JP2001151800A - Albumin polymer, method for producing the same and blood platelet substitute - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a method for preparing an albumin polymer capable of binding albumin molecules without using an irreversible heat denaturation and a cross-linking agent and controlling a polymerization reaction so as to provide a particle diameter of nanometers to micrometers and obtain a blood platelet substitute capable of recognizing a hemorrhagic site and aggregating using the albumin polymer as a carrier for supporting a blood platelet membrane protein and controlling the hemorrhage thereof. SOLUTION: This albumin polymer having 0.05-5 μm particle diameter in which spaces between albumin molecules are bound through disulfide linkages. The method for producing the albumin polymer is obtained. The blood platelet substitute comprises an albumin polymer-protein conjugate in which at least one kind of blood platelet membrane protein or a part of a structure thereof is bound to the albumin polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polymer having a controllable particle size obtained by polymerizing between albumin molecules, which are plasma proteins, by disulfide bonds, and a platelet substitute having a hemostatic ability having a platelet membrane protein bound to the particle surface. And a method for preparing the same.

[0002] The albumin polymer of the present invention whose particle size is adjustable can be applied as a carrier for new drugs, physiologically active substances or various receptor proteins. In the present invention, G, which is a platelet membrane protein, is used as the receptor protein.
By using PIb, GPIa / IIa, GPIIb / IIIa or a recognition site that is a part of the structure, a molecular assembly that recognizes a bleeding site and aggregates there can be constructed. In addition, if a blood coagulation-related protein such as fibrinogen or thrombin is simultaneously bound to an albumin aggregate in addition to these proteins, it can be used as a platelet substitute having hemostatic ability.

[0003]

2. Description of the Related Art At present, three types of platelet substitutes are being developed: liposome type, erythrocyte type and albumin type. In addition, expired platelets are freeze-thawed and fragmented by injectable platelet membrane (Infusible
Platelet Membrane) has been developed as a Cyplex, which assumes the use of human platelets and is not a substitute for platelets that do not use human origin.

[0004] Liposomal platelet substitutes are available from Rybak (Biomat. Art Cells, Immob. Biotech., 21,
101-118 (1993)) and Ikeda (Int. J. Haematol., 63,
301 (1996)) et al., In which a platelet membrane protein is immobilized on the surface of a phospholipid bilayer membrane (liposome) using a phospholipid having an aldehyde group. However, liposomes, which are molecular assemblies of phospholipids, are generally unstable, and gradually aggregate and fuse. Furthermore, when it is administered to the blood, it is quickly captured by the reticuloendothelial system, and the residence time in the blood is extremely short. Therefore, a method is often used in which surface modification is performed with, for example, a polyoxyethylene chain, which is a water-soluble polymer, so that liposomes are hardly recognized by the reticuloendothelial system (Klibanov et.al., FEBS Lett.,
268, 235-237 (1990); Woodle and Lasic, Biochim. B
iophys. Acta, 1113, 171-199 (1992)). However, surface modification with polyoxyethylene is expected to inhibit the function of platelet membrane protein, which is a receptor protein carried on the surface. In addition, there is a concern that the function of the reticuloendothelial system that has phagocytosed the liposomes may be reduced. In addition, the ratio of expensive phospholipids is large, so that the overall price is high, the preparation of liposomes with controlled particle size is complicated, and the effects of metabolism and the like are not sufficiently clarified. Have become barriers to practical use of these liposome-type platelet substitutes, and improvements thereof are currently being studied.

On the other hand, as an erythrocyte-type platelet substitute, one obtained by immobilizing fibrinogen on the surface of erythrocytes using formaldehyde has been constructed by Agam et al. (Agam et al., Eur. Clin. Invest., 2
2, 105-112 (1992)). In addition, the amino acid sequence recognized by fibrinogen, Ac-CGGGRD-
A thromboerythrocyte having NH2 immobilized on the surface of erythrocytes as a synthetic peptide has been developed by Coller et al. (Coller et al., J. Clin. Invest., 89,
546-555 (1992)). Erythrocyte-type platelet substitutes with a diameter of 8 μm are larger than platelets with a diameter of 1 μm and are thought to be advantageous for hemostasis. However, in the first place, the use of red blood cells always poses risks such as blood types and various infectious diseases. Follow me,
Can't eliminate problems from blood donation. Even formaldehyde, which is suspected to be carcinogenic, requires extreme caution when used in the synthesis of intravenous preparations.

[0006] On the other hand, albumin is a protein present in a large amount in plasma and has no problem in biocompatibility or metabolism. In recent years, recombinant mass production technology for albumin has been established, and safe human albumin can be produced in factories without depending on blood donation (Yokoyama, Omura, Clinical Molecular Medicine, 1,939 (1993)). Microspheres or large aggregates (macroaggregates) using human serum albumin have a particle size of a few micrometer to several hundred micrometers, and nanospheres have a particle size of several hundred nanometers. Internal structure. Most albumin microsphere preparation methods are suspension or emulsification (Gupta and Hung, J. Mi.
coencapsulation, 6, 427-462 (1989)). The most commonly used method is to first form a w / o emulsion by dropping an aqueous albumin solution into a suitable oil such as corn oil, olive oil, cottonseed oil, isooctane and the like and stirring to suspend. Then, heat denature and stabilize by heating at a high temperature of 100 ° C to 180 ° C for 2.5 minutes to 24 hours, or a suitable crosslinking agent such as formaldehyde, glutaraldehyde, 2,3-butanedione, or tetraphthaloyl. The constituent albumin molecules are crosslinked and stabilized using dichloride or the like. After that, use a volatile organic solvent such as ether, alcohol, acetone, etc. and centrifugal separation operation, or capture on a filter, wash and remove oil and cross-linking agent well, and disperse in powder form or organic solvent. At low temperature.

