JP4694114B2 - Amphoteric polymer substance having L-lysine residue excellent in antithrombotic property, antithrombotic agent comprising the polymer substance, and medical device having the antithrombotic agent fixed thereto - Google Patents

Description

The present invention relates to an amphoteric polymer substance having an L-lysine residue having excellent antithrombotic properties, an antithrombotic agent comprising the polymer substance, and a medical device to which the antithrombotic agent is fixed .

  In recent years, synthetic polymer materials have been widely used for medical materials such as artificial organs and catheters. Representative examples of the medical polymer material include hydrophobic polymers such as polyester, polyvinyl chloride, polystyrene, polymethyl methacrylate, polytetrafluoroethylene, polyvinyl alcohol, polyether urethane, 2-hydroxy methacrylate. It is a hydrophilic material such as ethyl, polyacrylamide, or cellulose. Most of these conventional materials are mainly used with attention paid to their physical and mechanical properties.

  On the other hand, with the advancement of medical technology, the opportunity for contact between a living tissue or blood and a material is increasing, and the biocompatibility of the material has become a time of great interest. Concerning the prevention of blood coagulation on the surface of the material, continuous administration of anticoagulants represented by heparin has been carried out, but recently, abnormal lipid metabolism, prolonged bleeding time, decreased platelets, allergic reactions, etc. The effects of long-term heparin administration are becoming a problem. In order to solve these problems, it is desired to develop a blood contact material having antithrombogenicity that does not require heparin or can reduce the amount of use.

  For the purpose of developing these blood contact materials, a method of applying surface modification having antithrombotic properties using a polymer as a base material on the surface of a medical device that comes into contact with blood has been conventionally performed. These modifications mainly involve coating a polymer material with physical and chemical characteristics on the surface, and a method for minimizing blood coagulation and a polymer material impregnated with an antithrombotic substance on the surface. From there, it is roughly classified into methods for slowly releasing antithrombotic substances. Examples of the former method include Patent Document 1, Patent Document 2, and Patent Document 3.

  In Patent Document 1, a synthetic polymer containing a monomer unit or N-alkyl (meth) acrylamide unit having a phosphorylcholine group excellent in blood compatibility and a lactam group excellent in hydrophilicity and crosslinkability in a synthetic polymer is physically used. It is focused on the fact that it is easily insolubilized by chemical and chemical treatment and becomes a material with excellent blood compatibility. 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer, which has the effect of suppressing platelet adhesion, aggregation, and plasma protein adhesion, is retained on the surface for a long time, and is effective in suppressing blood adhesion to a minimum. ing.

  In Patent Document 2, 100 parts by weight of at least one polymer having a repeating unit derived from at least one monomer selected from the group consisting of olefin monomers having 2 to 10 carbon atoms and diene monomers having 4 to 15 carbon atoms; The material containing a composition comprising 0.003 to 1.0 parts by weight of a nucleating agent has a microphase separation structure consisting of a crystalline region and an amorphous region on its surface, or is desorbed even if adsorbed It focuses on the feature that is easy. Similar to Patent Document 1, an effect of minimizing the adhesion of blood is shown.

  Patent Document 3 immobilizes fibrinolytic enzymes such as urokinase and tissue plasminogen activator (hereinafter abbreviated as t-PA) on a biocompatible porous crystallized glass mainly composed of calcium phosphate and titanium. Thus, the invention relates to a biocompatible material having a thrombolytic ability, which is excellent in biocompatibility and has a function capable of continuously converting plasminogen in blood into plasmin.

  As a method regarding the latter, patent document 4 is mentioned, for example. This is (2R, 4R) -4-methyl-1- [N2-((RS) -3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl) -L-arginyl] -2- Piperidine carboxylic acid hydrate (hereinafter referred to as argatroban <clinically used as an antithrombotic agent; unlike antithrombotic substances such as heparin and urokinase, it dissolves in organic solvents such as alcohol>) 2 It is noted that argatroban is gradually released from the surface when dissolved in a mixed solution of at least seeds and applied to a polymer substrate.

