 1 of Example 17 in phosphate-buffered saline (PBS, pH 7.4, [PSA] = 5 g / dL) were administered via the tail vein of male Wistar rats (7 weeks old, approximately 180 g) (200 mg-PSA / kg-rat). After 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days, 100 μL of blood was collected from the tail vein, centrifuged, and the resulting supernatant was stored at −80°C. After 28 days, each supernatant was subjected to indirect ELISA to quantify the amount of IgM antibody produced against PSA. In the albumin (PSA) administration group, the production of anti-PSA IgM antibodies was observed, peaking 4 days after administration. In the polyethylene glycol-conjugated albumin (PEG(5k)-eSM-PSA) administration group, the production of anti-PSA IgM antibodies was observed to be at the same level as in the albumin (PSA) administration group. In contrast, the production of anti-PSA IgM antibodies in the polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) administration group was significantly lower (see Figure 2). It was clear that polyoxazoline conjugation exhibits excellent immunological stealth properties.

[0128] Example 18 -Blood compatibility test- Blood was collected from a Wistar rat (male, 7 weeks old, approximately 230 g, Charles River) using an EDTA-containing vacuum blood collection tube and thoroughly mixed with EDTA by inversion. The collected blood was mixed with a solution of polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) obtained in Preparation Example 3 in Example 1 in phosphate-buffered saline (PBS, pH 7.4, [PSA] = 5 g / dL) so that the volume ratio of the polyoxazoline-conjugated albumin solution was 0, 10, 20, or 40% (total volume: 600 μL). The sample was placed in a thermostatic chamber at 37°C, and 50 μL of each aliquot was taken at 0 (immediately after mixing), 1, 2, 3, 4, 5, and 6 hours, and the red blood cell (RBC), white blood cell (WBC), and platelet (PLT) counts were measured using a multiparameter hematology analyzer (pocH-100iV Diff, Sysmex) (n=3). When the mixing ratio of polyoxazoline-conjugated albumin solution was 10, 20, and 40%, the blood cell counts were 90, 80, and 60% of the blood cell counts (reference values) in blood without polyoxazoline-conjugated albumin solution (see Figure 3). The blood cell counts did not change up to 6 hours later, demonstrating the high blood compatibility of polyoxazoline-conjugated albumin.

[0129] Example 19 -Blood retention measurement in rats- A male, 7-week-old, approximately 230 g, Charles River Wistar rat was anesthetized with sevoflurane (Maruishi Pharmaceutical) (5.0% in air) and then secured in a supine position on a heating pad (DC Temperature controller, Brain Science Idea) under inhalation anesthesia with sevoflurane (3.0-4.0% in air). A catheter (SP-31) was inserted approximately 3 cm into the right jugular vein, with the tip positioned into the right atrium. The opposite end of the catheter was tunneled subcutaneously and secured to the skin on the back. A solution of Cy5.5-fluorescently labeled polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) obtained in Preparation Example 3 of Example 1 in phosphate-buffered saline (PBS, pH 7.4, [PSA] = 5 g / dL) was administered (5% topload) via the right jugular vein (the dose was 5% of the rat's circulating blood volume (56 mL / kg) (140 mg / kg rat), fluorescently labeled:non-fluorescent labeled = 1:9). Three minutes after administration was designated time 0, and 200 μL of blood was collected from the right jugular vein at 0, 5, 15, 30, 1, 3, 6, 12, 18, and 24 hours after administration. The collected serum (100 μL) was centrifuged (6,000 rpm, 5 minutes) and stored in a refrigerator protected from light. 20 μL of serum components were mixed with 12 μL of TritonX-100 PB solution and 28 μL of PBS solution (serum components: 3-fold diluted, TritonX-100: 1% (w / v)), and left overnight in the dark under refrigeration. This solution was placed in a 3 mm microquartz cell (minimum sample volume: 50 μL), and the fluorescence spectrum of polyoxazoline-conjugated albumin fluorescently labeled with Cy5.5 was measured using a JASCO FP-8300 fluorescence spectrophotometer. The fluorescence intensity at 710 nm of the serum collected at 0 minutes was set to 100%, and the half-life of polyoxazoline-conjugated albumin disappearance in blood (t 1 / 2 ) was calculated. Similarly, polyoxazoline-conjugated albumin (POx(10k)-eSM-PSA) obtained in Preparation Example 2 in Example 3 and unmodified porcine serum albumin (PSA) were also tested. The elimination half-life (t 1 / 2 The blood elimination half-life (t 1 / 2The blood elimination half-life (t 1 / 2 The blood elimination half-life (t 1 / 2 ) was found to be extended.