In the above method, the particle size is determined by the structure of the stirrer, the stirring speed, the stirring time, the volume ratio of the aqueous solution to the oil, the respective viscosities, the surface tension, and the like. However, it is not suitable for preparing nanospheres of 1 micrometer or less. Furthermore, in this method, the degree of thermal denaturation of albumin and the degree of chemical denaturation due to the bonding of the cross-linking agent are large, and the method of completely removing the oil and the cross-linking agent is complicated. Further, removal of impurities adsorbed on the microsphere surface becomes complicated, such as using dimethylformamide or an aqueous buffer, and is not always preferred as a method for preparing an intravenous drug. Originally, polymerization by a cross-linking agent is a non-specific reaction with amino groups present on the protein surface in large numbers.
Although the degree of chemical modification is large, the polymerization efficiency is low and an excessive cross-linking agent is required for stabilization. In addition, since the surface of the microsphere prepared in oil is hydrophobic, it is necessary to further hydrophilize the surface with a surfactant or a modifier.

The preparation of albumin nanospheres is suspended using ultrasonic irradiation in the above emulsion method. It is said that the particle size can be controlled by the ultrasonic irradiation conditions at this time, but satisfactory accuracy cannot be obtained. Cleaning of nanospheres is extremely difficult. Therefore, a coacervation method is used in which acetone, ethanol, isopropyl alcohol and the like are added to an aqueous albumin solution to desorb the water of hydration of albumin to reduce the solubility (Ch.
en et al., J. Microencapsulation, 11, 395-407 (199
4), Ishizaka et al., J. Pharm.Sci., 74, 342-344.
(1985)). By heating the obtained coacervate at 75 ° C. for 15 minutes or more, stabilized albumin nanospheres having a particle diameter of 200 to 500 nm can be obtained. Alternatively, there is a method for producing stable nanospheres by adding a mixture of ethanol and a surfactant (US Pat. No. 5,049,322). Yen et al. Added a water-soluble lower alcohol to an aqueous solution of albumin to form colloidal particles, and added a stabilizer consisting of reducing agents, oxidizing agents, phosphorylated compounds, sulfur-containing compounds, polymers, and combinations thereof. Thus, a method for stabilizing albumin nanospheres without chemical crosslinking or denaturation is disclosed (PCT / US96 / 09458). In addition, as a method for obtaining nanospheres having a particle diameter of about 100 nanometers, there is a report combining pH control and coacervation method by adding acetone (Lin et al., J. Drug Targeting, 1, 237-243).
(1993)). However, this is not a method of controlling the particle diameter by greatly changing it from, for example, 100 nanometers to 1000 nanometers. Acetone and alcohol used in the above-described coacervation method are volatile solvents, but there is a concern that they may be affected by the process of complete removal or the residual. Although the surface has relatively high hydrophilicity, a stabilizer is required to obtain a satisfactory dispersion stability.

An aerosol of an aqueous albumin solution is dissolved in a hot oil or a hot oil by a spinning disk method or a spray drying method.
A method is known in which albumin is thermally denatured by spraying into a high-temperature gas phase at 0 to 220 ° C. to prepare microcapsules having a particle size of 0.1 to 50 μm. Sutton et al. Disclose a method of preparing hollow albumin fine particles of 1 to 10 micrometers by a spray drying method (US Pat. No. 5,955,108). The particle size controlled by the albumin concentration in the aqueous albumin solution is uniform. This method requires a special apparatus, and furthermore, there is a concern that the degree of denaturation is high due to exposure to high temperatures. Further, since the surface is prepared in the gas phase, the surface is considered to have high hydrophobicity.

[0010] Taplin et al. (J. Nuclear, Med., 5, 259, 196)
4) reports a method of obtaining aggregates by heating albumin at 80 ° C. on the alkaline side and then returning it to a neutral or weakly acidic side. However, it has not been found that a disulfide bridge is formed between albumin molecules using a thiol-disulfide exchange reaction, and that an albumin polymer having a controlled particle diameter can be obtained by controlling the disulfide bridge.

As a method for preparing an albumin polymer, a coacervation method and an emulsion method are known. In the former coacervation method, coacervate obtained by salting out is crosslinked and polymerized with glutaraldehyde via a Schiff base. In this case, since the amino group is consumed, it is disadvantageous for the subsequent reaction with a carboxyl group.
On the other hand, the emulsion method is suitable for preparing an albumin polymer having a particle diameter of 10 μm or more, but it is difficult to control the particle diameter to 5 μm or less that can pass through a capillary.

A system in which fibrinogen is immobilized on albumin microcapsules having a diameter of 3.5 to 4.5 μm prepared by spray drying has been proposed by Levi et al. (Nature Medicine 5, 107-111).
(1999)) However, the bleeding site cannot be recognized.

[0013]

An object of the present invention is to control albumin to (1) a particle size of from nanometer to micrometer, and (2) without using a crosslinking agent.
(3) Establish a method of polymerizing without the need for irreversible heat denaturation, and (4) securely bind platelet membrane proteins and blood coagulation-related proteins using this polymer as a carrier to achieve high biocompatibility. An object of the present invention is to construct a platelet substitute that has cost performance and can effectively recognize and aggregate a bleeding site and stop the bleeding.

[0014]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in view of the above situation, and as a result, focused on albumin aggregation near the equipotential point, and provided a heating operation with pH adjustment to allow albumin molecules to intersperse. Cross-linked by disulfide bonds,
In addition to establishing a method for preparing albumin polymers whose particle size can be adjusted from nanometers to micrometers, collagen and von Willebrand factor (von Wiebrand factor) exposed to the bleeding site for albumin aggregates of various particle sizes are established.
llebrand factor (vWF) recognizes platelet membrane proteins GPIbα and GPIa / IIa, and also binds blood coagulation-related proteins such as fibrinogen to successfully construct a molecular assembly system that has both bleeding part recognition ability and hemostasis ability. Thus, the present invention has been achieved.

That is, according to the present invention, the particle diameter of the disulfide bond between albumin molecules is from 0.05 to 0.05.
A 5 micron albumin polymer is provided.

According to the present invention, there is also provided a method for producing an albumin polymer having an intermolecular albumin disulfide bond, which comprises forming an intermolecular disulfide bond after cleaving the intramolecular disulfide bond of albumin. Is provided.

