JPH05507298A - Non-thrombogenic glycosaminoglycan copolymer - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Thrombogen Glycosaminoglycan Mukai Gokyokutai The present invention is biocompatible and non-thrombogenic in vitro and therefore Concerning the synthesis of glycosaminoglycan copolymers that can be used in medical devices. Ru.

11 to 5 The clinical use of devices or materials that come into contact with blood is the practice of modern medicine. It is well known that it is important. Generally speaking, biomedical All materials used in the device need to be biocompatible. biomedical equipment The use of a The main constraint is that the surface of the foreign body has a tendency to promote thrombosis. It is important to have a direction.

Although the use of anticoagulants has minimized thrombotic problems with extracorporeal devices, systemic Its use often causes complications such as bleeding tendency (Porter, J. and Tick, H. j　Am　MedAx 237 volumes. 879-1181 page. (1977). Because of these problems, heparin substitutes and analogs are being actively studied, but these compounds uniformly have lower anti-inflammatory properties than heparin. It showed blood clotting activity (Casu. B. dv Ca b d ate C am Bfochem Vol. 43, pp. 51-134, 1985).

Considerable efforts have been made to develop blood-compatible materials with which devices can be prepared. It has been done. Methods of interfering with surface thrombus formation in materials include placing the surfaces of the polymers against each other Designed to minimize the effect or selectively adsorb the passivating albumin layer. This includes: In addition, pharmacologically active drugs can be added to polymers by bulk dispersion. Efforts have been made to incorporate anticoagulants into polymers or to immobilize anticoagulants on surfaces.

Other methods include biological coatings or There is a method to create a pseudoneoplastic lining. Ru. However, no synthetic surface has ever been prepared that permanently resists clot formation. stomach.

In recent years, there have been numerous reports on the efforts made to synthesize polymers with improved biocompatibility. A notice has been issued. The hemocompatibility of polyurethanes has recently been reviewed (Ito , Y, and Imanishi, Y, , Crttic v’ev i B iocomat'b''tCRCPass.

5, pp. 45-104, 1989). Various physical properties of synthetic polymers How prosthetic materials (biomaterials) that come into contact with tissue can cause blood clots? have been tested to determine whether they are related, but so far these No one property or combination of properties can predict biocompatibility (Adda ms.

G, A,, Trans 1 antation m a tatio To a Volume 3, pages 45-56, 1986).

Eliminates the risks associated with systemic use of heparin while taking advantage of its anticoagulant properties. Attempts are being made to avoid this. When the surface of a biomaterial is heparinized, its The material can be made non-thrombogenic. slowly releases heparin Although it certainly protects anticoagulant properties when it is supplied, there is a limit to its supply [Iman ishi, Yl, Current TOoas’n Pamer 5cfen ce (edited by R, M, Ottenbrite et al.), volume 1, pages 58-711, 1987, Hansen Publishers, Munich].

Methods for attaching heparin to surfaces have also met with limited success. polymer The ionic binding of heparin to the polycationic surface of [Tanzi, M. , C, et al. l mars’n Med’cine II (E, Chie llint et al.), pp. 67-85 and 91-99, 1986, Ple num Press, two knee York]. Heparin also has covalent bonds on various surfaces. [Jozefowicz, M., and Jozefowicz, J., ol rae s’ Med’cLLILL (edited by E. Chieltni et al.), pp. 41-65, 1986, P. lenum Press, New York, USA]. For coating various surfaces The heparin-polyvinyl alcohol hydrogel used has improved biological properties. Compatibility has been reported (ip, W, F, et al., JBi.

119, pp. 1li1-178, 1985). this These materials are only suitable for use in short-term devices. Not enough potency, limited mixing, degradation by plasma heparinases, and species that neutralize heparin. There are problems due to separation and lack of platelet inhibitory effect.

All (different methods include chemical or radiochemical treatment of heparin) According to this method, the covalently incorporated heparin is combined with a monomer. Macro radicals are generated that produce phosphorus copolymers. But these things Some have anticoagulant activity, some do not, and heparin itself It is rather inferior compared to [Boffa, M, C, et al., Tromb Hae mostas vol. 41, p. 346, 1979; Salzman, E., et al. , Koumi I Soukou and Biolo of He art (R, Lundbl ad et al. (eds.), Elsevier, -1-x-York, p. 435, 191 10 years].

It was heparinized in an attempt to prepare a better nonthrombogenic surface. Other synthetic polymers have also met with limited success. Meto Beef Shiporiech Photographs of lene glycol methacrylate and dimethylaminoethyl methacrylate The graft copolymerization is carried out to give polyacrylonitrile, and then the resulting graft copolymer is A new antithrombotic polymer was synthesized by heparinizing M iyama, H, et al., lu"剋"■KU''■volume 20, pp. 895-902, 1 986). commercially available polyurethane surfaces for long-term biomedical applications. was grafted with polyethylene oxide and then modified to immobilize heparin. (Han, D, Ni, Ra, J B’omed, ate e 1 anal ■ Anus 23 (AI> volume, pp. 87-104, 1989: Park, ![-D, et al., n'ns, 5th ual Sac, 'omater, vol. 12, p. 26, (1989) Similarly to O, 2-hydroxyethyl methacrylate-styrene resin random copolymer (Lea, S, et al, Tan 5t nu Soc format e, vol. 12, p. 25, 1989), and polydimethyl containing heparin. Amphoteric Pro-Tsuku copolymer of siloxane and polyethylene oxide (Graing) er, D, W, et al., J BioIled Mater Res Volume 22, pp. 231-250, 1988; Piao, A., Z. et al., rans 1 5th Annual Sac iomater 12L 28L 1989 ), functionalized prepolymers, diisocyanates and derivatized It was prepared by a series of coupling reactions using parin. some of these has considerable difficulty associated with a series of derivatization reactions to prepare, and Palin's antithrombin ■ cofactor activity is sacrificed.

Other compounds exhibiting anticoagulant activity have been described in the literature. Platelets against polymer Inhibition or prevention of adhesion of polymers and prostaglandin-heparin conjugates (Kil, S, f, , Art'f Orans Vol. 11, pp. 228-236, 19117). Aini(, Pro Stacycline is extremely expensive and difficult to prepare; It is extremely unstable. A number of polymeric materials have some antithrombotic activity. , sulfonate group, carboxylic acid group, amino acid sulfamide group or amide group, Cross-linked polystyrene, polysaccharide or polystyrene-polyethylene graft copoly (Jozefovicz, M., et al.).

P 1 me s i Med’c’ e II, pp. 41-65, 190) . It was found that the anticoagulant activity of these substances depends on the amount and nature of the substituents. However, these agents were generally significantly less potent than heparin (Mauz ac, M., and Jozefovicz, J., Lyμrom 1B5, 30 1 page, 1984).

Recently, the outer surface of the lipid matrix of naturally occurring biological membranes () Iayvard, J. A. and Chapman, D., Biomaterials Volume 5, 1 35-142, 1984), phosphatidylcholine or its In particular, new products with a backbone of polyurethane structure with pendant groups containing congeners A new polymer was reported (Chapman, D. and Valencia, G, P., U.S. Pat. No. 4,589,386, 19117). phosphorylco Phosphorus is grafted or attached to the cellulose acetate membrane by radiation methods. (Jayasree, G., and Sharma, C.P., Tra. ns 15th annual Sac Biomater Volume 12, Page 185.

(1989) and a copolymer of phosphorylcholine and methyl methacrylate were also reported. (Nakabayashi, N, et al. - sister ■yu A nua Sa c Bformate, Vol. 12, p. 1g, 19119). However, platelet phosphorus The lipids, alkylacylphosphatidylcholines, are extremely potent platelet stimulators. It is well known that it is converted by enzymes into the platelet activating factor. Is Rukoto.

Covalent or graft attachment of heparin fragments with modified reducing terminal residues (reductive amination reaction): produced non-thrombogenic surface has been reported (Larm, O, et al., Biomater Med D ev Arttf Orans Volume 11, Page 161.

(1983). However, oligosaccharides derived from heparin are assembled into polymer backbone chains. This has not yet been attempted.