  The blood contact material described above is not sufficient from the viewpoint of an inexpensive material that can exhibit stable antithrombotic properties including thrombus dissolution performance. In addition, urokinase is very small in human urine, and t-PA is very small in human uterus, blood vessel wall, etc., so it is expensive to use and difficult to mass-produce.

JP-A-7-284528 JP-A-10-99426 JP 7-101828 A JP 2000-060960 A

The present invention relates to an amphoteric polymer substance having a lysine residue that exhibits stable antithrombotic properties including thrombolytic performance, an antithrombotic agent comprising the amphoteric polymer substance having the lysine residue, and the antithrombotic property The purpose is to provide a medical device in which an agent is fixed to the surface . In general, plasma proteins such as albumin and fibrinogen are adsorbed on the surface of a material in contact with blood, and the secondary structure of these proteins changes due to adsorption. This change in secondary structure promotes further protein adsorption, and as a result, multiple protein adsorption layers are formed on the material surface. When such a multiple protein adsorbing layer is formed, platelets in contact therewith are activated and finally coagulate blood.

  In blood coagulation, insoluble fibrin is formed by activation of the blood coagulation system reaction. The coagulation system includes an intrinsic system that is initiated by contact with blood, and an extrinsic system that is initiated by tissue factor flowing into the blood and binding to the coagulation factor. Formed by the enzyme thrombin activated from prothrombin. The formed thrombus is decomposed by the thrombus fibrinolysis system (thrombolysis), and its basic principle is the degradation of fibrin by the enzyme plasmin. Plasmin exists as a precursor plasminogen in the blood, and is activated by a plasminogen activator such as tissue plasminogen activator (t-PA) or urokinase plasminogen activator to form plasmin, which degrades fibrin.

As a result of a series of studies relating to antithrombogenic materials using synthetic polymers, the present inventors have found that synthetic polymers of ampholytes having amino acid groups that are non-toxic and inexpensive to living bodies, that is, L represented by Chemical Formula 1 or Chemical Formula 2 below. The amphoteric polymer substance having a lysine residue has a function of suppressing the adsorption of plasma proteins and a function of suppressing blood coagulation, and the amphoteric polymer substance having an L-lysine residue represented by Chemical Formula 2 In addition to each function, it discovered that it had thrombus dissolution performance, and was able to reach this invention. In the present invention, the term “antithrombogenic” refers to a substance capable of exhibiting the functions as described above .

（前式中、×はＨ、ＣＨ ₃ 基あるいはＣＨ ₂ ＣＨ ₃ 基を意味する。）

(In the above formula, x means H, CH ₃ group or CH ₂ CH ₃ group. )

（前式中、×はＨ、ＣＨ ₃ 基あるいはＣＨ ₂ ＣＨ ₃ 基を意味する。）

(In the above formula, x means H, CH ₃ group or CH ₂ CH ₃ group. )

The amphoteric polymer substance having an L-lysine residue of the present invention can be produced, for example, by the following production method .
A raw material in which the ε-amino group and carboxyl group of the amino acid L-lysine are protected is reacted with methacrylic acid chloride in a chloroform solvent using triethylamine (TEA) as a catalyst, the reaction solution is concentrated, washed, dried, filtered, concentrated, The precursor monomer was prepared by purification using a mixed solvent of petroleum ether: dichloromethane . This precursor monomer is dissolved in tetrahydrofuran (THF) together with 2,2 ′ azobisisobutyric acid dimethyl ester, subjected to freezing-degassing-nitrogen substitution, free radical polymerization under reduced pressure, washing with hot hexane, and aqueous trifluoroacetic acid solution. The reaction solution is concentrated and then washed with diethyl ether to obtain a solution. The solution is neutralized, dialyzed using a semipermeable membrane, and lyophilized to obtain an L-lysine residue represented by the following chemical formula 3. An amphoteric polymer substance having a group [hereinafter also referred to as P (α-LysMA)] could be produced .