According to the present invention, there is provided an albumin polymer-protein conjugate in which at least one platelet membrane protein or a part of its structure is bonded to an albumin polymer. Saga 0.05 ~
A platelet substitute that is 5 μm is provided.

Further, according to the present invention, there is provided a platelet substitute in which a blood coagulation-related protein such as fibrinogen is further bound to the albumin polymer site of the albumin polymer-protein conjugate.

In the present invention, the platelet membrane protein is GP
Ib, GPIa / IIa, GPIIb / IIIa, or a portion of their structure.

In the present invention, the bond between the albumin polymer and a platelet membrane protein or a part of its structure or a blood coagulation-related protein such as fibrinogen may include a disulfide bond. In the present invention, the binding between the albumin polymer and the platelet membrane protein or a part of the structure thereof, or a blood coagulation-related protein such as fibrinogen may include a polyoxyethylene chain. Furthermore, the bond between the albumin polymer and a platelet membrane protein or a part of its structure or a blood coagulation-related protein such as fibrinogen may include a disulfide bond and a polyoxyethylene chain.

Further, the present invention provides an albumin polymer-protein conjugate in which at least one platelet membrane protein or a part of the structure thereof is bound to an albumin polymer, wherein GPIb, GPIa / IIa,
GPIIb / IIIa, or a part of their structure. Also provided is an albumin polymer-protein conjugate wherein a blood coagulation-related protein such as fibrinogen is further bound to the albumin polymer site of the albumin polymer-protein conjugate. Here, the bond between the albumin polymer and a platelet membrane protein or a part of its structure or a blood coagulation-related protein such as fibrinogen may include a disulfide bond. Further, the binding of the albumin polymer to the platelet membrane protein or a part of its structure or a blood coagulation-related protein such as fibrinogen may include a polyoxyethylene chain.

Further, according to the present invention, there is provided a medicine comprising the above-mentioned albumin polymer-protein conjugate.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be described below.

Albumin used in the present invention is a plasma protein and is present in a large amount in blood. Any serum albumin can be used, such as human serum albumin (HSA), bovine serum albumin, recombinant human serum albumin. In that case, it is premised that infection is not caused and rejection by antigen-antibody reaction does not occur. Human serum albumin has 59 lysines on its surface and can efficiently bind a drug or a functional protein having a carboxyl group to its amino residue, and is therefore extremely useful as a carrier for drug delivery.

The albumin polymer of the present invention has a disulfide bond between albumin molecules having a particle size of 0.05.
It is an albumin polymer of 5 μm, which is obtained by cleaving the intramolecular disulfide bond of albumin and then forming an intermolecular disulfide bond.

The method for producing the albumin polymer of the present invention comprises the steps of:
Aggregation near the isoelectric point of albumin and albumin 17
An albumin polymer is prepared by utilizing the conversion of 34 sets of intramolecular disulfide bonds into intermolecular disulfide bonds. Specifically, after raising the pH of the albumin solution of about 1% to 10 or more, the mixture is heated to about 80 ° C. to cleave the intramolecular disulfide bond of albumin, and after cooling, the pH is set to around the equipotential point, Preferably 5.7-
6.3. At a reaction temperature of 4 to 50 ° C., preferably 25 to 40 ° C., aggregation of albumin and formation of disulfide bonds between albumin molecules are performed. The particle size of the polymer obtained by forming a disulfide bond between albumin molecules in this way can be controlled by pH and reaction time, and is useful as a carrier for a drug carrier because the original amino group is retained. It is. For example, when control is performed by stirring at 40 ° C. near the isoelectric point, an albumin polymer having a particle size of several hundred nanometers to several micrometers is obtained by stirring at pH 5.7 to 5.9 and about 5 to 7 hours. Is obtained. Further, according to the method for producing an albumin polymer of the present invention, it is possible to synthesize an albumin polymer having a larger particle diameter by controlling the temperature, pH, and reaction time during the thiol-disulfide exchange reaction. . For example, after adjusting the pH to 6.0,
By stirring for 8 hours, an albumin polymer having a particle size of 5 micrometers or more can be obtained. However, the conditions for controlling the particle size of the albumin polymer are not limited by these conditions. In the present invention, the preferred size of the albumin polymer is 0.05 to 5 μm,
More preferably, it is 0.2 to 2 μm.

Since the albumin polymer prepared as described above has an amino group, it can be bound to a plasma membrane protein or a part of its structure in various ways. Three typical examples are shown below, but the present invention is not limited to these.

The simplest method is a method of binding by adding glutaraldehyde.

The second method is to bind a protein having a free thiol group such as cysteine to an albumin polymer. In that case, first, the albumin polymer is reacted with N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) to introduce a pyridyldithio group into the albumin polymer, and then a thiol-disulfide exchange reaction is performed. It can bind to proteins with groups. Here, the succinimidyl group reacts with the amino group of albumin, while the pyridyldithio group reacts specifically with the thiol group.

The third method is a case where a protein having no free thiol group is bound to an albumin polymer. In this case, the protein is reacted with SPDP to introduce a pyridyldithio group, which is reduced with dithiothreitol to a thiol group, and mixed with the albumin polymer into which the pyridyldithio group has been introduced obtained in the second method. Then, the two are bonded by a thiol-disulfide exchange reaction, or the albumin polymer into which the pyridyldithio group obtained by the second method is introduced is reduced with dithiothreitol to form a thiol group, and then the pyridyldithio group is introduced. The two can be mixed with each other by a thiol-disulfide exchange reaction. In this case, crosslinking can be performed with the minimum necessary chemical modification.

In the present invention, a platelet membrane protein is used as a target to be bound to the albumin polymer. Such platelet membrane proteins include GPIa / IIa, GPIb (including GPIb / IX, etc.) or a part of its structure (GPIbα), GPIIb
/ IIIa. The albumin polymer-platelet membrane protein conjugate of the present invention has, in its albumin polymer site,
A blood coagulation-related protein such as fibrinogen can be further bound.

GPIa / IIa is a platelet-recognizing glycoprotein that interacts with collagen exposed at the site of bleeding. GPIb also recognizes von Willebrand factor (vWF) which adheres to collagen. Therefore, by binding them to the albumin polymer, the albumin polymer can be accumulated at the bleeding site. Fibrinogen is a hemostatic factor that is converted into fibrin by thrombin to form fibers and to make hemostasis firm.

