JPS5941656B2 - Method for imparting anticoagulant properties to regenerated cellulose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for imparting anticoagulant properties to regenerated cellulose or its derivatives.

Such regenerated cellulose-based materials are suitably used as medical polymer materials. It has been known to immobilize heparin on regenerated cellulose or its derivatives to obtain medical polymeric materials with anti-blood coagulation properties.

For example, a method of fixing heparin after treating a cellulose acetate membrane with bromine cyanide is proposed in Transactions of the American Society for Artificial Internal Organs, Volume 22, Page 654 (1976). A method for fixing heparin by treating cellulose with lead tetraacetate, periodic acid, or a salt thereof is disclosed in JP-A-53-57288. However, in these conventional cellulose heparinization methods, heparin is directly bonded and immobilized to the cellulose substrate, so the amount of heparin immobilized is small, and highly toxic substances are used to treat cellulose. Therefore, there were problems such as increased cleaning costs, and the method was impractical.

As a result of various studies regarding the above-mentioned problems, the present inventors have found that hydrogen peroxide-F
We have already proposed a method of graft polymerizing glycidyl acrylate or glycidyl methacrylate using a polymerization initiator such as e21 system or cerium salt, and then converting it into heparin (Japanese Patent Application Laid-open No. 18704/1983). (hereinafter abbreviated as GA) or glycidyl methacrylate (hereinafter abbreviated as GMA) is reacted with heparin in the presence of a polymerization inhibitor. He proposed a heparin derivative that exhibits coagulability (Japanese Patent Application Laid-Open No. 14780-1980)
2).

Based on these findings, we have further added heparin and GA or GMA.
By reacting the heparin derivative obtained by bonding with one or more types of polymerizable vinyl monomer and regenerated cellulose (including regenerated cellulose derivatives having a hydroxyl group) using a graft polymerization initiator to the regenerated cellulose, It was found that a material with extremely high anticoagulant properties can be obtained by bonding heparin to regenerated cellulose, and the present invention was completed. That is, the present invention provides a heparin derivative having a polymerizable double bond obtained by reacting heparin with an epoxide monomer having a polymerizable double bond such as GA or GMA, and one or more vinyl monomers in an aqueous solvent in the presence of a polymerization initiator,
The present invention provides a method for imparting anti-blood coagulability to regenerated cellulose or a derivative thereof by grafting it onto regenerated cellulose or a regenerated cellulose derivative having substantially hydroxyl groups. Examples of regenerated cellulose used in the present invention include cuprammonium rayon, viscose rayon, and cellophane. The regenerated cellulose derivative may be one that substantially has a hydroxyl group and can be graft-polymerized with a cerium salt or monoferrous hydrogen peroxide based polymerization initiator.A typical example of such a derivative is acetic acid. There are cellulose, carboxymethylcellulose, carboxyethylcellulose, methylcellulose, hydroxyethylcellulose, cellulose acetate propionate, cellulose acetate butyrate, cyanoethylcellulose, nitrocellulose, etc. Among these, cellulose acetate is particularly important as a medical molded product. be. The heparin derivatives used in the present invention are disclosed in, for example, JP-A-56-1478.
As shown in the specification, it is obtained by reacting GA or GMA and heheparin in an aqueous solution in the presence of a radical polymerization inhibitor, and its physical and chemical properties,
And its biological activity is almost the same as that of heparin.

Heparinization rate (which indicates the amount of heparin binding in the present invention)
Its definition is shown in Example 1.

) varies depending on the reaction conditions, the shape of the reaction target, etc.
For example, thickness 18 μm 1 diameter 50 m 77! When a Kyupammonium rayon film is used, it becomes about 5% or more. The vinyl monomer used in the present invention can be grafted onto regenerated cellulose or its derivatives in an aqueous solvent in the presence of a polymerization initiator such as ceric ammonium nitrate or hydrogen peroxide monosulfate. There are no particular restrictions as long as it is.

This polymerizable vinyl monomer has the function of binding regenerated cellulose or its derivative and the heparin derivative of GA or GMA. Therefore, hydrophilic properties are desirable in reactions with heparin derivatives, regenerated cellulose, and the like. Examples of such vinyl monomers include acrylamide, acrylic acid and its salts, 2-hydroxyethyl methacrylate, acrylonitrile, N-vinylpyrrolidone, sodium p-styrenesulfonate, methyl acrylate,
Examples include methyl methacrylate, vinyl acetate, sodium acrylic sulfonate, GA, and GMAl.

In the method of the present invention, the preferred aqueous solvent in the graft copolymerization reaction is water.