The raw material protecting the α-amino group and carboxyl group of the amino acid L-lysine is reacted with methacrylic acid chloride in N, N-dimethylformamide (DMF) solvent using triethylamine (TEA) as a catalyst, and the reaction solution is filtered and reduced in pressure. After concentration, the precursor monomer ( solid substance ) was prepared by washing with hexane . This precursor monomer is dissolved in 2,2 'azobisisobutyric acid dimethyl ester in ethyl alcohol, subjected to freezing-degassing-nitrogen substitution, free radical polymerization under reduced pressure, washed with hot hexane, and reacted with an aqueous sodium hydroxide solution. The reaction solution was neutralized, dialyzed using a semipermeable membrane, freeze-dried, and an amphoteric polymer substance having an L-lysine residue represented by the following chemical formula 4 (hereinafter also referred to as P (ε-LysMA)). Could be manufactured .

Conventionally, anticoagulants such as heparin and thrombolytic agents such as urokinase are known. However, these have side effects, are difficult to synthesize, purify or isolate, and are difficult to fix to the surface of a base material constituting a medical device with good durability. There was. On the other hand, the antithrombotic agent comprising an amphoteric polymer substance having an L-lysine residue represented by Chemical Formula 1 or Chemical Formula 2 of the present invention combines plasma protein adsorption suppression, blood coagulation inhibition, and thrombolytic properties. It has no side effects on the human body and can be widely applied to medical instruments that come into contact with blood. Further, the polymer itself represented by Chemical Formula 1 or Chemical Formula 2 can be synthesized by a simple synthesis method, and the obtained polymer can be easily separated and purified. Further, the antithrombotic agent comprising the polymer can be easily and excellently durable by polymerizing the precursor monomer on the substrate constituting the medical device as shown in Example 2 below. Can be fixed on the surface. Therefore, the medical device in which the antithrombotic agent of the present invention is fixed to the surface of the medical device based on the antithrombotic agent of the present invention and a polymer material that is in contact with the living body is not only economical and safe, but also marketable. Also excellent .

Hereinafter, although an Example is described based on P ((alpha) -LysMA) and P ((epsilon) -LysMA) , this invention is not limited to these.

A raw material obtained by protecting the ε-amino group of the amino acid L-lysine with a tBu group and a carboxyl group with a Boc group was reacted with methacrylic acid chloride in a solvent in chloroform using triethylamine (TEA) as a catalyst, and the reaction solution was concentrated. The reaction mixture was washed several times with% hydrochloric acid and water, dried over anhydrous magnesium sulfate, the reaction solution was filtered, and the solid obtained by concentration was columned with a mixed solvent of petroleum ether: dichloromethane (50:50 vol%). Purify by chromatography to obtain a white solid. The obtained white solid and 2,2 ′ azobisbutyric acid dimethyl ester are dissolved in tetrahydrofuran (THF) in a glass polymerization tube, freeze-degas-nitrogen substitution is performed several times in liquid nitrogen, and the mixture is sealed under reduced pressure. After free radical polymerization at 60 ° C. for 20 hours, it is washed with a large amount of hot hexane to obtain a white solid. The obtained white solid is reacted with an aqueous trifluoroacetic acid solution at 50 ° C. for 12 hours, and then the reaction solution is concentrated and washed with diethyl ether. The solution was neutralized with sodium hydroxide solution or hydrochloric acid, and dialyzed with acetyl cellulosic semipermeable membrane, and lyophilized was possible to produce the P (α-LysMA) ampholyte. This manufacturing method is shown in FIG .

The raw material obtained by protecting the α-amino group of amino acid L-lysine with Ac group and carboxyl group with Me group is reacted with methacrylic acid chloride in N, N-dimethylformamide solvent using triethylamine (TEA) as a catalyst, and the reaction solution is filtered. After concentration under reduced pressure, washing with hexane is repeated to obtain a white solid. The obtained white solid and 2,2′-azobisbutyric acid dimethyl ester were dissolved in ethyl alcohol in a glass polymerization tube, freeze-deaerated-nitrogen replacement was performed several times in liquid nitrogen, and sealed under reduced pressure. After free radical polymerization at 60 ° C. for 20 hours, it is washed with a large amount of hot hexane to obtain a white solid. The obtained white solid was dissolved in 2N aqueous sodium hydroxide solution and reacted at room temperature for 12 hours. The reaction solution was neutralized with hydrochloric acid or aqueous sodium hydroxide solution, dialyzed using an acetylcellulose semipermeable membrane, and frozen. The amphoteric electrolyte P (ε-LysMA) could be produced by drying. This manufacturing method is shown in FIG.