Since proteins such as GPIbα have one free cysteine, they can be bound to an albumin polymer by the above-mentioned second method. GPIa / IIa
Since fibrinogen and the like do not have free cysteine, they can be bound by the third method.
That is, if a blood coagulation-related protein such as fibrinogen is bound to an albumin polymer together with a recognition protein such as GPIa / IIa, a platelet substitute that can recognize, accumulate, and stop bleeding can be constructed.

When a recombinant form of the platelet membrane glycoprotein in which only the water-soluble active site is constructed is used, the spacer portion of the carbohydrate is not included, and instead, a polysaccharide having a molecular weight of 1,000 to 10,000 is used as the spacer. An albumin polymer can be bound to the recognition protein using an oxyethylene (POE) derivative. Further, a POE derivative having a carboxyl group at the terminal and a succinimidyl group at the other terminal is obtained by adding 1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide (EDC)
And N-hydroxysulfosuccinimide (Sulfo-
After activation with NHS), GPIa / GPIa / is added to the albumin polymer via the spacer POE chain if used in the same manner as SPDP.
IIa, GPIbα and blood coagulation-related proteins can be bound.

Thus, a platelet substitute derived from an albumin polymer having a bleeding site recognition ability and a hemostatic ability can be constructed.

In platelet dysfunction, congenital and acquired thrombocytopenia, platelet dysfunction or a decrease in the number of platelets causes the hemostasis function of the platelets to not work normally and causes a bleeding tendency. For such diseases, platelet transfusion for the purpose of hemostasis is performed, but the platelet substitute of the present invention can be used instead of blood transfusion.

When the platelet substitute of the present invention is used as a medicine, the administration route includes intravenous infusion or intravenous administration, and it is usually used as a suspension in a salt solution or a physiologically compatible solution. The dose is appropriately selected depending on the condition, age, body weight, etc., and a maximum of 100 g per day is administered to an adult once or in multiple doses. When an ointment, a liniment or a patch is prepared, it is used by mixing with a pharmacologically acceptable excipient or the like.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0039]

EXAMPLE 1 1% by weight of human serum albumin (H
SA) 800 μL of 0.1N NaOH aqueous solution was added to 25 mL of the solution (pH 6.65, 24.8 ° C.), and then 2
The mixture was stirred for 0 minutes to reach pH 10.5 (26.2 ° C). After heating at 80 ° C. for 20 minutes in a water bath, the mixture was gradually cooled to room temperature to obtain an HSA solution having a pH of 10.32 (21.0 ° C.). A total of 800 μL of a 0.1N HCl aqueous solution was added to the solution at intervals of 10 minutes, and the pH was adjusted to 5.9, followed by stirring at 40 ° C. for 5 hours in a water bath. Dialysis was performed for 12 hours against phosphate buffered saline (PBS), and the particles having a large particle diameter were centrifuged (500 rpm, 2 rpm).
Min) to obtain an albumin polymer having a particle size of 358 ± 110 nm.

Example 2 1% by weight HSA solution (pH 6.6) diluted with physiological saline
(5, 24.8 ° C.) 800 mL of 0.1N NaOH aqueous solution was added to 25 mL, and the mixture was stirred for 20 minutes.
6.2 ° C). After heating in a water bath at 80 ° C. for 20 minutes, the mixture was gradually cooled to room temperature, and pH 10.32 (21.0 ° C.) H
An SA solution was obtained. 0.1N H at 10 minute intervals
A total of 900 μL of a Cl aqueous solution was added, and a total of 500 μL of a 0.1 N HCl aqueous solution was added at 10-minute intervals in total of 100 μL. . Dialysis was performed for 12 hours against PBS, and the polymer having a large particle size was centrifuged (500
(2 min. rpm), an albumin polymer having a size almost equivalent to platelets (particle size: 2860 ± 680 nm) was obtained.

Example 3 As in Example 1, the HSA solution was heated under alkaline conditions, and then gradually cooled to room temperature to obtain an HSA solution having a pH of 10.32 (21.0 ° C.). To this solution, 900 μL of 0.1N HCl aqueous solution was gradually dropped, and after adjusting the pH to 6.02 (24.6 ° C.), the mixture was stirred in a water bath at 25 ° C. for 48 hours, and then 10 mg of iodoacetamide was added. PB
Dialysis against S for 24 hours, particle size 54000 ± 3200nm
An albumin polymer was obtained.

Example 4 6 mg of N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) was dissolved in 3 mL of a 99.5% ethanol solution to give a SP of 6.4 × 10 ^-3 mol / L.
A DP solution was obtained. This 6.4 was added to 19.5 mL of the albumin polymer solution of Example 1 adjusted to a concentration of 0.37% by weight.
172 μL of a × 10 ⁻³ mol / L SPDP solution was added, and the mixture was stirred at 23 ° C. for 2 hours. The albumin polymer and unreacted SPDP were separated by gel permeation chromatography (GPC) (Sephacryl S-400), and the volume of the pyridyldithio (PD) group-bound albumin polymer solution was adjusted to 4 mL. Next, after replacing this albumin polymer solution with nitrogen, 40 μL of a 7.2 wt% GPIbα solution was added, and 5 ° C.
For 30 hours. Unreacted GPIbα is separated by GPC (Sephacryl S-400), and albumin polymer
A GPIbα conjugate was obtained. Albumin polymer and GPIb
is bonded to fluorescein isothiocyanate (F
(ITC) and tetramethylrhodamine isothiocyanate (TRITC) were confirmed by a double staining method. The binding ratio between the two was measured by a secondary antibody method of GPIbα, and albumin molecules on the albumin polymer surface / GPIbα = 9/1.
Met.

Further, an anti-human GPIbα antibody GUR20-
By flow cytometry using No. 5, it was confirmed that GPIbα was immobilized on the albumin polymer surface.