The structural requirements for significant heparin anticoagulant activity are similar to those for antithrombin. It was shown that a specific trisaccharide sequence of heparin constitutes the smallest binding site. Significant evidence can be found in the literature (Casu, B., Danlta Bee Volume 43, 5l-134Ji, 1985). This specific heparin segment is sufficient for antithrombin-mediated inhibition of factor Xa, but Insufficient for effective inhibition of thrombin and development of sufficient anticoagulant activity . In fact, antithrombin-mediated inhibition of thrombin involves At least four (trisulfated) trisaccharide uni-nod segments in addition to the trisaccharide sequence of the sex site (Lane, D. A., et al., Biochem J 21a Vol., pp. 725-732, 1984). However, these heparin fragments The size of Therefore, the above fraction contains platelet adhesion/clumps and other plasma components. ◎Therefore, the smaller heparinase with anti-Xa activity is responsible for causing the binding. Incorporating the nanosegments into the polymer backbone provides a permanently antithrombotic and long-lasting , nonthrombogenic (thromboresistant), or antithrombinic biomaterial will be provided.

The advantage of using less heparin segments is the high anticoagulant properties of regular heparin. Prevent thrombosis while reducing or eliminating the risk of bleeding associated with hemodynamic activity It is to be.

Biomaterials incorporating such heparin fragments into the polymer backbone terial may be useful in preventing superficial thrombosis in a more effective manner. The reason The reason is that these components, such as lipoproteins and fibrinogen, are present on the surface of blood plasma. There are various types of hepatopathols, including antithrombin (which is contained in much larger amounts than antithrombin). Less susceptible to competitive interactions with phosphorus-neutralizing or heparin-degrading species It is the body. This surface also promotes platelet adhesion and platelet aggregation; fibronectin interacts with cell surface components such as laminin. Probably less. Moreover, the substance has a negative polarity somewhat similar to that of the vascular endothelium. A non-thrombogenic surface with a charge and a binding site for antithrombin Although essential, it does not interfere with the interaction between fibrinogen or platelets and thrombogen. By mimicking the inappropriate sulfate group arrangement for antithrombin, will provide affinity.

Oitomo 81jo Useful for biomedical devices that come into contact with blood and are thromboresistant and antithrombotic There continues to be a demand for new materials, and a large number of studies are being conducted. There are two basic methods It has been adopted. One method is to prevent the initiation of blood clot formation on the surface of the medical device. A method of administering blood thinners such as heparin at or around the same time to stop It is. Other methods include adding blood thinners such as heparin to devices that come into contact with blood. This method involves direct derivatization of the moiety at the base. The latter method requires a rather complex derivatization method is often used. In addition, both of the above methods require heparinization under physiological conditions. limited by lack of long-term stability.

The present invention provides an anticoagulant and antithrombotic glycosaminoglycan fragment. As a nomer unit, it is a non-grid unit that provides the necessary physical properties to make the device Xli. By incorporating it into a copolymer with a cosaminoglycan monomer unit , solves the above problems. Therefore, the device of the invention reduces the part that comes into contact with blood. It is prepared from a copolymer that itself has anticoagulant, nonthrombogenic, and antithrombotic properties. can be done. In addition, these copolymers are suitable for devices that come into contact with biological substances such as blood. It can be used to coat the surface of the device. Finally, water-soluble copolymers , to treat clotting disorders or certain immune disorders, cardiovascular diseases and viral diseases. It is useful as a pharmacological agent used to

In addition, the copolymer of the present invention can be synthesized in the presence of a crosslinking agent, if desired, to make it harder. Copolymer forms with physical properties can be provided. This copolymer is Utilizing monomer units with certain functional groups that can be further derivatized It can be further derivatized into additional moieties by doing so. This derivatization may include, for example, affinity ligands, labels, or additions that promote anticoagulation. Includes derivatization to glycosaminoglycan units.

Accordingly, in one aspect, the present invention comprises a biomedical device in contact with blood. and/or biologically compatible copolymers suitable for coating. child The copolymers of at least one monomer that is a glycosadi/glycan fragment capable of unit, as well as the appropriate physical characteristics necessary to configure and code the above equipment. at least one added non-glycosaminoglycan monomer unit that confers It can be prepared by copolymerizing with

Of course, mixtures and/or copolymerization of glycosaminoglycan fragments Mixtures of monomer units can be used to prepare the copolymer. Obtained Depending on the physical properties required for the copolymer, non-glycosaminoglycan monomers are The choice of what to make of the unit is decided. This physical property also depends on the crosslinking used It is also affected by the type and amount of the agent.

Water-soluble forms of the copolymers may also be used in the present invention, in the case of derivatized forms of the above-mentioned copolymers. Included in light In another aspect, the invention provides that individual monomer units or components thereof can be copolymerized. Relating to a method of preparing the copolymers of the present invention, which comprises mixing under conditions in which polymerization occurs. do.

In yet another aspect, the present invention provides related to medical devices that have been

Figure-Plane fl Nest 3JA-Tsui Figure 1 shows heparin decomposition reaction, (A) nitrite cleavage reaction, and (B) heparin decomposition reaction. Typical products from enzyme digestion are shown.

Figure 2 shows acrylamide and heparinase produced by typical (A) heparinase digestion. Lin fragment (Nl performed), (B) allylglycyl of the product of hevarinase digestion Coside derivatives and (C) aminoethyl methacrylate products of nitrous acid cleavage reaction Figure 2 shows a diagram of copolymerization with the derivative (Example 2).

Figure 3 shows the relationship between acrylamide and heparin + glycosides obtained from heparinase digestion. This is a proton NMR spectrum at D20 of "Constant Copolymer" (Preparation Method A).

This copolymer (prepared in Example 1 and named CH3-139) was produced using Sephadex Desalting with a column of G-25, then dialysis against 50mM EDTA, and finally I 7.9% by weight heparin flag after purification by dialysis against hard ice Contains ment. The inset shows the proton NMR spectrum of the corresponding heparin glycoside. Indicates the vector.

Figure 4 shows the result of a limited deamination cleavage reaction between acrylamide and nitrous acid. D20 of a copolymer of heparin decasaccharide with an aminoethyl methacrylate derivative Preparation method B) shows a typical proton NMR spectrum. This copolymer ( C1111-32B) prepared in Example 2 elutes at 0.2M NaCl After purification on a Sepharose 4B column and dialysis against pure water, 28 wt. contains fragments.

Figure 5 shows the opening by tolylene diinocyanate and ethylene glycol with nitrous acid. Prepared by copolymerizing with low molecular weight heparin produced by cleft reaction. Proton NMR spectrum of polyurethane type polymer in deuterated DMSO shows.

This copolymer (CF3-22C prepared in Example 6) was prepared using chloroform, ethanol, Wash with alcohol and finally water to remove unreacted heparin and other monomers. It contains 0.7% by weight of heparin after thorough washing.

Figure 6 shows the relationship between acrylonitrile and heparin glycosides (by hevarinase digestion). The proton NMR spectrum in deuterated DMSO of the copolymer of is shown. this The polymer was prepared in Example 11 and contains 8% by weight heparin.

Figure 7 shows tolylene-2,4-diisocyanate prepared in Example 18 and tris - Low molecular weight heparin (4 Typical FTIR spectra of polyurethane-type copolymers with Showing the spectrum.

This copolymer contains 5.24 μg heparin/mg solids.

Figure 8 shows the copolymerization of copolyurethane synthesis with low molecular weight heparin fragments. Show the diagram.

“2 for The present invention is useful for constructing and/or coating medical devices that come into contact with blood. Relating to polymers. These copolymers have biocompatible physical properties that make blood non-biocompatible. Used in contact with blood without the thrombotic effects that normally occur when in contact with synthetic surfaces It is advantageous because it has physical properties that allow it to be used.

As used in this application, the term "non-thrombogenic" refers to substances that themselves are thrombogenic. and does not cause blood clotting. ``antithrombotic'' and ``anticoagulant'' The term ``sexual'' means that the substance itself does not cause clot occlusion, but also that other stimuli means actively preventing the formation of blood clots promoted by Copolymerization of the present invention The conjugate is antithrombotic and nonthrombogenic.

These physical properties give the copolymer the antithrombotic and nonthrombotic properties of glycosaminoglycans. conferred by incorporating a genetic fragment. The fragment is one Generally prepared by depolymerizing appropriate glycosaminoglycans, It is not entirely possible to prepare these fragments synthetically. current time In this respect, it is more economical to prepare by depolymerization.

Glycosaminoglycans (GAGs) are generally sulfated biopolymers that It is a repeating trisaccharide unit of uronic acid-glycosamine disaccharide. GAG is classified The divisions are dependent on the type of these subunits. Heparin is probably the best ( Some of the known GAGs and available hydroxyl and amino positions Most of the repeating subunits of glucosamine iduronate are sulfated. It is configured. Dermatan sulfate is coupled to galactosamine residues, Iduronic acid or sulfated iduronic acid or It is composed of trisaccharide units that are glucuronic acids.

Other GAGs useful in the present invention include hyaluronic acid, heparan sulfate and chondramide. There are two types of leucine sulfate, particularly type A and type C.