The structure of the obtained precursor monomers in the production process of the P (α-LysMA) was confirmed by NMR and elemental analysis. 1H-NMR (CDCl3): 1.4 [s, 9H, -C (CH3) 3], 1.45 [s, 9H, -C (CH 3) 3], 1.5 [s, 2H, -CH _{2 -], 1.7 [d,} 2H, -CH2 -], 1.9 [d, 2H, -CH 2 -], 2.0 [s, 3H, -CH 3], 3.1 [d, _{2H, -CH 2 -], 4.5} [s, 1H, -CH <], 5.3 [s, 1H, -NH -], 5.4,5.8 [s, 2H, = CH2], Elemental analysis: As C19N2H34, calculated value C: N: H = 61.61%: 7.60%: 9.19%, measured value C: N: H = 62.28%: 7.46%: 9.43 %
From the confirmation data, it was confirmed that the precursor monomer had a structure represented by the following chemical formula 5 .

The structure of the obtained precursor monomers in the production process of the P (ε-LysMA) was confirmed by NMR and elemental analysis. _{1H-NMR (CDCl3): 1.4} [s, 2H, -CH 2 -], 1.6 [s, 2H, -CH 2 -], 1.8 [d, 2H, -CH 2 -], 1 _{.9 [s, 3H, -CH3]} , 2.0 [s, 3H, -CH 3], 3.3 [d, 2H, -CH 2 -], 3.75 [s, 3H, -CH 3] 4.5 [s, 1 H, —CH <], 5.4, 5.8 [s, 2 H, ═CH ₂ ], 6.1 [s, 1 H, —NH—], 6.5 [s, 1H, -CONH-], elemental analysis: calculated as C19N2H34, C: N: H = 57.79%: 8.14%: 10.37%, measured value C: N: H = 57.10%: 8 .32%: 10.63%
From the confirmation data, it was confirmed that the precursor monomer had a structure represented by the following chemical formula 6 .

The fact that P (α-LysMA) and P (ε-LysMA) became polymer materials was confirmed by disappearance of the double bond peak from ¹ H-NMR. Moreover, it was confirmed by ¹ H-NMR and ¹³ C-NMR that the protective groups for P (α-LysMA) and P (ε-LysMA) were removed. It was confirmed by isoelectric point measurement by an acid-base titration method that P (α-LysMA) and P (ε-LysMA) have an amphoteric electrolyte structure (FIG. 2). In FIG. 2, ● represents the measurement result of P (α-LysMA), and ■ represents the measurement result of P (ε-LysMA).

Immobilization of an amphoteric polymer substance having a lysine residue represented by Chemical Formula 1 or 2 on the base material surface of a synthetic resin, which is a medical material, was performed by an argon plasma-post-post polymerization method. A base material adjusted to an arbitrary size is placed on the bottom of the separable flask of the plasma irradiation apparatus, and the pressure is adjusted to 1.3 Pascal by adjusting the flow rate of argon. Using a capacitive load type for high-frequency load, high-frequency power to the copper plate 2 places of wound separable flask (manufactured by JEOL Ltd. JRF-300 type) Lysine (frequency of 13.56 MHz) indicated a by Formula 5 or 6 10 ml of tetrahydrofuran containing a precursor monomer of an amphoteric polymer substance having a residue and a substrate are placed. The polymerization tube is repeatedly frozen and degassed in a liquid nitrogen bath to remove dissolved oxygen, sealed under reduced pressure, and post-polymerized for a predetermined time while shaking at 60 ° C. Substrates obtained by immersing the post-polymerized base material in a large amount of tetrahydrofuran for 24 hours, washing with tetrahydrofuran and then vacuum-drying for 24 hours to graft an amphoteric polymer substance having a lysine residue represented by Chemical Formula 1 or 2 Get.

There is no problem if the base material of the medical device for immobilizing the antithrombotic agent of the present invention is a polymer material such as a synthetic resin, which is a known medical material. Even if the material does not have a functional group on the surface, the polymer of the present invention can be immobilized by surface treatment. Examples of such medical materials include catheters and blood packs made of polyurethane or polyvinyl chloride, artificial organs made of polytetrafluoroethylene, syringes made of polyolefin such as polypropylene, nylon, etc. Artificial blood vessels made of these polyamides.