Further, the albumin polymer-GPIbα conjugate (final concentration: 60 μg GPIbα protein / mL) was
F (final concentration 50 μg / mL)
The function of the albumin polymer-GPIbα conjugate can be confirmed by adding g / mL of ristocetin and observing the aggregation of this system. The present albumin polymer-GPIb
The α-conjugate did not aggregate with vWF alone, but aggregated with the addition of ristocetin. This aggregation was specifically suppressed by the anti-vWF monoclonal antibody NMC-4.

Example 5 6 mg of SPDP was dissolved in 3 mL of a 99.5% ethanol solution to obtain an SPDP solution having a concentration of 6.4 × 10 ^-3 mol / L. This 6.4 × 10 ^-3 mol / l was added to 19.5 mL of the albumin polymer solution of Example 2 prepared to a concentration of 0.37% by weight.
172 μL of L concentration SPDP solution was added, and the mixture was stirred at 23 ° C. for 2 hours. The albumin polymer and unreacted SPDP were separated by centrifugation (3000 rpm, 2 minutes), and the resulting PD-bound albumin polymer solution was made up to 4 mL. Next, after replacing this PD-bound albumin polymer solution with nitrogen, 40 μL of a 7.2% by weight GPIbα solution was added, followed by stirring at 5 ° C. for 30 hours. Centrifugation (2000 rpm
m, 2 minutes) to separate unreacted GPIbα to obtain an albumin polymer-GPIbα conjugate. The binding between the albumin polymer and GPIbα was confirmed in the same manner as in Example 4, and the binding ratio was determined by the ratio of albumin molecule / albumin molecule on the albumin polymer surface
GPIbα = 15/1. Also, anti-human GPIb
From the results of flow cytometry using the α antibody GUR20-5, it was confirmed that GPIbα was immobilized on the surface of the albumin polymer.

Further, the obtained albumin polymer-GP
Ibα conjugate (final concentration 60 μg GPIbα protein / m
L) was mixed with vWF (final concentration 50 μg / mL), ristocetin having a final concentration of 1.0 mg / mL was added, and the aggregation of this system was observed. As a result, ristocetin aggregation was observed. This aggregation was also caused by the anti-vWF monoclonal antibody NMC-4.
Was specifically suppressed.

Example 6 7.2% by weight of GP was added to 1 mL of PBS purged with nitrogen for 30 minutes.
40 μL of the Ibα solution was added, and 10 μL of the SPDP solution having a concentration of 6.4 × 10 ⁻³ mol / L was added thereto, followed by stirring at 5 ° C. for 1 hour to obtain a PD-bound GPIbα solution.

On the other hand, 0.37% by weight prepared in Example 2
172 μL of 6.4 × 10 ⁻³ mol / L SPDP solution was added to 19.5 mL of the albumin polymer solution, and the mixture was added at 23 ° C. for 2 hours.
Stirred for hours. The albumin polymer and unreacted SPDP were separated by centrifugation (3000 rpm, 2 minutes), and the resulting PD-bound albumin polymer solution was made up to 4 mL. To this was added 3.1 mg of dithiothreitol, and added at 5 ° C.
And then centrifuged (2000 rpm, 2 rpm).
Min) to separate unreacted dithiothreitol and by-products, and to obtain 3 mL of the SH-bound albumin polymer solution obtained.
Fixed volume. To the SH-bound albumin polymer solution, the PD-bound GPIbα solution was added, and the mixture was stirred at 5 ° C. for 24 hours. Then, unreacted GPIbα was separated by centrifugation (2000 rpm, 2 minutes), and albumin polymer-GPIbα.
The conjugate was obtained. The binding ratio of this albumin polymer-GPIbα conjugate is defined as the ratio of albumin molecule / albumin molecule on the albumin polymer surface.
GPIbα = 12/1. Also, anti-human GPIb
From the results of flow cytometry using α antibody GUR20-5, it was confirmed that GPIbα was immobilized on the albumin polymer surface. Further, a ristocetin agglutination test of the albumin polymer-GPIbα conjugate was conducted in the same manner as in Example 4, and as a result, agglutination occurred.

Example 7 GPIa / IIa (5 mg) was added to PBS (1 mL), and 6.4 ×
10 ^-3 mol / L SPDP solution (10 μL) was added, and
The mixture was stirred at C for 1 hour to obtain a PD-bound GPIa / IIa solution.

On the other hand, 0.37% by weight prepared in Example 2
172 μL of a 6.4 × 10 ⁻³ mol / L SPDP solution was added to 19.5 mL of the albumin polymer solution, and the mixture was stirred at 23 ° C. for 2 hours. The albumin polymer and unreacted SPDP were separated by centrifugation (3000 rpm, 2 minutes).
The D-linked albumin polymer solution was made up to 4 mL. After adding 3.1 mg of dithiothreitol to the mixture and stirring at 5 ° C. for 10 minutes, unreacted dithiothreitol and by-products were separated by centrifugation (2000 rpm, 2 minutes), and the reduced SPDP-bound albumin thus obtained was obtained. 3 mL of polymer solution
Fixed volume. To the SH-bound albumin polymer solution, the PD-bound GPIa / IIa solution was added, and the mixture was stirred at 5 ° C. for 24 hours. Then, unreacted GPIa / IIa was separated by centrifugation (2000 rpm, 2 minutes), and the albumin polymer- G
A PIa / IIa conjugate was obtained. The binding ratio of this conjugate is defined as the albumin molecule / GPIa / IIa =
13/1.

GPIa / IIa adheres to collagen. When the albumin polymer-GPIA / IIa conjugate is placed in a container coated with collagen and mixed, the mass is increased by the quartz oscillator method, that is, the albumin polymer-GP
Adhesion of the Ia / IIa conjugate to collagen was confirmed.
This result indicates that the albumin polymer-GPIa / IIa conjugate has the ability to recognize a bleeding site.

Example 8 7.2% by weight of GP was added to 1 mL of PBS purged with nitrogen for 30 minutes.
40 μL of the Ibα solution was added, 7 μL of a 6.4 × 10 ⁻³ mol / L SPDP solution was added, and the mixture was stirred at 5 ° C. for 1 hour to obtain a PD-bound GPIbα solution.