Regardless of the type, GAGs can be degraded using appropriate enzymatic or chemical decomposition methods. Can be easily depolymerized. For example, heparin is digested by heparinase. Therefore, it is easily decomposed, while fundroitin sulfate is decomposed by chondroitinase. It will be done.

Chemical decomposition is most commonly carried out using nitrite or periodate. minutes The type of decomposition product depends on the mode of decomposition. Furthermore, the type of decomposition method determine the availability of functional groups for different polymerization reactions of low molecular weight GAGs.

Generally, if the reducing terminal aldehyde is retained, it will be derivatized after decomposition. to obtain allyl derivatives that can participate in addition polymerization reactions with unsaturated non-GAG monomers. can be done. The added carbonyl moiety can also be generated by some decomposition methods. These can be further derivatized and used directly in polymerization reactions.

Available carbonyl groups are alcohols that can participate in polymerization reactions with isothiocyanates. can be reduced to (By some reaction methods, carbon-carbon unsaturated bonds are formed in sugar residues.) generated directly. The available functional groups of the decomposition products are also optionally suitable for polymerization reactions. It may also be provided with a linker that binds to a functional group.

A schematic for heparin illustrating the degradation of GAGs is shown in FIG.

As shown in Figure IA, reducing glucosamine terminals are Deamination of the residue and ring reduction occur to yield 2゜5-anhydromannose. . The free aldehyde in the resulting reducing end group can form a copolymer. is a convenient reactive group for derivatization into substances that can be by periodate The decomposition reaction also generates aldehyde functional groups, and this free aldehyde is also reduced. Anhydromannitol can be obtained.

As shown in Figure IB, the decomposition reaction by hevarinase produces 4 ．． 5-D-uronate is formed, which can be used directly for addition polymerization.

The above suggests that the functionality of aldehydes or uronates can be directly influenced by the formation of copolymers. It does not mean that it must be used directly or indirectly. But this The use of these functionalities confines copolymerization to these sites, but e.g. Hydroxyl or carboxyl groups can be used for polymer coupling. Contains internal sugar residues of the fragment.

Figure 2 shows some of the copolymerization methods of GAG fragments and additional monomer units. Here is an example. As shown in Figure 2A, e.g. 4,5 obtained by hevarinase digestion - Uronate is reacted with acrylamide and incorporated into the resulting copolymer. Addition polymers with phosphorus fragments can be obtained.

As shown in the figure, multiple acrylamide residues are linked to glycosaminoglycan fragments. components can form “monomer units” that are copolymerized (and ). Therefore, the term "monomer unit" used in this application This term refers to glycosaminoglycans in heterogeneous copolymers in general copolymerization reactions. lycan and non-glycosaminoglycan subunits, respectively. Therefore Therefore, the heparinase fragment is separated by an unsaturated bond at the non-reducing end. It is theoretically possible to merize or trimerize, but the acrylamide component It is extremely likely that oligomers of Therefore, it is called “component” The term is used to refer to individual monomers, and the term “monomer unit” Used to indicate individual GAG or non-GAG regions in a polymer.1. % can be done.

Figure IB shows the presence of unsaturated bonds in GAG fragments to participate in addition polymerization reactions. Showing different ways to give. Its reducing end is shown in this diagram for the production of allyl glycosides. As shown in the synthesis reaction (this can be easily derivatized with an unsaturated moiety) . The reaction for producing allyl derivatives is a very common reaction in the technical field. This induction The non-GAG monomer unit exemplified in this application and the acrylamide Can be polymerized. or (as previously defined monomer unit, Oligomeric units are produced from acrylamide. The figure shows an allyl group on the polymer. The polymerization reaction was performed in a similar manner to that shown in Figure 2Al. Of course, it is also possible to use the non-reducing end.

Figure 2C shows that the decomposition products of heparin due to nitrous acid are combined with the amination of aldehydes. to give the desired unsaturated bond, in this case induction into aminoethyl methacrylate. Here is yet another way to perform incarnation. Unsaturated bonds in methacrylate! ±, Ak The addition monomer provided by 1-noramide is convenient for combining uni-nod81. It is a part.

Suitable for incorporating GAG fragments within the addition copolymer, as shown in Figure 2. Additional functionality can be imparted by derivatizing convenient functional groups on this fragment. You can get it. In general, carbon-carbon double bonds may be aldehydes. The hemiacetal is derivatized into a reducing end group of the fragment due to its reactivity. obtain. Allyl groups can be directly derivatized as shown in the figure. or Residues with carbon-carbon double bonds, such as acrylic acid or its derivatives, are reducible. Coupling can be done with the free aldehyde at the end or with the ring-opened form.

Moreover, as a result of the decomposition reaction at the non-reducing end, the carbon atoms present in the GAG itself Double bonds can also be used directly in copolymerization reactions.

In addition to the example shown in Figure 2, the addition polymer is diincyanate as shown in Figure 8. It can be generated using In this example, the appropriate reactive group is the hydroxyl group. This is an amino group. The addition is carried out on the N-C double bond of the incyanate. It will be done. GAG fragments often have multiple hydroxyl groups. , this method selects compared to the majority of secondary hydroxyl groups in the fragment. It would be most advantageous to be able to provide an eastern alcohol that is reactive. Nitrous acid solution The products of polymerization reactions are easily reduced to primary alcohols using, for example, borohydride. provides the aldehyde that is produced. In the depolymerization reaction of heparin, 2,5-anhydro Mannitol is formed, which can be conveniently reduced to the corresponding anhydromannitol. can be done. In the depolymerization reaction of dermatan sulfate, 2,5-anhydrotallow a gas is formed, which can be reduced to the corresponding anhydrotalitol. return The East-m alcohol provided by Yuan can be utilized for reaction with isocyanate. and the resulting fragment is incorporated into the addition polymerization reaction.

In addition, in the above copolymerization reaction, the non-GAG r monomer unit is first It can be modified by including a diol that polymerizes with the annate.

Therefore, for example, ethylene glycol, propylene glycol, etc. may be present in the copolymerization mixture. diisocyanate, 1,3-propanediol, etc. A copolymer with a diol having a fragment is obtained.

As is clear from the above discussion, the non-GAG monomers used in this copolymer are "knits" are composed of various monomers, tiemers, trimers and oligomers. These monomer units may be The material may be uniform or non-uniform. Non-GAGs in the copolymers of the invention Candidates for components constituting the monomer unit include acrylic acid, methacrylic acid, and Included are its carboxyl derivatives, such as its amides and esters. the amide may also be a primary amide as shown in Figure 2 or, for example, a 1-6C alkyl Mid is also fine. Similarly, the ester may be a 1-6C alkyl ester, or The alkyl group of the or ester preferably has a hydrophilic substituent such as a hydroxyl group. It may be substituted with a group. Therefore, the components of non-GAG monomer units and Hydroxyalkyl esters of acrylic or methacrylic acid are suitable as aminoalkyl esters, aminoalkyl esters, and glyceryl esters. In addition, the present invention of esters such as vinyl alcohol, allyl alcohol, and acrylonitrile. Other hydrophilic alkylenes are also useful, such as. Vinyl alcohol ester esthetics After polymerization, the group can be hydrolyzed if desired. In some examples, Low hydrophilic non-GAGs such as tyrene, styrene sulfonic acid or polypropylene ingredients are available.

In general, if non-GAG subnits are produced from unsaturated monomers, The nomer component is represented by the general formula: CEI2=CXY.

However, in the formula, X is H or CH3r; Y is -COOR, -CONH2, -C 0NHR, -CONH2, -CONH(CH2)1.1OH--CON[1(C I'12),,OR--Coo(CH2)n.

H, -Coo(CH2), OR, -0COR, -OR, -Ct, -CN%-C6 H5, substituted monolayer, Hs, -0CO(CH2), OR, -0CO(CH2 ),,OR,-0CO(CH2)nNH2,-0Co(CH2)nNHR - and -0CO(CH2)n　NR2, and these groups n is an integer of 1 to 4, and R is a 1 to 4C straight or branched alkyl It is the basis. Another reaction may be performed on Y after the polymerization has taken place. For example - 〇COR may be hydrolyzed and strained for 10 years. The preferred value of n is 2, and the value of R is Preferred embodiments are methyl and ethyl.

Non-GAG “monomer units” contain copolymers of many of these components. Therefore, for example, hydroxyethyl methacrylate and vinyl alcohol Copolymers of esters could be used. As used in this application, the term "many" , two or more different GAG or non-GAG components are monomer units, whole It means to be used for copolymers as or both.