However, if the monomer is insufficiently dissolved, an organic solvent can be added to the water to the extent that the heparin derivative is dissolved. For example, a mixed solvent with an organic solvent can be used up to a ratio of acetone to water of about 1:2 (volume ratio) and a ratio of formamide to water of about 1:9 (volume ratio). Other hydrophilic organic solvents such as N,N-dimethylacetamide, N,N'-dimethylformamide, methyl ethyl ketone, and dioxane can also be used as a mixed solvent with water. If these mixed solvents are used, even monomers such as styrene, which are not hydrophilic, can be graft copolymerized in small amounts. Examples of the polymerization initiator used in the present invention include cerium salt-based initiators represented by ceric ammonium nitrate, hydrogen peroxide monometallic salt-based initiators represented by ferrous hydrogen peroxide monosulfate, etc. There is. The regenerated cellulose or its derivative used in the present invention is preferably shaped into a flat membrane, hollow fiber, or fiber. The reason for this is that in the case of an unformed product, the reaction solvent is extremely limited, and it is extremely difficult to find a solvent that can dissolve the heparin derivative, and even if it is possible to dissolve the heparin derivative, the heparin activity will be lost. Furthermore, even if it is sufficient to fix heparin only on the surface, if an unmolded product is used, it will be fixed uniformly to the inside, which is extremely uneconomical. For example, although heparin derivatives such as GA or GMA are dissolved in cupric ammonium solution, which is the solvent for cupric ammonium rayon, most of the heparin activity and anticoagulant properties are lost due to its strong alkalinity. There is a big possibility. In a preferred embodiment of the present invention, an appropriate amount of one or more vinyl monomers having a polymerizable double bond and a heparin derivative bonded with GA or GMA are added to the aqueous solvent within a range in which they are dissolved, and then regenerated. A molded product of cellulose or a derivative thereof is added, a polymerization initiator and, if necessary, an acid such as nitric acid are added, and the mixture is completely replaced with nitrogen to react.

The aqueous solvent is preferably kept in advance at a temperature above the freezing point and below 90°C, preferably above the freezing point and below 60°C. Generally speaking, the lower the temperature, the more heparin binds, and the higher the heparin activity, the longer the reaction time will be.On the other hand, if the temperature is high, the reaction rate will be fast, but if it is too fast, it will be difficult to control. Heparin degradation also increases and the activity of bound heparin decreases. The charging ratio for the reaction is as follows, based on 1 part by weight of the solvent at around 20'C.

The lower limit of the amount of regenerated cellulose is arbitrary, but the upper limit can be up to 0.06 parts by weight based on experience. The amount of heparin derivative is 0.001 to 0.5 parts by weight, preferably 0.05 parts by weight.
~0.3 parts by weight. The lower limit of this range is determined by reaction rate, reaction amount, etc., and the upper limit is determined by solubility and viscosity. The amount of heavy vinyl monomer is 0.0001 to 0.1
The amount is between parts by weight, and is determined by experiment, mainly within a range that maximizes the heparinization rate.

For both cerium salt-based and hydrogen peroxide-based polymerization initiators, it is easier to wash away residual polymerization initiators after the reaction if the amount is as small as possible, and the heparinization rate tends to be higher at the same grafting rate. Therefore, it is advantageous. However, if it is too small, the grafting rate will be too small. The desirable amounts for both hydrogen peroxide and cerium salt are 0.01 to 1 per 11 parts of the solvent.
0 mm011, preferably about 0.1 to 2 mm01. Further, the amount of iron salt relative to hydrogen peroxide and the amount of nitric acid relative to ceric ammonium nitrate are preferably in the range of about % to 5 times, respectively. If regenerated cellulose or its derivatives are hollow fibers and a heparin derivative is bonded only to the inner surface, a module with a form similar to a completely sealable hollow fiber artificial kidney is made, and a flask that is also sealable is prepared. The reaction can be carried out by introducing and circulating an aqueous solution containing a heparin derivative, a vinyl monomer, a polymerization initiator, and a small amount of acid.