GPIa / IIa5m was added to PBS1mL.
g of SPD having a concentration of 6.4 × 10 ⁻³ mol / L.
10 μL of the P solution was added, and the mixture was stirred at 5 ° C. for 1 hour to obtain a PD-bound GPIa / IIa solution.

Further, 6.4 × 10 ^-3 was added to 19.5 mL of the 0.37% by weight albumin polymer solution prepared in Example 2.
172 μL of a SPDP solution having a mol / L concentration was added, and the mixture was added at 23 ° C.
For 2 hours. Centrifugation (3000 rpm, 2 minutes)
To separate the unreacted SPDP from the albumin polymer,
The volume of the obtained PD-bound albumin polymer solution was adjusted to 4 mL. To this was added 3.1 mg of dithiothreitol, and 5
After stirring at 10 ° C. for 10 minutes, centrifugation (2000 rpm,
2 minutes) to separate unreacted dithiothreitol and by-products. The resulting SH-bound albumin polymer solution was 3 m long.
L. To the SH-bound albumin polymer solution, add the PD-bound GPIbα solution, further add the PD-bound GPIa / IIa solution, stir at 5 ° C. for 24 hours, and centrifuge (2000 rpm, 2 minutes) to remove unreacted GPIbα. GPIa / IIa was separated to obtain an albumin polymer-GPIbα; GPIa / IIa conjugate. The binding ratio of this conjugate was albumin molecule / GPIbα / GPIa / IIa on the albumin polymer surface = 13/1/1. Flow cytometry using the anti-human GPIbα antibody GUR20-5 also confirmed that GPIbα was immobilized on the albumin polymer surface.

This albumin polymer-GPIbα; GP
The Ia / IIa conjugate was subjected to a ristocetin aggregation test according to Example 4, and as a result, specific aggregation was confirmed. Example 7
Similarly to the above, adhesion of the albumin polymer-GPIbα; GPIa / IIa conjugate to collagen was observed in a system using a quartz oscillator. The results indicated the ability of the albumin polymer-GPIbα; GPIa / IIa conjugate to recognize the bleeding site.

Example 9 4.7 mg of human fibrinogen was dissolved in 1 mL of PBS, and a SPDP solution having a concentration of 6.4 × 10 ⁻³ mol / L was obtained.
μL was added and the mixture was stirred at 23 ° C. for 1 hour to obtain a PD-bound human fibrinogen solution.

GPIa / IIa5m was added to PBS1mL.
g, and then add 10 μL of a 6.4 × 10 ⁻³ mol / L SPDP solution, and stir at 5 ° C. for 1 hour to obtain PD-bound GPI
a / IIa solution was obtained.

Further, 6.4 × 10 ^-3 was added to 19.5 mL of the 0.37% by weight albumin polymer solution prepared in Example 2.
172 μL of a SPDP solution having a mol / L concentration was added, and the mixture was added at 23 ° C.
For 2 hours. Centrifugation (3000 rpm, 2 minutes)
To separate the unreacted SPDP from the albumin polymer,
The volume of the obtained PD-bound albumin polymer solution was adjusted to 4 mL. To this was added 3.1 mg of dithiothreitol, and 5
After stirring at 10 ° C. for 10 minutes, centrifugation (2000 rpm,
2 minutes) to separate unreacted dithiothreitol and by-products. The resulting SH-bound albumin polymer solution was 3 m long.
L. To the SH-bound albumin polymer solution was added the PD-bound fibrinogen solution,
After adding D-linked GPIa / IIa solution and stirring at 5 ° C. for 24 hours, unreacted fibrinogen and GPIa / IIa were separated by centrifugation (2000 rpm, 2 minutes), and albumin polymer-fibrinogen; GPIa / IIa-bound I got a body. The binding ratio of this conjugate is determined by the albumin molecule / fibrinogen / GPIA / II on the albumin polymer surface.
a = 15/1/1. Since fibrinogen, which is a soluble protein, is changed to fibrin, which is an insoluble protein, by adding thrombin, the function of this system is changed by adding thrombin to an albumin polymer-fibrinogen; GPIa / IIa conjugate to change into a gel state. You can check. Further, according to Example 7, adhesion of the albumin polymer-fibrinogen; GPIa / IIa conjugate to collagen was confirmed. The above results strongly support that the albumin polymer-fibrinogen; GPIa / IIa conjugate also has a bleeding site recognition ability and a hemostatic function.

Example 10 A conventional method (Thomas Haselgrubler et al, Bioconjugate Ch.
emistry, 1995, 6, 242-248) 5 mg of polyoxyethylene (POE) having a carboxyl group at one end and a pyridyldithio group at the other end and having a molecular weight of 1900.
(2.63 mmol) was dissolved in 100 mL of pure water.
Activated with ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). on the other hand,
7.2 wt% GP in 1 mL of PBS purged with nitrogen for 30 minutes
40 μL of Ibα solution (4.53 × 10 ⁻⁸ mol) was added,
After sufficient deoxygenation, 1.9 μL of the above POE solution (4.93 ×
10 ⁻⁸ mol) and stirred at 5 ° C. for 12 hours.

6.4 × 10 ^-3 mol / L was added to 19.5 mL of the 0.37% by weight albumin polymer solution prepared in Example 2.
172 μL of the concentrated SPDP solution was added, followed by stirring at 23 ° C. for 2 hours. The albumin polymer and unreacted SPDP were separated by centrifugation (3000 rpm, 2 minutes), and the resulting PD-bound albumin polymer solution was made up to 4 mL. To this was added 3.1 mg of dithiothreitol, and 10
After stirring for 2 minutes, unreacted dithiothreitol and by-products were separated by centrifugation (2000 rpm, 2 minutes).
The volume of the obtained SH-bound albumin polymer solution was adjusted to 3 mL. The albumin polymer solution was added to the PD-bound GPI
After adding the bα solution and stirring at 5 ° C. for 30 hours, unreacted GPIbα was separated by centrifugation (2,000 rpm, 2 minutes) to obtain an albumin polymer-GPIbα conjugate bonded via a POE spacer. The binding ratio of this conjugate is defined as the albumin molecule on the albumin polymer surface / GPIbα =
15/1. This albumin polymer-GPIbα
FITC labeling of the conjugate followed by flow cytometry showed the interaction between GPIbα and vWF.