As mentioned above, non-GAG fragments are urethane-type monomer units. You can. Urethane type uni-nod number, condensation of diol and diisocyanate obtained by letting Useful diincyanates include tolylene diisocyanates. Contains annates and diisocyanate derivatives of various alkylene diamines. Ru.

The copolymerization reaction of GAG subunits and non-GAG subunits It is carried out under conditions suitable for the ingredients. Non-GAG fragments are pre-oligomerized or more typically; more preferably the components of the fragment are copolymerized. It is fed as monomer in the reaction mixture. General conditions for such polymerization reactions are known to those skilled in the art.

As mentioned above, a single type of GAG or non-GAG component or multiple types of Either one or both of the components may be used. Therefore G.A. G component G1 is copolymerized with non-GAG component NGI to produce a copolymer of the following form, for example: can be obtained.

-Gl-(NGlh-Gl-(NGI)s-Gl, or -NGI-Gl-N G1-Gl-NGI-1 or -Gl-(NGI)+5-(Gl)2-(NGI )3-.

-Gl-(NGlh-02-<NGI)+1l-G2- can be obtained, or or using two (multiple) non-GAG components, e.g. -Gl-<NGI)3-Gl-(NGI-NG2-NGI-NG2)-Gl, ma (±-Gl-(NG2)a-Gl-(NGI-NG2-NG2-NGI)-G l, l again Gl-(NG2-NGI)-Gl-<NG2-NG2-NGI-N GI)-Gl, etc. can be obtained.

The typical molecular weight of the resulting copolymers is large enough to allow for the construction of desired medical devices. or have suitable physical properties that are acceptable for coating. For devices like this This includes catheters, transport lines for extracorporeal treatment, endoscopic devices, etc. Also water soluble Copolymers are particularly useful for treating coagulation disorders or certain immune disorders, 'L' vascular diseases and It can be used as a pharmacological agent for the treatment of viral diseases.

11ヱ The copolymers of the present invention can also be prepared in the presence of crosslinking agents to achieve specific intended uses. A product with more desirable physical properties can be obtained. addition type polymer Typical crosslinkers for use in t1 are monomer components with two centers of unsaturated bonds, e.g. Evabis type acrylamide or hydroxyalkyl J rare acrylate esthetics Contains . The part that performs crosslinking! ±, non-GAG monomer components in the polymerization mixture 5% to 15%, preferably about 10% by weight.

A's The copolymers of the present invention can be used, for example, with affinity ligands, camphor or especially with low adducts. It can be derivatized with other useful moieties, including molecular weight GAGs. This kind of temptation To perform conductorization, it must not react under polymerization conditions but be subsequently derivatized with a copolymer. In order to prepare copolymers containing additional functional groups that can react with functional groups in the It is advantageous to provide the same monomer. For example, an amino acid in the non-GAG moiety The use of methyl methacrylate provides a free amino group, which can be used to reduce molecular weight. It can be derivatized with the aldehyde group of palin. Any convenient coupling The method used depends on the type of material to be derivatized and the functionality of the resulting copolymer. can be used.

Finally, in some instances, the backbone copolymer contains only non-GAG components and is completely Contains low molecular weight GAGs as all derivatized ligands. in this case , functional groups inert to polymerization are included in the monomer components, and only one functional group is present. It is used to selectively bind functional groups, such as reducing end groups of GAG. Main departure This form of light is illustrated in Examples 13-15.17 and 19-21 below. Ru.

Ino f mountain The following examples are intended to illustrate the invention and are not intended to limit the invention. isn't it.

-A: Flavobacterium Hepa Alimentation of heparin fragments of heparin by heparinase of ar'num A, heparin to alcohol, A, method of Grant et al. (Anal Bi och ra, vol. 137, 25-32L 1984) Therium's heparinase digests it. 1 g of commercially available porcine mucosal heparin (Sigma company a), 25+*M sodium acetate, 0.25@M calcium, pl! 7.0 Dissolve in 5.0 ml of. Commercial heparinum derived from Flavobacterium heparinum 1,250 units of 50% sodium phosphate (also obtained from Sigma) were added to 50mM sodium phosphate. Dissolve in 5.0 mL of thorium, pn'r, 4. Combine these two solutions Mix well and incubate overnight in a water bath at 30°C. Decomposition by enzymes The product was extracted at 232 nm of 20 μl of the digestion mixture in 2.0 mL of 3 hM HCL. Monitor periodically by measuring absorbance at .

Approximately 20 le 24 Divide into 5 x 2 mL portions (each containing 200 mg heparin). Each part Separately, two 100cm x 2 in series. 5cm Sefatefukus G-50 Inject onto a superfine column (Linker, A. O and Hovin gh, P. ,'oc competition'st volume 11, pages 553-572, 1972 Year b). Collect 15 minute fractions using 0.2M NaCl and 10% ethanol. It elutes at a flow rate of 0.4 gL/min. All fractions were measured by absorbance at 232rv. assayed for heparin by measuring the degree of Heparin fragments of various sizes (clear pins corresponding to trisaccharides ~+ptt=> was analyzed in detail.

The heparin fragments in the column fractions are then separately dialyzed against solid ice and frozen. Freeze drying was performed.

B. Derivatization of heparin fragments to allyl glycosides: Should be derivatized 45,011 g of fragments were placed in a cold trap in the pipeline for 24 hours under reduced pressure. It dries completely. This is dissolved in water-free allyl alcohol and then , water, methanol, allyl alcohol and finally allyl alcohol without water Cationic (acidic) amberlite resin (IR- 12011). This mixture is allowed to react at room temperature with vigorous stirring. . After 96 hours, the reaction mixture was filtered and the resin was dissolved in 50+50 ethanol/water and Wash 5 times with 98% ethanol. Pool the filtrate and washings and concentrate to a small volume. and inject into a reversed phase silica gel column (ZGcx 1.5c+e). this The column was washed sequentially with 50+ mL of water, 50 aL of methanol, then heparin. The aqueous portion containing the allyl glycoside of the phosphorus fragment is lyophilized.

°　B:　by　Amino　, by Hepari 2 Danko 1 Emperor B. depolymerization of heparin. The method of Ca5u et al. (Lzka 1) Volume 197.

Pages 599-609. (1981). 1g commercially available porcine mucosal hepa Dissolve phosphorus in 400+aL of Type 1 water to make a 0.25% solution. 2. By adding 14aL of concentrated sulfuric acid, O. Make the concentration 1M sulfuric acid . The mixture was cooled to 10 °C in a water bath and 5.55 g of NaNO2 was added into the mixture. By dissolving to a concentration of 0.2M NaNO2. 2 at 10℃ Let it react for a minute. Next, IOM NaO was added until the pH stabilized at 7.0 to 7.2. Neutralize the solution by adding H. Concentrate the mixture to 500 molecules Partially desalt using a volume cut-off Amlcon membrane and lyophilize. This drying The heparin fragments were then redissolved in a small amount of hard ice and then prepared as described in Preparation A. Analyze using a Sephadex G-50 superfine column. T. Bftter and 8. M. Muir's uronic acid assay method (■■Yu Crypitation■ Volume 4) , pp. 330-334, 1962), the fractions were analyzed for heparin content. Test. Heparin fragments of different sizes (trisaccharides) present in column fractions ˜caat sugar) are separately dialyzed against Type I water and then lyophilized.

(Margin below) back 1” low Acrylamide with heparin solution 45-\ uronide Heparin fragments A, A typical polymerization mixture consists of 4,5-unsaturated bond-bearing henophosphorus fragments. (Hexasaccharide ~ Naana sugar) (prepared in the same manner as described in Preparation method A ) IQmg, acrylamide 11.6 g and TEMED (N, N, N', N'-tetramethylethylenediamine) 0.725 μL, 180 Contain in μL of water. This mixture was degassed for 30 minutes, then 5 μL 0.18 μg of ammonium persulfate in water is added. Polymerization reaction at 4℃ for 18 hours Do it for a while. Cefazo Nox G-25 desalting column and/or 50mM EDT Extensive dialysis against A Separate the copolymer from the mer. The henokurin-containing fraction is analyzed for uronic acid. mosquito Detected by the Rubazol method (Bitter and Muir, 1962). fraction Pool and finally! Dialyze against 1 cup of water and freeze-dry. It is shown in Figure 3. Sea urchin, NMR svector cata, contains both polyacrylamide and henocrine in the product. It is confirmed that there is one.

B: of this example, (raggraph A) using the same general method as described here: Several copolymers were prepared with the following phosphorus fragments of various sizes: Ta.