Of course, it is preferable to carry out the reaction by completely purging the entire system including the module, flask, and reaction solution with nitrogen. If the reaction temperature is within the above range, it is advantageous from the two aspects of maintaining heparin activity and controlling the reaction rate, and the reaction time is linked to the temperature, initiator concentration, monomer concentration, monomer type, and heparin derivative. GA or GM
Although it varies depending on the amount of A, etc., it is carried out for about 10 minutes to about 10 hours. The amounts of reacted heparin derivatives and monomers can be accurately estimated from the weight difference between the regenerated cellulose or its derivatives before and after the reaction, and from estimating the amount of heparin from measuring the sulfur content by the henomarin nephelometric method, sulfur combustion method, etc. I can do it. To qualitatively examine the binding of heparin, it is sufficient to stain with Azure A and observe the metachromatic coloration to deep blue. The effectiveness of heparin bound to regenerated cellulose or its derivatives can be determined by clotting time measurements using a Lindform cell method using canine whole blood. The measurement method in this case can be carried out by a method called Lindholm test, the outline of which is as follows. First, a film bound with about 1 gram of henomarin by dry weight was immersed in 500ml of 25% by weight saline for 10 hours, and the saline was replaced and this operation was repeated twice to eliminate ionic bonds and similar dissociation. Easy to remove heparin. After washing this film with a large amount of physiological saline, place it on a glass plate, press it with a 5 mm thick silicone gasket with a hole of 20 ψ in the center, and then cover it with a glass plate with a hole of the same diameter. Assemble the Lindholm cell by pressing down and tightening it with a board. After discarding the most 0.5 TLe from the dog's jugular vein, collect about 5 me of whole blood and immediately spread exactly 0.5 TLe per sample evenly on the film, every 5 minutes for the first 20 minutes. After 20 minutes, tilt the tube 452 every 2 minutes to measure the time until the blood coagulates and stops moving. 1 each for 3 dogs
The blood clotting time was measured twice and is shown as the average value of the blood clotting time of 3 dogs. The anticoagulant regenerated cellulose or its derivatives obtained by the present invention may contain small amounts of polymerization initiators and vinyl monomers as impurities, but these can be relatively easily removed by washing with water.

Furthermore, since relatively inexpensive vinyl monomers can be used, the cost is low and the practicality is high. Furthermore, compared to the method of directly bonding heparin to regenerated cellulose or its derivatives, the method of the present invention is presumed to have heparin bonded to some of the branch molecules of the graft polymer. Since heparin is immobilized in large quantities and extremely efficiently, it exhibits better anticoagulability. The regenerated cellulose or its derivatives obtained by the present invention can be applied as a flat membrane or hollow fiber, or as a membrane for dialysis or filtration, if appropriate reaction conditions are selected, and if it is made in the form of fibers, it can be used in surgical procedures. It can also be used as a surgical suture. Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Heparin sodium (Handon Chemicals, 1501U/M7
) Dissolve 10 grams in distilled water to make a 10% by weight solution.

Add to this 0.1 P-methoxyphenol as a polymerization inhibitor.
GMA577ll with (f) added! and stirred for 50 minutes.
'C and react for 16 hours. After the reaction, the reaction solution was concentrated using a rotary evaporator and poured into 11 methanol with stirring to obtain a white powder precipitate. The obtained precipitate was collected, thoroughly washed with methanol, dissolved again in a small amount of water, the same operation was performed, the precipitate was collected, and it was dried in a vacuum dryer until it reached a constant weight. The resulting material is a white, hygroscopic powder with roughly the same physical properties as the starting material heparin, soluble in water and insoluble in organic solvents such as methanol and acetone. When this product was dissolved in water and gel permeation chromatography was performed, it showed a chromatography that was almost completely the same as that of the starting material, indicating that the double bonds of GMA bound to heparin were not polymerized. I solved it. Furthermore, it was confirmed from the infrared spectrum and NMR spectra that GMA and heparin were bonded. The amount of bound GMA determined from the infrared spectrum was about 0.2 mm01 for heparin 19. This heparin derivative was designated as [1] and used in the next reaction. 500m
Fill the following four flasks with 1.0 g (approximately 30 sheets) of circular copper ammonia rayon film 18 μm thick and 50 mm in diameter using the composition shown below, and repeat the method of sealing and reducing the pressure three times to purge with nitrogen. After that, the reaction was carried out.

The reaction temperature was 30°C. Copper ammonia rayon (1
8/111176 approx. 30 pieces) 1.0g50m77! After ψ nitrogen substitution, the reaction was stopped with a magnetic stirrer, and the reaction was stopped after 1 hour. A copper ammonia rayon film was poured into a large amount of water and washed, and the grafting rate, heparinization rate, Lindholm test, etc. , compared with unreacted film.

The grafting rate and heparinization rate were defined by the following formulas.
The amount of heparin bound by the reaction was measured by extracting the sulfate radicals of the reactant by acid hydrolysis, adding a gelatin solution of barium chloride to this, and estimating the amount of sulfate radicals measured by turbidimetry. It is something. Next, the measurement results are shown.
These results show that 8.1 x 10-5 (g) of heparin is bound, resulting in a significant prolongation of the coagulation time.