Example 11 10.2 mg of SPDP was dissolved in 3 mL of a 99.5% ethanol solution to give 10.9 mM
An SPDP solution was obtained. 10.9 mM PDP in 5 mL of albumin polymer dispersion prepared in Example 1 (concentration in HSA 16 mg / mL)
50 mL of the solution was added and the mixture was stirred at 23 ° C. for 30 minutes. Gel filtration (Se
Remove unreacted SPDP and by-products with phadexG25)
11 mL of a pyridyldithio (PD) group-bound albumin polymer (PD-albumin polymer, [HSA] = 9.0 mg / mL) was obtained. GPI
Dissolve 100 mL of ba (74 mg / mL) in PBS and add 10.9 mM MSPDP to 20 mL.
mL was added and shaken at 22 ° C for 30 minutes. Subsequently, 10 mL of a 1 M dithiothreitol solution was added, and the mixture was shaken at 22 ° C. for 10 minutes. Then, 2.2 mg / mL thiol group-bonded GPIba (SH-GPI
2.3 ml of ba) were obtained. PD-albumin polymer and SH-GPIb
After mixing a and shaking at 25 ° C for 10 hours, ultracentrifuge (33000)
The supernatant was replaced with PBS, and the same procedure was repeated three times. After that, GPIba was quantified by ELISA, and GP
Iba-albumin polymer ([GPIba] = 27 mg / mL, [HSA]
= 15 mg / mL).

GPIba-albumin polymer ([GPIb
a] = 4 mg / mL, [HSA] = 1.0 mg / mL), vWf (50 mg / mL), and aggregation induced by the addition of ristocetin (1.0 mg / mL). The function can be confirmed. This GPIba-albumin polymer did not aggregate with vWf alone, but aggregated with the addition of ristocetin, and was specifically inhibited by the addition of GUR20-5, a GPIba monoclonal antibody. FITC-labeled GPIba-albumin polymer ([GPIba] = 4mg / mL, [HSA] = 1.0mg / mL)
When the interaction with immobilized vWf (10 mg / mL) was observed under a flow microscope under a flow condition, the GPIba-albumin polymer adhered to the immobilized vWf and GUR20-5
Was suppressed.

Example 12 5.7 mg of SPDP was dissolved in 5 mL of a 99.5% ethanol solution to give 3.65 mM MS
A PDP solution was obtained. 3.65 mM PDP in 8 mL of the albumin polymer dispersion prepared in Example 2 (HSA equivalent concentration: 15 mg / mL)
164 mL of the solution was added, and the mixture was stirred at 23 ° C. for 30 minutes. Gel filtration (S
ephadex G25) to remove unreacted SPDP and by-products.
24 mL of a PD-albumin polymer (s [HSA] = 4.0 mg / mL) was obtained. Recombinant GPIaIIa (rGIaIIa, 10mg / mL) 40mL
Was dissolved in 45 mL of PBS, and 5 mL of 3.65 mM PDP was added.
Shake for a minute. Continue with 1 mL of 1M dithiothreitol solution
After shaking at 22 ° C for 10 minutes, separate by gel filtration,
Mix PD-albumin polymer and SH-GPIaIIa,
After shaking at ℃ for 10 hours, the supernatant was replaced with PBS in an ultracentrifuge (33000 rpm, 20 minutes), and the same operation was performed three times.
GPIa / IIa is quantified by Method A, and GPIa / IIa is determined.
a-albumin polymer ([GPIA / IIa] = 1.0 mg / mL, [H
[SA] = 8.8 mg / mL).

Under the flow conditions of a FITC-labeled GPIa / IIa-albumin polymer ([GPIA / IIa] = 1.0 mg / mL, [HSA] = 8.8 mg / mL), the reaction with the immobilized collagen (10 mg / mL) was performed. When the interaction was observed with a fluorescence microscope, the GPIa / IIa-albumin polymer adhered to the immobilized collagen, and the GPIa / IIa monoclonal antibody Gi
It was suppressed by the addition of 9.

Example 13 5.7 mg of SPDP was dissolved in 4.3 mL of a 99.5% ethanol solution to obtain 4.2 mM
An SPDP solution was obtained. To 5 mL of the albumin polymer dispersion prepared in Example 2 (18 mg / mL in HSA equivalent concentration), 64 mL of a 4.2 mM PDP solution was added, followed by stirring at 23 ° C. for 30 minutes. Gel filtration (Seph
adexG25) to remove unreacted SPDP and by-products
-7 mL of an albumin polymer ([HSA] = 12 mg / mL) was obtained. 4.2mMSP in 500mL of fibrinogen (24mg / mL, dispersion medium PBS)
35 mL of DP was added and shaken at 22 ° C for 30 minutes. Then add 10 mL of 1M dithiothreitol solution, shake at 22 ° C for 10 minutes, separate by gel filtration, mix PD-albumin polymer and SH-fibrinogen, shake at 25 ° C for 10 hours, The supernatant was replaced with PBS in a centrifuge (33000 rpm, 20 minutes), and the same procedure was performed three times. After that, fibrinogen was quantified by ELISA, and a fibrinogen-albumin polymer ([fibrinogen] = 1.0 mg / mL) , [HSA] = 8.0mg / mL)
I got

FITC-labeled fibrinogen-albumin polymer ([fibrinogen] = 1.0 mg / mL, [HSA] = 8.0 mg / m
L) under the flow conditions of immobilized collagen (10mg / m
The interaction with L) was observed with a fluorescence microscope, and it was confirmed by double staining that the fibrinogen-albumin polymer was bound to the platelets adhered to the immobilized collagen.