(Margin below) Copolymer y ir iy / Copolymer used Heha in the copolymer ゛Rin no Tsun 2　Hehe゛゛Nfu゛Men　%CH6-131D　Hesugar Body 3.6 13CH6-131E + Glycoside 7.3 26CBS-131F Thirteen saccharides 10.4 39 ml 4-1 by acrylamide and Aminoethyl methylate of purified heparin fragment A A, low molecular weight heparin (LMWII) obtained by deamination cleavage reaction with nitrous acid (Sigma's 1115640. Average molecular weight 4.000-6.00 (+ Dal Heat 1.25 iL of an aqueous solution (20 mg/mL) of . To this was added 6.25 mg of 2-aminoethyl methacrylate (AEM).

1.25 μL of an aqueous solution containing 18513 from Odak is added. hepa The phosphorus derivatization is carried out at 50° C. for 1 hour with stirring.

B, copolymerization with acrylamide, 2.5IIL of acrylamide (BioRa d Company, 161-0100.56 ming) was added, degassed, and then , 50 μL of N, N, N', N'-tetramethylethylenediamine (T EMED, BioRad, 161-0880) and 10+*L persulfuric acid Storage of ammonium (BioRad, 161-0700.0.5mg/μL) This is done by adding the existing solution. Polymerization was continued for 10 minutes at 50°C with stirring. Ru.

A 2-fold molar excess of NaBH4 relative to AEM is then added to complete the aldehyde-imine bond. The combined reduction is carried out for 30 minutes at room temperature. Sepharose 4B column (1,5c+mX 80cm) and elute with 0.2M NaCl at a flow rate of 0.27sL/min. Chromatography separates the copolymer from unreacted monomers. 15 minutes each fractions for uronic acids using the carbazole method (Bitter and Muir, (1962) to detect the copolymer peak. copolymer elutes near the total volume of the column, V. The NMR spectrum shows that the copolymer is Confirmed that it is composed of both phosphorus and polyacrylamide, typical An example is shown in Figure 4.

C1 Following the same method in paragraphs A and B above, the paragraphs mentioned above Using different heparin fragments obtained in Rough B, several A copolymer was prepared.

Copolymer 15') "Int/In the copolymer used Hepan in the copolymer To the σn board χCH3-23D LMWB (Si gma) 6.8 22C■8-32B Glycoside 9.5 28C118-3 6A + glycoside 10.0 351! Lm Amino made from styrene 4 of the aminoethyl methylate of the heparin fragment of Low molecular weight heparin (Stgga E15640. Average molecular weight 4,000-6,0 00 daltons) with aminoethyl methacrylate (AEM). 36 Dissolve mg of heparin in 1.8 L of water to make a 2% solution and heat to 50°C. Ru. Add 1.8 μL of 5 mg/mL AEM solution to the above heated heparin solution. Ru. This solution is reacted at 50°C for 1 hour.

Heparin 42B derivatized with AEM was heated to 70°C to produce 24 Dissolve in 0 wL of formamide. The resulting heparin solution was added to the reaction mixture at a concentration of 0. The concentration of sodium dodecyl sulfate was reduced to 0 by adding 27 mg of SDS. ．． Make it 1%. Degas the solution by bubbling N2 for 20 min. I do. Next, 0.81g of AIBN (2,2'-7zobi-inbutyronite) Add 5 μL of a 5 mg/IL potassium persulfate solution. Contains no inhibitors Add 24 μL of fresh styrene and stir the resulting mixture vigorously at 60°C for 24 hours. Ru. The resulting copolymer is washed several times with water and freeze-dried. P, ni, Si+it Metachromatic toluidine blue dye binding method of H et al. 6-473, 1980), 1.3% by weight of heparin was copolymerized. It was found that it was incorporated into the compound (designated CR2-39).

Emperor 11”- Hydroxy ether route M 13.5 mg of heparin allylglyphsitide (described in Section B of 31 Manufacturing Process A) ) was dissolved in 400 μL of methanol:water (1:1) and Add 100 μL of inhibitor-free HEMA to the solution. Add N2 to this solution The mixture is degassed by bubbling for 20 minutes. Next, 1 μL of TEMED (N, N, N', N'-tetramethylethylenediamine) and 100 5 μL of mg/mL ammonium persulfate solution was added and the reaction mixture was incubated at room temperature for 24 hours. Let stand for a while. The copolymer on the obtained jelly was dissolved in I) Hlo, O (1: 1. Improper tool: water) and pH 3.0 solution (water) alternately 3 times for 30 minutes (M, R, van de Mark et al., dvaces in i m’ca o mers, E, A, Gebelinmli collection, Pol ymer 5science and Technology, Volume 35, 373~ 380 pages, 19117, New York, PIenu+e Press (done in accordance with the law). After a final wash, the copolymer was lyophilized and heparinized. As a result of testing the content, the copolymer (named CH2-42) contained 0.1% by weight of Palin was present.

Real J111 Made with acrylonitrile Δ of heparin fragment 44 mg Noheparin Disaccharide Disaccharide Process A, as described in paragraph A. preparation) in 500 μL of water. Acrylonitrile 52μ without inhibitor TEMED the mixture by adding L and then 5.5 μL of reagent. Set the a degree to 1%. By slowly bubbling N2 for 20 minutes, Degas the solution. 110 mg/mL ammonium persulfate in DMSO (di 5 μL of methyl sulfoxide) solution was added after degassing and the copolymerization mixture was stirred. Heat to 70"C with vigorous stirring. After 2 hours at 70"C, the mixture is heated to 70"C. Remove from the water bath, centrifuge the precipitate, wash with water three times, and then dry. (named CHII-11P).

Five branch doctors Tolylene diisocyanate and Amino by ethylenedi1fur - Heparin fragment of - A, low molecular weight heparin (Sigma [5640, average molecular weight 4.000~a , ooo Dalton) is reduced with sodium borohydride. 110+mg he Dissolve Parin in 2 L of water and add 76 g of NaBH in 1 mL water-cooled water to the solution. Add 4 solutions. The anhydromannosyl moiety of heparin was reduced overnight (approximately 20 time) at room temperature. The resulting reaction mixture was soaked in ice vinegar until the pa stabilized at 5.2. Acidify with acid.

Remove excess borohydride by repeated evaporation (3 times) with methanol Finally, the reduced heparin is lyophilized.

8. Heat 101 mg of reduced heparin (approximately 17 to 25 OL) to 70°C. Dissolve in 5 μL of formamide by: Add 100μ of this solution Mix with ethylene glycol (18 mmol), then add 5 μL of dimethyl sulfochloride. Dilute with cid. Cool this mixture in a water bath and add excess Add LmL of tolylene-2,4-diisocyanate (7 mmol). Mixed Copolymerization was carried out for about 64 hours while stirring while gradually heating the mixture to room temperature (20°C). Continue reacting. Copolymerize by pouring the resulting viscous mixture into chloroform. Isolate the coalescence. Chloropholate the precipitate until there is no unreacted heparin in the supernatant. Thoroughly wash with lumen, ethanol and finally water. In addition, unreacted heparin is Measured by the carbazole method that measures rononic acid. Freeze-dry the washed copolymer, Heparin is assayed by the dye binding method (Sn+ith et al., 19110). The test is It shows that the product contains 0.7% by weight heparin. its copolymerization (named CF3-22G) is insoluble in water but insoluble in dimethyl sulfoxide. It dissolves easily, and its NMR spectrum shows that the polyurethane structure is M is closely related to the aromatic residues of heparin and some new bands assigned to heparin. ing.

C, 93 mg reduced heparin (approximately 19imo1) in 3 L dimethyl sulfoxide ．． Add 46.5 μL of ethylene glycol to the solution containing (prepared in paragraph A) (0.83 mmol) and further diluted with 3+*L dimethyl sulfoxide. The solution is prepared in the same manner as described in paragraph B above, except that:

The solution was then added with 121 μL of tolylene-2,4-diincyanate (0,85 m+ so1. (equivalent mole) to the total amount of reduced heparin and ethylene glycol. , cooled in a water bath and exposed to N2 atmosphere while the mixture gradually approached room temperature (21 °C). The reaction is allowed to continue for 24 hours while stirring. The produced copolymer (CR2-2IC After proper washing, drying and assay, the product contained 1.97% heparin by weight. Indicate quantity.

Actual 1u ahead L The heparin fragments were absorbed into a small group of eight. The copolymer was tested for anticoagulant and antithrombotic activity by APTT (activated partial thrombosis). Plastin time, Sig+ea Procedure No, A766g) and anti-Xa factor (Sigma Tecbnfcal Bulletin No. 870) No., Heparfn in Plasma) assay method. The latter method is , neutralizes activated factor X (Xa) by its plasma inhibitor (antithrombin) It is based on Anti-Xa:APTT ratio is calculated from these test results be done.