[0130] Example 20 -Efficacy evaluation in a rat 50% hemorrhagic shock model- A male, 7-week-old, approximately 230 g, Charles River rat was anesthetized with sevoflurane (Maruishi Pharmaceutical) (5.0% in air) and placed in a supine position on a heating pad (DC Temperature Controller, Brain Science Idea) under inhalation anesthesia with sevoflurane (3.0% in air). A catheter (SP-31, inner diameter: 0.5 mm, outer diameter: 0.8 mm, Natsume Seisakusho) was inserted centrally into the right carotid artery for blood pressure measurement and blood withdrawal, and the opposite end was connected to a blood pressure measurement device (PAS-101, Star Medical). The same catheter was inserted into the right jugular vein for sample administration. A tracheal cannula was inserted, and respiratory management was performed using an artificial ventilator. Hemorrhagic shock was induced by withdrawing 50% of the total blood volume (56 mL / kg) through the arterial catheter at a rate of 1 mL / min. After 15 minutes, the animals were resuscitated by administering (1 mL / min) via the venous catheter a solution of polyoxazoline-conjugated albumin (POx(5k)-eSM-PSA) prepared in Preparation Example 3 of Example 1 in phosphate buffered saline (PBS, pH 7.4, [PSA] = 5 g / dL) (n = 6) and an aqueous solution of hydroxyethyl starch (Otsuka Pharmaceutical Co., Ltd., Volven Infusion 6%) (n = 6). The amount administered was equivalent to 30% of the total blood volume (56 mL / kg). Vital signs (mean arterial blood pressure (MAP), heart rate (HR), respiratory rate, and rectal temperature) were recorded at the following 10 time points: (1) before 50% blood removal, (2) immediately after 50% blood removal, (3) immediately before sample administration, (4) immediately after sample administration, (5) 5 minutes after administration, (6) 15 minutes after administration, (7) 30 minutes after administration, (8) 1 hour after administration, (9) 1.5 hours after administration, and (10) 2 hours after administration. Mean arterial blood pressure (MAP), which was approximately 100 mmHg, decreased to approximately 30 mmHg after blood removal, but increased with administration of polyoxazoline-conjugated albumin solution, recovering to approximately 90 mmHg 2 h after administration (see Figure 4(A), **p<0.01 vs. hydroxyethyl starch). In contrast, in the hydroxyethyl starch-treated group, MAP only increased to approximately 60 mmHg 2 h after administration. Heart rate (HR), which was approximately 400 beats / min, decreased to approximately 300 beats / min after blood removal, but increased with administration of polyoxazoline-conjugated albumin solution, recovering to approximately 400 beats / min 2 h after administration (see Figure 4(B), *p<0.05, **p<0.01 vs. hydroxyethyl starch). In contrast, in the hydroxyethyl starch-treated group, MAP only increased to approximately 320 beats / min 2 h after administration. Administration of polyoxazoline-conjugated albumin solution was found to be effective in resuscitating patients from hemorrhagic shock. Other vital signs and blood gas parameters also returned to their initial values ​​after administration of polyoxazoline-conjugated albumin solution. [Industrial Applicability]