Example 14 10.2 mg of SPDP was dissolved in 3 mL of a 99.5% ethanol solution to give 10.9 mM SPDP.
An SPDP solution was obtained. 10.9 mM PDP in 5 mL of albumin polymer dispersion prepared in Example 2 (concentration in HSA 16 mg / mL)
50 mL of the solution was added and the mixture was stirred at 23 ° C. for 30 minutes. Gel filtration (Se
After removing unreacted SPDP and by-products with phadexG25), P
11 mL of a D-albumin polymer ([HSA] = 9.0 mg / mL) was obtained.
Dissolve 50mL of GPIba (74mg / mL) in 50mL of PBS and add 10.9m
10 mL of MSPDP was added, and the mixture was shaken at 22 ° C. for 30 minutes. Subsequently, 5 mL of a 1 M dithiothreitol solution was added, and the mixture was shaken at 22 ° C. for 10 minutes. Then, SH-GPIba was obtained by gel filtration. GPIa /
Dissolve 20 mL of IIa (10 mg / mL) in 25 mL of PBS and add 3.65 mM PDP
And then add 5 mL of 1 M dithiothreitol solution
After shaking at 22 ° C for 10 minutes, use SH-GPIA / II by gel filtration.
a was obtained. And PD-albumin polymer and SH-GPIb
a, SH-GPIa / IIa was mixed, shaken at 25 ° C for 10 hours, and the supernatant was replaced with PBS using an ultracentrifuge (33000 rpm, 20 minutes). GPIb
a, Quantification of GPIa / IIa, GPIba, GPI
a / IIa-albumin polymer ([GPIba] = 10 mg / mL,
[GPIa / IIa] = 1 mg / mL, [HSA] = 15 mg / mL).

GPIba, GPIa / IIa-albumin polymer ([GPIba] = 10 mg / mL, [GPIa / IIa] = 1 mg / mL
, [HSA] = 15mg / mL), vWf (50mg / mL) and ristocetin (1.0mg / mL) were added to observe the aggregation induced by GPIba, GPIa / IIa-albumin polymer. You can check. This GPIba, GPI
The a / IIa-albumin polymer did not aggregate with vWf alone, but aggregated with the addition of ristocetin, and was specifically inhibited by the addition of GUR20-5, a GPIba monoclonal antibody. F
ITC-labeled GPIba, GPIa / IIa-albumin polymer ([GPIba] = 10 mg / mL, [GPIa / IIa] = 1 mg / mL,
[HSA] = 15mg / mL) under the flow condition,
When the interaction with GPIba-albumin polymer was observed with a fluorescence microscope, the GPIba-albumin polymer adhered to the immobilized vWf and was inhibited by the addition of GUR20-5. Also,
GPIba, GPIa / I for immobilized collagen
The adhesion of the Ia-albumin polymer to the collagen was suppressed by the addition of GUR20-5 and Gi9.

[0069]

As described above, according to the present invention, an albumin polymer having a disulfide bond between albumin molecules, which is useful as a carrier for supporting platelet membrane proteins,
A production method thereof, and a platelet substitute capable of recognizing and coagulating a bleeding site and stopping the bleeding by reliably binding a platelet membrane protein or a blood coagulation factor to the albumin polymer are provided.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 19/00 A61K 37/02 (72) Inventor Yuji Teramura 3-36-5 Takadanobaba, Shinjuku-ku, Tokyo 101

Claims (20)

[Claims] An albumin polymer having a disulfide bond between albumin molecules and having a particle diameter of 0.05 to 5 micrometers. 2. A platelet substitute obtained by binding at least one platelet membrane protein or a part of its structure to the albumin polymer according to claim 1. 3. The method according to claim 1, wherein the platelet membrane protein is GPIb, GPIa /
3. The platelet substitute of claim 2, which is IIa, GPIIb / IIIa, or a part of their structure. 4. The platelet substitute according to claim 2, wherein a blood coagulation-related protein is further bound to the albumin polymer. 5. The platelet substitute according to claim 4, wherein the blood coagulation-related protein is fibrinogen. 6. The platelet substitute according to claim 2, wherein the bond between the albumin polymer and the platelet membrane protein or a part of the structure thereof comprises a disulfide bond. 7. The platelet substitute according to claim 4, wherein the bond between the albumin polymer and the blood coagulation-related protein includes a disulfide bond. 8. The platelet substitute according to claim 2, wherein the bond between the albumin polymer and the platelet membrane protein or a part of the structure thereof comprises a polyoxyethylene chain. 9. The platelet substitute according to claim 2, wherein the bond between the albumin polymer and the blood coagulation-related protein comprises a polyoxyethylene chain. 10. An albumin polymer-protein conjugate obtained by binding at least one kind of platelet membrane protein or a part of its structure to the albumin polymer according to claim 1. 11. The platelet membrane protein is GPIb or GPIa.
11. The albumin polymer-protein conjugate of claim 10, wherein the conjugate is part of / IIa, GPIIb / IIIa, or a structure thereof. 12. The albumin polymer-protein conjugate according to claim 10, wherein a blood coagulation-related protein is further bound to the albumin polymer. 13. The albumin polymer-protein conjugate according to claim 12, wherein the blood coagulation-related protein is fibrinogen. 14. The albumin polymer-protein conjugate according to claim 10, wherein the bond between the albumin polymer and the platelet membrane protein or a part of its structure contains a disulfide bond. 15. The bond between the albumin polymer and the blood coagulation-related protein contains a disulfide bond.
An albumin polymer-protein conjugate according to claim 1. 16. The albumin polymer-protein conjugate according to claim 10, wherein the bond between the albumin polymer and the platelet membrane protein or a part of the structure thereof comprises a polyoxyethylene chain. 17. The bond between the albumin polymer and the blood coagulation-related protein contains a polyoxyethylene chain.
17. The albumin polymer-protein conjugate according to item 16. 18. A pharmaceutical comprising the albumin polymer-protein conjugate according to claim 10. 19. A method for producing an albumin polymer having an intermolecular albumin disulfide bond, comprising forming an intermolecular disulfide bond after cleaving the intramolecular disulfide bond of albumin. 20. The albumin polymer having a particle size of 0.05
20. The method according to claim 19, wherein an intermolecular disulfide bond of albumin is formed at a pH of 5.7 to 6.3 and a reaction temperature of 4 to 50C.