The anti-Xa activity:APTT ratio measures the antithrombotic power of a substance in terms of its anticoagulant activity. important for determining Substances with a high proportion are more antithrombotic and Less risk of blood. This suggests that anti-Xa assays target early stages of the coagulation cascade. Measures the ability of substances to block the production of thrombin, while APTT measures the progress of blood clotting. This is because many steps measure the ability of a substance to stop blood clotting. follow Therefore, anti-Xa activity is specifically related to local antithrombotic power, whereas APTT is related to global antithrombotic power. It is a measure of anticoagulant activity. Commercially available heparin has an anti-Xa/APTT ratio of approximately 1. The same ratio for low molecular weight heparin obtained from Sigma, Inc. is approximately 2.

Table I shows the analytical results of the copolymers prepared as described in Examples 1 and 2. child Since these copolymers are soluble in water, they cannot be directly assayed using the APTT method and anti-Xa method. I can do it. Analytical results of these copolymers prepared with small heparin fragments The fruit is that these copolymers generally contain short oligosaccharides used to prepare the copolymers. This shows that it maintains a characteristically high anti-Xa/APTT ratio. for example , heparin fragments of heparin size of hesaccharides, decasaccharides and decasaccharides according to preparation method A The anti-Xa/APTT ratios are 4J, 7.4 and 6.5, respectively. similarly , anti-Xa heparin fragments of heparin size of heglycosides and decasaccharides according to Preparation Method B. /APTT ratios are 10.4 and 10.0, respectively. One of the copolymers, sana Specifically, the fragment named CH-36A has a higher anti-X content than the corresponding fragment. a/A PTT ratio, which is probably due to fragmentation due to different preparation methods. This may be due to variation in the intrinsic activity of the components, or due to the fact that they are separated from each other and the copolymer backbone such as active fragments that are randomly incorporated as pendant moieties into the chain. This is probably due to its superior usage.

(Margin below) Permanent work (11 (Hikietsu example 1) cH’-13” A, /X’) n) 9 11.0 55 5.0 (Fathers Imari ],) (γl-92) To αtoco 2B 1. Fl) Nheo stop-2811912710,7 (il i) [γii! Example 2-) Back Ju11 Heparin produced by Acrylamide rubber Heparin fragments were prepared as described in Preparation Method A (par) A). -Prepared by digestion with heparinase derived from Heparinum. common common In polymerization methods, an aqueous solution of monomers and an organic phase are prepared. The monomer phase is 60 mg of heparin fragments (size ranges from hemoglycosides to anaglycosides), 240 mg of aca. Acrylamide, 60 mg polyvinylpyrrolidone, and 20 mg N, N' - Dissolve methylene-bis-acrylamide (Bis) in 1.6 mL of water. Contains many things, one-sided education 8! The phases were 11 mL toluene, 5 mL chloroform. and 80 μL of a mixture of 5pan85. Place the monomer phase in a water bath to about 0°C and then add 160 μL of 50 mg/mL ammonium persulfate solution or and 20 μL of TEMED (N, N, N’, N’-tetramethylethyl (Diamine). This solution was stirred into a paddle stirrer (padate-st. The organic phase contained in the Sanro round bottom flask is rapidly stirred with a trrer. , add with a pipette. Brush the flask with nitrogen until all monomer phase has been added. Gas was passed in slowly. The mixture was stirred at high speed for 30 minutes at 25°C. React and then react at 65° C. for 60 minutes with low speed stirring. reaction mixture Transfer the material to a glass funnel and wash with solid ice, acetone and finally methanol.

The resulting copolymer particles were suspended and immersed for 5 days to be mixed with polyvinyl pyroton and unreacted. excluding the corresponding ingredients. Place the copolymer particles again on a glass funnel with a large amount of hard ice and methane. Wash with a towel.

The obtained solid copolymer is dried under reduced pressure at 60° C. for 4 to 5 hours.

This was purified using the toluidine blue dye binding method of Sm1th et al. (1980). As a result of assaying for parin content, 2 mg of heparin derivative particles (0.2 g weight % heparin) content.

fruit” [column” − A. Low molecular weight hebaria obtained from Sigma Chemical Co. (4,000-6,0OODa) in Example 2 (Paragraph A) and Example 3 Derivatized with aminoethyl methacrylate (AEM) according to the method described. Ru. The obtained heparin derivative was heated to 3,500 MWCO5 pectrapore. Further purify the excess aminoethyl meth by dialysis in a tube. lyophilize the internal dialysate.

B. A typical copolymerization mixture is 30 mg acrylamide, 30 mg AEM inducer. Conductive low molecular weight Heparin/(AEM-LM, WH) and 2.5 mg Bis-A Contains crylamide, and 1.0 mL of these! Dissolve in water in mold and then in water bath for approx. Cool to 0°C. Add 160 μL of 50 mg/ml ammonium persulfate solution to this. and 20 μL of TEMED. Mix the resulting solution thoroughly and sparge with nitrogen gas. Slowly (rebubble) into the reaction mixture. Continue polymerization overnight at room temperature. make it worse The resulting reaction mixture was placed in 50 m of 3,500 MWCO tubing. Dialyze against M EDTA followed by type I water. internal dialysate Lyophilize and then apply a column of Sephadex G-150 Super Fine Gel. Filter through using 0°2M NaCl as running buffer.

fractions (15 min each) were collected at a flow rate of approximately 1 mL/min for uronic acid. The fraction containing the copolymer is fixed. These containing copolymers Fractions are pooled, desalted and lyophilized. of uronic acid, APTT and anti-factor Xa. Assays are performed on the copolymers to determine heparin content and biological activity. Test results The copolymer has a heparin content of 17%, an APTT activity of 2.9 U/mg, and It shows that the anti-Xa activity is 6.63 U/mg.

Real if I”- Bis-acrylamide Description of 4 Preparation Method A (Paragraph B) of Allylhepain Acrylamide First, we prepared allyl glycosides of heparin fragments obtained by enzymatic digestion. Allyl-heparin (saccharide) instead of OAEM-heparin The copolymerization followed essentially the same procedure as in Example 9, except that 30 mg was used. cormorant. The heparin content of the suitably purified and lyophilized copolymer was 11%, with APT The T activity is only 0.62 U/ig, and there is virtually no anti-Xa activity.

1st floor craftsmanship Allylhebarin A enzymatically produced heparin saccharide with acrylonide del The body derivatizes to allyl glycosides as before. A product that does not contain inhibitors Allyl-heparin zoBt-dissolved in 200 μL of crylonitrile, I'! AIBN (2,2'-azo-bis-isobutylnitrile) in 150 μL of water Mix with 5mg. Stir this mixture in an oil bath at 70°C for 4.5 hours. Ru. A solid copolymer is produced, which is recovered by centrifugation and washed thoroughly with I condensate. Ru. This copolymer was dried, and using the toluidine blue dye binding method, heparin-containing Analyze abundance. Biological activity is measured with APTT and anti-Factor Xa assays.

The above assay results show that the heparin content is 8%, and the APTT and anti-Xa activities are respectively 0.1511/mg solid and 1. Indicates QU/mg solid. Figure 6 shows the copolymer A typical NMR spectrum of . This spectrum is clearly compatible with the allyl group. This indicates that there is no corresponding characteristic signal.

Uratategoko 1 Description of AEM-Hepatone System Example 9 (Paragraph A) Dissolve the AEM-heparin, prepared as described, in water. in a 100mL round bottom flask. AEM-heparin solution (10 mg/L) 25a+I, was adjusted to temperature using a thermostat. Heat to 70°C in a controlled oil bath. Acryloni without inhibitors A solution of 7 mg of AIBNl in Trill SmL was added to the flask and the temperature was increased to 75°C. Raise it with When this mixture was allowed to react for 3 hours while stirring, it formed inside the flask. A white precipitate forms. Remove this precipitate and thoroughly wash it with I condensate in a glass funnel. Wash.

This is resuspended in I condensate and freeze-dried. Heparin content is 0.5%, copolymerized The body exhibits high anticoagulant activity.

L-supervisor Aminoethyl methylate, amylonitrile, and heparin Add 2.5 g of AEM to 10 mL of dimethyl sulfoxide (DMSO) and inhibitor-free acrylonitrile (AEM and acrylonitrile). 1 iL (1:1 molar ratio) and 3 g of AIBNo are added to this solution. stirring While heating the solution to 70° C., the solution is allowed to react for 4 hours. The reaction mixture is a viscous solution. This reaction mixture was added dropwise to the methylene chloride solution IL while stirring strongly. Then, the copolymer is precipitated. Filter the precipitated copolymer with a glass funnel and remove the salt. Wash thoroughly with methylene chloride. This copolymer was dried in a vacuum oven at 70°C. , crushed into powder.