The above-mentioned antithrombotic agent can be fixed to a base material of a sex medical device by a known method . In particular , fixing using plasma treatment can process a large amount of medical devices in a short time. Furthermore, since the treatment is performed in a gas, it is preferable that cleanliness can be maintained.

In general, plasma proteins such as albumin and fibrinogen are adsorbed and denatured on the surface of a material in contact with blood, and blood is coagulated by activating platelets. Therefore, fluorescence measurement using plasma protein was performed to quantitatively measure the adsorption of plasma protein in the presence of the amphoteric polymer substance having the lysine residue . Albumin in the blood as a plasma protein to be used for measuring, gamma chromatography globulin, using fibrinogen, plasmin, plasminogen. In addition, the same test was performed when polyethylene glycol (hereinafter referred to as PEG) whose biocompatibility is known as a comparative substance and when neither the plasma protein nor PEG was used . In this test, the plasma protein and the amphoteric polymer substance having the lysine residue were used at the same concentration. The results are shown in FIGS. A decrease in fluorescence intensity indicates adsorption or denaturation of the material and protein.

3 is a fluorescence measurement of plasma protein albumin, FIG. 4 is a fluorescence measurement of plasma protein γ-globulin, FIG. 5 is a fluorescence measurement of plasma protein fibrinogen, FIG. 6 is a fluorescence measurement of plasma protein plasmin, and FIG. 7 is a plasma protein plasminogen. It is the result of fluorescence measurement of. Each figure is a plot of the time course and the maximum fluorescence intensity around 350 nm, and a decrease in fluorescence intensity indicates adsorption or denaturation of the material and protein. When P (α-LysMA) and P (ε-LysMA) were used in each figure, no decrease in fluorescence intensity was observed since the fluorescence wavelength did not shift. In addition, albumin, γ-globulin, fibrinogen and plasmin were adsorbed somewhat, but the adsorbed amount was not so different from that of PEG. P (α-LysMA) interacted only with plasminogen. 3 to 7, ■ indicates that nothing is fixed to the base material (none), □ indicates that P (α-LysMA) is fixed to the base material, ○ P indicates that P (ε-LysMA) is the base material And ● indicate the test results when PEG is adsorbed on the substrate .

The interaction between plasminogen, which is greatly involved in the thrombolytic action, and the amphoteric polymer substance having a lysine residue was evaluated using a biomolecular interaction analyzer (hereinafter abbreviated as IAsys). Two minutes after the start of measurement on the chemically immobilized plasminogen surface, an amphoteric polymer substance having a predetermined concentration of lysine residue is added to the plasminogen-immobilized surface, and the change in the resonance angle of light is traced. Thus, the interaction was analyzed in real time, and the change in the resonance angle of light with time was plotted. The curve in the addition of an amphoteric polymeric substance having a lysine residue shows a specific interaction with plasminogen. The result is shown in FIG.

The reason for the amphoteric polymer material having a lysine residue of the present invention suppressed the adsorption of plasma proteins, the structure of the amphoteric polymer materials. Usually, when an artificial material and a biological component come into contact with each other, the plasma protein adsorption action proceeds first. Because it is a synthetic polymer, it is considered a stealth material that is difficult to recognize as an artificial material for biological components.

In order to examine the effect of the amphoteric polymer substance having a lysine residue of the present invention on blood coagulation, thrombin activity using a chromogenic synthetic substrate was quantitatively examined. The experimental method is as follows. After adding the amphoteric polymer substance having a predetermined concentration of thrombin and the lysine residue of the present invention and stirring for 10 minutes, a substrate S-2238 that specifically recognizes thrombin and exhibits color development is added to start from S-2238. The absorbance of liberated p-nitroaniline was followed for 120 minutes using a fluorescence microplate reader, and the change in absorbance of p-nitroaniline over time was plotted. A decrease in the slope of the resulting curve indicates inhibition of blood clotting activity. The results are shown in FIG. (2) shows the test results when nothing is added (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