The water permeability rate of this film has a pressure difference of 50m77! /Hg, the unreacted film was 3 at 37°C.

2 (Me/Hrm2・MmHg), compared to 1.
95 (d/Hrm2·M77!Hg), and has high applicability as a dialysis type and filtration type artificial kidney membrane.

Example 2 5g of heparin sodium was dissolved in distilled water to make a 5% solution, and GA2 containing 0.5% of P-methoxyphenol was added to the solution.
Me was added and reacted at 40°C for 24 hours.

The reaction product was recovered and purified in the same manner as for heparin derivative [1] in Example 1. This product was confirmed from an infrared spectrum to be a heparin derivative bound to GA having a double bond. This product was made into a heparin derivative [], and the following formulation was placed in a constant temperature bath kept at 40°C, and charged into a 500 d four-hole flask. [Approximately 30 sheets, 18 μm thick, 50 m long] The above materials were charged, replaced with nitrogen, and reacted and washed in the same manner as in Example 1.

The reaction temperature was 25°C and the reaction time was 1.5 hours. A test was conducted in the same manner as in Example 1, and the following results were obtained.
The above results demonstrate that the method of the present invention was able to sufficiently bind heparin and produce a film with high anticoagulant properties.

When the water permeation rate of this film was measured at a pressure of 50 mnHg, it was 2.25 ((4-1rm2・MmHg).The unreacted film was 3.2 (MlAlrm2・MmHg).
), it was found that the possibility of application as a dialysis membrane for artificial kidneys is large enough. Example 3 Copper ammonia rayon hollow fiber (Asahi Kasei product, inner diameter 200
μm1 (outer diameter 226 μm), and using urethane as a potting agent, it was assembled into a hollow fiber type artificial kidney module (distance between dialysate inlet and outlet 9.0?, hollow fiber 5000 mm).
Dialyzer inner diameter: 2.6CTL) Connect one end of the dialysate inlet/outlet of each of the two modules thus made to a nitrogen source, and connect the other end to a rubber tube with a pinch stopper. The pinch bottle was closed and sealed.

Also, one of the blood inlets and outlets of the two modules is connected to one opening of the 21 four-loop flasks with a tube, and the other one is connected to the other opening of the four-loop flask via a roller pump, respectively. Connect them so that one of the parts joins. The other two openings in the four-ring flask were connected to a nitrogen source for nitrogen purging of the flask and circuit, one used as a thermometer insertion port and the other for nitrogen purging of the flask and circuit. With this circuit configuration, the reaction solution in the flask was allowed to return to the flask through the hollow fibers in the module. The passage speed of the reaction liquid inside the module is 10me/M
L. I adjusted it so that The reaction solution was charged in four Lof flasks according to the following composition, and immediately the cycle of reducing pressure and introducing nitrogen into the module was repeated three times to purge with nitrogen. Next, the temperature was maintained at 40°C and the roller pump was heated. After reacting for 40 minutes, the module was taken out and washed with a large amount of distilled water.

[Composition]

One of the washed modules was destroyed, the hollow fiber was taken out, and the fiber was immersed in 500 me of 25% saline twice for 10 hours, changing the saline twice, and then thoroughly washed with physiological saline. , Put the blood that has just been collected from the jugular vein of the vehicle dog using the double syringe method, close both ends, and
It was left sealed in a polyethylene bag in an incubator at 7°C.

At the same time, blood was added to the unreacted hollow fiber using the same procedure for a comparative test. The hollow fibers bound with heparin did not cause blood clots even after 240 minutes, whereas the unreacted hollow fibers showed blood clots after 60 minutes. In addition, when the water permeation rate was measured for another module at 400 mmHg for the membrane,
It was 2.50 (Me/Hr.m2.mTLHg).

Claims (1)

[Scope of Claims] 1. A heparin derivative obtained by bonding glycidyl acrylate or glycidyl methacrylate to heparin, one or more polymerizable vinyl monomers, and regenerated cellulose or its derivative are combined in water in the presence of a polymerization initiator. A method for imparting anticoagulant properties to regenerated cellulose, which comprises reacting in a solvent. 2 Claim 1 in which the polymerization initiator is hydrogen peroxide-Fe^2^+ type or ceric ammonium nitrate
The method described in section. 3. The method according to claim 1, wherein the aqueous solvent is water. 4. The method according to claim 1, wherein the reaction temperature is above the freezing point and below 60°C. 5. The method according to claim 1, wherein the regenerated cellulose is steel ammonia rayon, viscose rayon, or cellophane. 6. The method according to claim 1, wherein the regenerated cellulose derivative is cellulose acetate.