Dissolve 250 g of AEM-acrylonitrile copolymer in 14 m of 5% phenol. low molecular weight heparin (4, 6 to 6) dissolved in 5% phenol. ,0OODa) 63 g is added to this. Let this mixture sit gently at room temperature for 24 hours. Stir in crab. Next, add sodium cyanopohydride Long (p[I5) , the reaction is continued for an additional 48 hours. The derivatized copolymer is a, ooo to 8. ■ Dialyzed against hard ice in OOOMWCO Spectrabore tube and freeze-dried dry

The heparin content is 5.5% as determined using the uronic acid assay. AP Biological activity was determined by TT and anti-factor Xa assays, each at 8.8 U/I. 1g and 16.70/*g.

Sogogogojodama Hepatinol ζte′-゛aminoermyl t-rate 2-hydroxyethyl lume i rate loca person day boshi friend 1kin AEM2. Sag and HEMA2 abuse, DMS O2g, 5mLk: Dissolve and add IZO-67 (2,2'-7 zobisumethylene) Add 1155 mg of Rubutyronitrile). This flask is a turbidity condenser. and place it in an oil bath heated to 70℃. Pour this mixture at this temperature. Incubate for 4 hours at ℃. While stirring vigorously, add 50% dichloromethane to this reaction solution. When added dropwise to OsL, the copolymer precipitates, and is then filtered through a glass funnel. child The copolymer is redissolved in DMSO, reprecipitated with dichloromethane, and filtered again. . This procedure is repeated once more and the copolymer is dried in a vacuum oven at 60°C.

100 g of AEM-HEMA copolymer and low molecular weight heparin (4,000 to 6,0 00Da) 25℃ 1g was dissolved in 9i+L of I condensate and mixed gently overnight at room temperature. do. Since this solution is slightly acidic (pH 6), sodium cyanoborohydride I Add OB to this solution and continue stirring at room temperature overnight. 6. Reaction mixture. Goo~ 8. Dialyze against ice in a 0001 fl CO tube and freeze the dialysate inside. It freezes and dries. The uronic acid assay showed that the heparin content was 5.1%, and the APTT and and anti-Xa activity are 17.6 and 118 U/ig, respectively.

L55 upper e Heparin contains aminoethyl ester 4-styrene sulfonate. 4AEMQ, 5g and 0.52g of 4-styrenesulfonic acid were covered with water. Dissolve in 25 mL. Dissolve 75g of I/AZO-670 in the mixture and start the reaction. The solution contains 3% of the agent. This solution was heated to 70'C and kept at this temperature for 4 hours. Allow to react while stirring. A yellow precipitate is obtained, so separate it from the reaction mixture. Let go. Add this to 50 mL of O, 1M sodium phosphate buffer (pH 10,9). After reconstitution and dialysis against I solid ice in a 3.5OOlffCO tube, this Lyophilize the copolymer.

AEM-styrene sulfonic acid copolymer 4G+*g and low molecular weight heparin (4,0 00-6. QOODa) 10mg, type I water 3. Dissolve in SmL and stir at room temperature for 2 Mix slowly for 4 hours. Next, add sodium hydroxide to a slightly acidic solution. Dissolve 10 g of Lido and mix for an additional 24 hours at room temperature. This mixture is a, o Unbound heparin was dialyzed against solid ice in a OO~8,0OOIFlCO tube. The internal dialysate was freeze-dried. In the uronic acid assay, induction The heparin content of the incorporated copolymer is 22%. APTT and anti-Xa activity , 9.8 and 140 U/ag, respectively.

L Bloody Heroes Δ between heparin aminoethyl metacrude and 4-styrene sulfone Low molecular weight barrier (4,000-6 ,0OODa) NoAEM derivative is prepared. AEM-heparin aqueous solution (10m g/mL), dissolve 4oOmg of 4-styrenesulfonic acid solution in 10mL. V Add 300 mg of AZO-67 and raise the temperature to 70”C. The polymerization reaction is carried out overnight. Next, add this solution to 6.000 to 11. ooOMWcochu Dialyze in a tube against 50mM EDTA followed by condensate and freeze-dry. . According to the uronic acid assay, the heparin content of the dried copolymer is 12.7%. A The biological activity using PTT and anti-Xa methods was 16 U/mg and It is 160U/mg.

Hynyl acetate (1,75 g) and AEM (0,25 g) were dissolved in DMP (25 mL). Dissolve and add VAZO-67 (2G+*g) to this solution under a nitrogen atmosphere. I can do it.

This solution is heated in a 70° C. stellar oil bath for 20 hours. The resulting melt The liquid is concentrated by distillation under reduced pressure, and then the viscous solution is reduced with vigorous stirring. When suspended in water, a copolymer precipitates out. Filter this precipitate and wash quickly with water After that, it was freeze-dried. Following the same general procedure as described in Examples 13-15, DM This copolymer is derivatized using LMWH (Sigma) in F solution. The heparin content of is 0.26% (by weight).

Branch hill Gokami 1 Tolylene diisocyanate and heparin LMWH and tris-hydro bovine dimethyl Ami/Methane 9” U1 included A, low molecular weight heparin (4,000-6,000 Da, ) are obtained from Sigma Chemical Co., U, S, A. Hydrogenation triglyceride in a 20- to 25-fold molar excess of formamide solution in the presence of sodium boron. This is prederivatized by reductive amination using a solution. In general, Tris 5 0+*g, LMWH101.2mg and NaBH45.5mg were Dissolve in 5 mL (total amount) of Midin and leave this solution at room temperature overnight (16-22 hours). do. Transfer the mixture to a small round-bottomed flask set up in a water bath and seal with a rubber stopper. , passing nitrogen gas through it. After about 15 minutes, add tolylene-2,4-diiso using a syringe. Add cyanate (large excess) and dilute the solution with 5IIL of dimethyl sulfoxide. do. Copolymerization is continued overnight (approximately 18 hours) at 4°C under N2 atmosphere with stirring. . The resulting copolymer was dissolved in methanol and chloroform (100 mL each). Add the mixture and precipitate. This precipitate was filtered, and chloroform, methanol, methanol, Thoroughly wash with tanol-water and finally with pure water. This copolymer was heated to 70°C. After drying in a vacuum open, 5sith et al. toluidine blue dye binding method (198 According to 0), the heparin content is 5.24 μg/mg solid. APTT and The biological activity measured by anti-factor Xa assay was 7.3 mU/mg solid and and 8.3511 IU/mg solids. Figure 7 shows the hydroxyl group at 3400 cm-'. Typical FTI showing strong absorption bands of urethane bonds at and 1700 cm-' The R spectrum is shown.

B, then L! For prederivatization of ff[T, LMWHL14rsg, f- 121.4 mg of Lis and 12.61 g of NaBI were dissolved in dimethylformamide. The above pattern was dissolved in a mixture of 2 mL (DMF) and 6 mL formamide. Perform the same procedure as described for Lagraph A. Dibutyltin 100 used as catalyst ALL and 145 μL of tolylene-2,4-diincyanate (amount of 1-lith and The copolymerization was carried out under exactly the same conditions as described above, except for the presence of cormorant. The reaction is continued for 5 hours in a water bath and then for 16 hours at room temperature. with chloroform Precipitate the copolymer, wash thoroughly with methanol and water, and dry in an oven at 60°C. do. Now the APTT and anti-Xa activities are 13IIU/*g and 14 lotus U/mg, yielding 0.4 μg/mg heparin solid.

LMWH81+gg, I” squirrel 49.6B and NaB11g 5.1mg, Same procedure as described in Example 18, except for dissolving in 3 mL of Formua, Mido. Follow. Derivatization is carried out using Tris overnight (19 hours) at room temperature. Then high In Bohr containing 01ypol) 2000 (1.6meq NC07g) cyanate-terminated prepolymer) and dimethyl sulfoxy Transfer to a round bottom flask under N2 atmosphere. The reaction was carried out at 4°C for 22 After 6 hours, add chloroform to reduce the viscosity of the mixture. last A semi-solid mass was obtained, which was diffused into 200 mL of chloroform and a large amount (approximately 8 00 mL) of methanol. Precipitate washed thoroughly with methanol and water Dry the item as before. The heparin content of this copolymer is 05gg/* g solid, APTT activity is 8.4raU/Hg, anti-Xa activity is 7.2mU/mg be.