The main component of the thrombus is fibrin formed by activation from fibrinogen. Therefore, the influence of lysine methacrylamide [P (α-LysMA) and P (ε-LysMA)] on fibrin formation was examined. The experimental method is as follows. After adding an amphoteric polymer substance having a predetermined concentration of thrombin and lysine residue and stirring for 10 minutes, the turbidity change of fibrin formed by adding fibrinogen is traced for 120 minutes using a fluorescent microplate reader. The change in turbidity with the course and fibrin formation was plotted. A decrease in the slope of the resulting curve indicates inhibition of blood clotting activity. The results are shown in FIG. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

The effect of addition of P (α-LysMA) and P (ε-LysMA) on thrombin activity inhibition was examined in terms of enzyme kinetics. The results are shown in FIG. In the figure, the inverse of the substrate concentration and the inverse of the reaction rate are plotted. The suppression of the activity of the coagulation factor thrombin in the presence of P (α-LysMA) and P (ε-LysMA) can be compared to the thrombin activity suppression of the biopolymer hirudin. Hirudin binds to thrombin at the N-terminal or C-terminal of hirudin. In particular, blood coagulation is suppressed because the C-terminal site is the same as the fibrinogen binding site of thrombin. Moreover, the result of having examined the addition effect of Ri P ((alpha) -LysMA) and P ((epsilon) -LysMA) in thrombin activity inhibition from an enzyme reaction kinetics. P (α-LysMA) and P (ε-LysMA) inhibited thrombin activity anti-competitively. Therefore, it is considered that P (α-LysMA) and P (ε-LysMA) directly interact with the blood coagulation active site of thrombin, thereby inhibiting the binding of fibrinogen and thrombin and suppressing blood coagulation. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

To examine the effect of P (α-LysMA) and P (ε-LysMA) on thrombolysis, plasmin activity using a chromogenic synthetic substrate was quantitatively examined. The experimental method is as follows. After adding a predetermined concentration of plasmin and polymer and stirring for 10 minutes, the substrate S-2251 was added and the absorbance of p-nitroaniline released from S-2251 was traced for 120 minutes using a fluorescent microplate reader. The change in absorbance of p-nitroaniline over time was plotted. An increase in the slope of the resulting curve indicates an increase in enzyme activity. The results are shown in FIG. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

Fibrin is artificially prepared by reacting blood concentrations of fibrinogen and thrombin for 40 minutes, and P (α-LysMA) or P (ε-LysMA) and plasmin are added to the fibrin, and the turbidity generated by fibrin degradation is increased . Changes were followed for 240 minutes. It shows that fibrin degradation is accelerated | stimulated, so that the fall of the turbidity after plasmin addition is large. The results are shown in FIG. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

Plasminogen / t-PA activity using S-2251 was quantitatively examined. The experimental method is as follows. Plasminogen, P (α-LysMA) and P (ε-LysMA) were added and stirred for 10 minutes, S-2251 and t-PA were added, and the absorbance of p-nitroaniline was measured using a fluorescence microplate reader. Followed for 240 minutes. An increase in the slope of the resulting curve indicates an increase in enzyme activity. The results are shown in FIG. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

Plasminogen / t-PA activity was measured using artificially prepared fibrin. After adding P (α-LysMA) or P (ε-LysMA) and plasminogen to artificially prepared fibrin and stirring for 10 minutes, t-PA was added to change the turbidity caused by fibrin degradation. Tracked minutes. The larger the slope of the straight line obtained after the addition of t-PA, the more the fibrin degradation in plasminogen / t-PA activity is promoted. The results are shown in FIG. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

The effect of addition of lysine methacrylamide [P (α-LysMA) and P (ε-LysMA) in the plasmin reaction system was examined from the enzyme kinetics. The results are shown in FIG. Plot the reciprocal of the substrate concentration on the horizontal axis and the reciprocal of the reaction rate on the vertical axis, Lineweaver-Burk
Evaluated from Plot. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

The effect of polymer addition in the plasminogen / t-PA reaction system was examined from the enzyme kinetics. The results are shown in FIG. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added . Plot the reciprocal of the substrate concentration on the horizontal axis and the reciprocal of the reaction rate on the vertical axis, Lineweaver-Burk
Evaluated from Plot. (2) shows the test results when nothing is added to the substrate (none), ▲ shows the results when P (α-LysMA) is added, and ● shows the results when P (ε-LysMA) is added .