[0131] The artificial plasma expander of the present invention, which contains polyoxazoline-conjugated albumin as an active ingredient, can be used as a highly safe plasma substitute when administered in vivo. Its target is not limited to humans; it can also be administered to animals (pets such as dogs and cats, livestock, etc.). The artificial plasma expander of the present invention, which contains polyoxazoline-conjugated albumin as an active ingredient, can be administered to cases of hypoalbuminemia caused by bleeding, increased capillary permeability, decreased hepatic albumin synthesis, excessive excretion from the kidneys or intestines, increased metabolism, or dilution by intraoperative fluid infusion. Specifically, it can be expected to be used as a therapeutic agent for hemorrhagic shock, sepsis, cardiac surgery using cardiopulmonary bypass, extracorporeal circulation with unstable hemodynamics, severe burns, pregnancy-induced hypertension, and other conditions, including refractory ascites associated with liver cirrhosis, refractory edema, nephrotic syndrome with pulmonary edema, and protein-losing enteropathy. [Explanation of symbols]

[0132] 100: Polyoxazoline-conjugated albumin 10: Albumin 20: Polyoxazoline

Claims

1. having albumin as a core and polyoxazoline as a shell covalently bonded to the albumin via a crosslinker, The covalent bond via the crosslinker comprises the following structure (2): A polyoxazoline-conjugated albumin, characterized in that the binding site of the albumin to the crosslinking agent is lysine or a primary amine at the protein terminal. 【Chemistry 1】 [In structure (2), R １ represents any one of the following general formula (4), the following general formula (5), the following chemical formula (1), and the following chemical formula (2). 【Chemistry 2】 [In general formula (4), n represents an integer of 1 to 10.] 【Transformation 3】 [In general formula (5), n represents an integer of 2, 4, 6, 8, 10, or 12.] 【Chemistry 4】 【Transformation 5】

2. 2. The polyoxazoline-conjugated albumin according to claim 1, wherein the bonding site of the polyoxazoline with the crosslinking agent is a terminal hydroxyl group or amino group of the polyoxazoline represented by the following general formula (1): 【Transformation 6】 [In general formula (1), R １ represents a hydrocarbon group having 1 to 8 carbon atoms, and R ２ is a hydroxyl group, an amino group, or -NH-(CH ２ ) ２ represents —OH, and n represents the number of repeating monomer units.

3. The polyoxazoline-conjugated albumin according to claim 1 or 2, wherein the covalent bond via the crosslinking agent comprises a structure derived from a maleimide group-introducing agent.

4. The polyoxazoline-conjugated albumin according to claim 3, wherein the maleimide group introducing agent comprises at least one compound selected from the group consisting of compounds represented by the following general formula (2) or the following general formula (3): 【Transformation 7】 【Transformation 8】 [In general formula (2), R ２ is a hydrogen atom or SO ３ － Na ＋ represents R １ represents any one of the following general formula (4), the following general formula (5), the following chemical formula (1), and the following chemical formula (2). ３ represents the following general formula (4), and R ４ represents OH or Cl. 【Chemistry 9】 [In general formula (4), n represents an integer of 1 to 10.] 【Chemistry 10】 [In general formula (5), n represents an integer of 2, 4, 6, 8, 10, or 12.] 【Chemistry 11】 【Chemistry 12】

5. the covalent bond via the crosslinking agent further includes a structure derived from a thiol group introducing agent, The polyoxazoline-conjugated albumin according to claim 3 or 4, wherein the thiol group introducing agent is at least one compound selected from the group consisting of compounds represented by the following chemical formula (3), the following general formula (6), or the following general formula (7): 【Chemistry 13】 【Chemistry 14】 [In general formula (6), n represents an integer of 1 to 10.] 【Chemistry 15】 [In general formula (7), R １ represents OH or Cl, and n and m represent integers of 1 to 10.

6. A polyoxazoline-conjugated albumin described in any one of claims 1 to 5, wherein the covalent bond via the crosslinking agent comprises the following structure (3) or structure (4): 【Chemistry 16】 【Chemistry 17】 [In Structure (3) and Structure (4), m represents an integer of 1 to 10.]

7. The polyoxazoline-conjugated albumin according to any one of claims 1 to 6, wherein the polyoxazoline has a weight average molecular weight of 500 to 100,000 daltons.

8. An artificial plasma expander, characterized by containing the polyoxazoline-conjugated albumin described in any one of claims 1 to 7.

9. A resuscitation solution for hemorrhagic shock, characterized by containing the polyoxazoline-conjugated albumin described in any one of claims 1 to 7.

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