L55 main character Tolylene diisocyanate se1ol 4 and hepan 1 were present in A. Dissolve 1 mol (127.4 mg) of serinol hydrochloride in dimethylacetamide and add Rylene-2,4-diisocyanate 142 μL (equimolar) and dibutyltindi Add 50 μL of laurate (catalyst). In a rotary evaporator at 65℃ The mixture is allowed to react for about 30 minutes and then for 18 hours at room temperature (21° C.). This mixture The combined solution was suspended in 200 mL of chloroform to precipitate the copolymer, and then mixed with methanol and water. After thorough washing and filtration, dry in a vacuum oven. Dry copolymer 35.6 mg of LMWH51,3B, also in formamide, was dissolved in formamide. NaBHt 8. hag and this solution to make a total volume of 4 mL. copolymer For derivatization, this mixture was left at room temperature overnight (about 17 hours), and then the precipitated and rinse with water.

When this precipitate was dried under reduced pressure, heparin was removed using the toluidine blue dye method. ．． APTT and anti-Xa activities were 30 mU each. /mg and 240m/rag.

Plan Ju1 Yu”- Tolulene diisocyanate and toluene hydroxymethylamic acid Hepa-rich Δ except that 120 mg (1 mmol) of Tris was dissolved in dimethylformamide. , using the same procedure as described in Example 20. Equimolar amount of tolylene-2,4-di Add socyanate (142 μL) and the presence of 50 μL dibutyltin dilaurate. Copolymerization is carried out below. As before, this copolymer was precipitated, washed, and placed in a vacuum oven. Bung dry. The dry copolymer (361 g) was mixed with low molecular weight heparin (61.3 m g) and sodium borohydride (If 4 mg) together with formaldehyde. (4 mL). Derivatization of the copolymer is continued overnight at room temperature. As a result The product produced by e,ooo~8. QOO1flco Spectrapore Dialysis Chi Dialyze thoroughly against pure water using a tube. Dialyzed on Rotovap Concentrate the solution and dry in open vacuum at 70°C. This product contains heparin 64μ g/mg solids and exhibits high anti-Xa activity.

072w -2,5-Unhuman +-D-77 No-human~6-stone iJl 4,5 -D- Laminato A B abstract A novel biocompatible glycosaminoglycan combination that is antithrombotic and nonthrombogenic. The polymer is expected to maintain its anticoagulant properties for a long period of time or permanently. of biomedical applications. The novel copolymer of the present invention is Small glycosaminoglycans, such as heparin, copolymerized with monomer components. Contains fragments or segments. The present invention provides glycosaurin, such as heparin. Small segments of minoglycan have antithrombotic activity that inhibits or prevents thrombosis. It takes advantage of the fact that Anticoagulant fragments are It is easily produced by elementary or chemical means and copolymerized with synthetic monomers.

International search report PCT/CA 91100120+me increase---＾-ebta+ van PCT/CA 91100120 International Search Report C^9100120 S^　46217

Claims (26)

[Claims] 1. Biocompatible, suitable for constructing or coating biomedical devices that come into contact with blood wherein the copolymer is antithrombotic and nonthrombogenic; a monomeric unit copolymerized with at least one non-glycosaminoglycan monomer unit. containing at least one monomer unit of a glycosaminoglycan fragment. A biocompatible copolymer. 2. The glycosaminoglycan fragment is a high molecular weight glycosaminoglycan prepared as an enzymatic or chemical degradation product of Claim 1, wherein the minoglycan fragment is antithrombotic and nonthrombogenic. Copolymer described in. 3. The glycosaminoglycan fragments include heparin, dermatan sulfate, and Derived from droitin sulfate type A, chondroitin sulfate type C or hyaluronic acid The copolymer according to claim 2. 4. Claim wherein said glycosaminoglycan fragment is derived from heparin. 3. The copolymer described in 3. 5. The glycosaminoglycan fragments depolymerize heparin and form pentasaccharides. Obtained by recovering deca-saccharide-sized fragments from the body, claims Item 4. Copolymer according to item 4. 6. The depolymerization is performed by digestion with heparinase, treatment with nitrous acid or periodic acid. 6. A copolymer according to claim 5, which is carried out by acid cleavage. 7. The monomer unit of the non-glycosaminoglycan is an unsaturated monomer or The copolymer according to claim 1, which is an oligomer thereof. 8. At least one of the unsaturated monomers is a monomer represented by the formula CH2=CXY - in which X is H or CH3, and Y is -COOR, -C ONH2, -CONHR, -CONR2, -CONH(CH2)nOH, -CO NH(CH2)nOR, -COO(CH2)nOH, -COO(CH2)nOR , -OCOR, -OR, -Cl, -CN, -C6H5, substituted -C6H5, -OCO(CH2)nOH, -OCO(CH2)nOR, -OCO(CH2)n From NH2, -OCO(CH2)nNHR, and -OCO(CH2)nNR2 n in the formula of these groups is an integer from 1 to 4, and R is 1-4. 8. The copolymer according to claim 7, wherein C is a linear or branched alkyl group. 9. The unsaturated monomer is acrylamide, styrene, 4-styrene sulfone. acid hydroxyethyl methacrylate, acrylonitrile, or aminoethyl methacrylate 9. The copolymer of claim 8, which is a tacrylate. 10. The unsaturated monomer is a diisocyanate or a diisocyanate and a diisocyanate. The copolymer according to claim 7, which is an oligomer of ol. 11. The glycosaminoglycan fragment is permeabilized with a non-reducing end. 2. The copolymer of claim 1, which is a low molecular weight heparin. 12. the heparin derivative is an allyl derivative or the aminoethyl methacrylate The copolymer according to claim 11, which is a derivative of 13. The copolymer according to claim 1, further comprising crosslinking. 14. 14. The copolymer of claim 13, wherein said crosslinking is provided by a diunsaturated crosslinking agent. Combined. 15. The monomer units of the glycosaminoglycan fragment are non-glycosylated. The copolymer according to claim 1, which is provided after the monomer component of the saminoglycan has been polymerized. Polymer. 16. Is the copolymer a large number of monomer units of different glycosaminoglycans? The copolymer according to claim 1, which is formed from. 17. The copolymer comprises a large number of different non-glycosaminoglycan monomer units. The copolymer of claim 1 formed from. 18. A blood contacting product constituted or coated using the copolymer according to claim 1. Medical devices suitable for applications involving touch. 19. Blood antiseptic, suitable for composing or coating biomedical devices that come into contact with blood A method of preparing a biocompatible copolymer that is pluggable and nonthrombogenic, comprising: , at least one glycosaminoglycan fragment and at least one The process involves reacting non-glycosaminoglycan components under conditions that effect copolymerization. method. 20. 20. According to claim 19, the reaction is carried out in the presence of at least one crosslinking agent. Method described. 21. The glycosaminoglycan fragment is a high molecular weight glycosaminoglycan fragment. prepared as an enzymatic or chemical degradation product of Claim that the cosaminoglycan fragment is antithrombotic and nonthrombogenic The method according to item 19. 22. The glycosaminoglycan fragments depolymerize heparin and This product is obtained by recovering fragments of the size of hexasaccharides from glycosides. The method according to claim 19. 23. The monomer unit of the non-glycosaminoglycan is an unsaturated monomer or 20. The method of claim 19, wherein is an oligomer thereof. 24. The unsaturated monomer is a monomer represented by the formula CH2=CXY, and the unsaturated monomer is a monomer represented by the formula CH2=CXY, where X is H or CH3, and Y is -COOR, -CONH2, -C ONHR, -CONR2, -CONH(CH2)nOH, -CONH(CH2) nOR, -COO(CH2)nOH, -COO(CH2)nOR, -OCOR, -OR, -Cl, -CH, -C6H5, substituted -C6H5, -OCO(CH 2) nOH, -OCO(CH2)nOR, -OCO(CH2)nNH2, -OC O(CH2)nNHR, and '-OCO(CH2)nNR2. selected, and in the formula of these groups, n is an integer from 1 to 4 and R is a 1-4C straight chain or 24. The method of claim 23, wherein is a branched alkyl group. 25. Antibiotics suitable for constructing or coating biomedical devices that come into contact with blood. A method of preparing a thrombogenic and non-thrombogenic biocompatible copolymer comprising: at least one low molecular weight glycosaminoglycan and a prepolymerized non-glycan A prepolymer of cosaminoglycan is added via the reducing end of the glycosaminoglycan. and reacting with the prepolymer under conditions for derivatizing the glycosaminoglycan. A method that includes: 26. Water soluble, antithrombotic and nonthrombogenic, useful as a pharmacological agent at least one non-glycosaminoglycan. at least one glycosaminyl copolymerized with the monomer units of the fragment A water-soluble copolymer containing monomer units of noglycan fragments.