Hereinafter, a mechanism in which P (α-LysMA) of the present invention specifically interacts with plasminogen in blood to increase the thrombolytic property will be described.
The plasminogen present in the blood has a site (lysine binding site) that specifically recognizes the ε-amino group of the amino acid L-lysine of fibrin, and plasminogen binds to fibrin through the lysine binding site. And exhibits thrombolytic properties. In addition, when thrombolytic action occurs on fibrin, plasmin does not receive inhibition of thrombolytic action because the lysine binding site, which is the binding site with α2-plasmin inhibitor, which is a plasmin inhibitor, is occupied by fibrin. It can degrade fibrin. Therefore, since P (α-LysMA) is free of the ε-amino group and carboxyl group of L-lysine, it interacts specifically with the lysine binding site of plasminogen and increases thrombosolubility. However, P (ε-LysMA) does not specifically interact with the lysine binding site of plasminogen because the α-amino group and carboxyl group of L-lysine are free. The solubility is not considered to be directly affected.

Moreover, although each said test result was performed about P ((alpha) -LysMA) and P ((epsilon) -LysMA), in said chemical formula 1 and chemical formula 2, even if X is structures, such as H and -CH2CH3, for example. It is considered that the same performance in antithrombogenicity is exhibited.

It is a figure which shows the manufacturing method of the amphoteric polymer substance which has a lysine residue of this invention. It is a figure which shows the measurement result of the isoelectric point measurement of the polymer composition by an acid base determination method. It is a figure which shows the result of the fluorescence measurement of plasma protein albumin. It is a figure which shows the result of the fluorescence measurement of plasma protein (gamma) -globulin. It is a figure which shows the result of the fluorescence measurement of plasma protein fibrinogen. It is a figure which shows the result of the fluorescence measurement of plasma protein plasmin. It is a figure which shows the result of the fluorescence measurement of plasma protein plasminogen. It is a figure which shows the result of the molecular interaction analysis by IAsys measurement. It is a figure which shows the result of thrombin activity inhibition evaluation using the substrate S-2238. It is a figure which shows the result of the fibrin formation inhibition test of the polymer composition of this invention. It is a figure which shows the result of the kinetic analysis of thrombin activity inhibition using the substrate S-2238. It is a figure which shows the result of plasmin activity evaluation using substrate S-2251. The degradation of artificial fibrin by plasmin activity was evaluated by following turbidity change. It is a figure which shows plasminogen / t-PA activity evaluation using substrate S-2251. It is a figure which shows the result of the evaluation which evaluated degradation of the artificial fibrin in plasminogen / t-PA activity by tracking turbidity change. It is a figure which shows the result of the enzyme reaction kinetic analysis in plasmin activity. It is a figure which shows the result of the enzyme kinetic analysis in plasminogen / t-PA activity.

Claims (5)

An antithrombotic agent comprising an amphoteric polymer substance having an L-lysine residue represented by the following chemical formula 1 .

(In the above formula, X means H, CH ₃ group or CH ₂ CH ₃ group.) An antithrombotic agent comprising an amphoteric polymer substance having an L-lysine residue represented by the following chemical formula 2 .

(In the above formula, X means H, CH ₃ group or CH ₂ CH ₃ group.) A medical device, wherein the antithrombotic agent according to claim 1 or 2 is fixed to a surface of a medical device based on a polymer material that comes into contact with a living body. The antithrombotic agent according to claim 1 or 2 is applied to the substrate surface of the medical device by polymerizing a precursor monomer of the amphoteric polymer substance represented by Chemical Formula 1 or Chemical Formula 2 on the surface of the base material of the medical device. The medical instrument according to claim 3 , wherein the medical instrument is fixed. A precursor monomer obtained by reacting an amino acid L-lysine in which an ε-amino group and a carboxyl group are protected with a protecting group with methacrylic acid chloride is polymerized, and the protecting group is removed after the polymerization. A method for producing an amphoteric polymer substance having a lysine residue represented by the following chemical formula 3.

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