JPH0744950B2 - Antithrombogenic material and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an antithrombotic material and a method for producing the same, more specifically, a material that is relatively inexpensive, can be easily produced, and has a high antithrombotic property. The present invention relates to a manufacturing method thereof.

(Prior Art) Research on the application of heparin to antithrombotic materials is based on blending
It is roughly divided into coating method, ionic bonding method and covalent bonding method.

The blending method and the coating method are based on the idea that heparin is consumed stoichiometrically to inhibit thrombus formation on the material surface. The coating method is
Heparin is simply applied to the surface of the material, and its effect lasts little. Further, in the blending method, even if the heparin is directly kneaded with the resin, it is impossible to uniformly disperse the resin, and thus it is extremely difficult to maintain its sustained release property. Therefore, heparin was adsorbed on graphite and dispersed in silicone rubber (Hufnagel CA, et al., Ann NY Acad Sci., Vol.146, 262 (1
968)) or heparin adsorbed on silica and dispersed in polyethylene resin (Yamashita et al., Chem. Expr.
ess, vol 1, NO.10,611 (1986)), or one in which heparin is embedded in a hydrogel and the hydrogel is dispersed in a resin (Akashi et al., JP-A 1-232968). It was However, in any of the materials, a carrier for adsorbing and accumulating heparin is indispensable, and the safety of this carrier must be carefully considered.

On the other hand, studies have also been actively conducted in which heparin is bound to a base material by an ionic bond and then released. For example, after the base material is molded and processed, it is immersed in a solution of heparin to accumulate heparin in the base material at a high concentration by ionic bonding (Nagaoka et al., JP-A-55-60461, JP-A-57-119756). It is said that this material is excellent in long-term sustained release of heparin. However, there are problems that the manufacturing method is complicated, the manufacturing time is long, the activity of heparin is lowered due to the relatively high temperature treatment, and the cost is high.

(Problems to be Solved by the Invention) The object of the present invention is to say that it has been said to be extremely difficult until now, does not include a carrier for adsorbing and accumulating heparin at all, and is composed of heparin and a matrix polymer. And a highly safe heparin sustained release material,
It is to provide it easily and cheaply.

(Means for Solving the Problems) The present invention has a particle size of 0.1 in a water-insoluble polymer matrix.
Heparin particles of ~ 30μm are dispersed 5-40wt%,
By contact with blood at 37 ℃, 10 ^-1 to 10 ^-3 μg / cm
Heparin is gradually released at a rate of ² min, which is an antithrombotic material characterized by inhibiting thrombus formation on the polymer surface for 24-150 hours, and further, 5 wt% or more and 30 wt% or less. A heparin aqueous solution having a concentration of 3 to 100 times the volume of the aqueous solution is poured into the precipitating agent while stirring the precipitating agent at a speed of 50 rpm or more to reprecipitate heparin, and centrifuge at 4,000 G or more and 10,000 G or less. 10 at acceleration
Ultracentrifugation for ~ 60 minutes to precipitate heparin, then discard the supernatant, and pour approximately the same amount of organic solvent as the precipitating agent into 0.1 μl of heparin while the precipitated heparin is not dried.
m or more and 30 μm or less in the form of fine particles, in which the water-insoluble polymer soluble in the organic solvent is dissolved, and finally the weight of heparin relative to the total weight of the water-insoluble polymer and heparin Of 1 to 40 wt%, and finally, the prepared solution is molded by a casting method or by coating on the surface of a substrate by a coating method and then drying. It is a method for producing an antithrombotic material.

The present inventors have found that the particle size of heparin particles to be dispersed and its content, and three factors of hydrophilicity of the matrix polymer govern the sustained release of heparin,
Further investigations have led to the completion of the present invention. Hereinafter, the present invention will be described in more detail.

In the present invention, the size of heparin particles dispersed in the matrix polymer is 0.1 to 30 μm, preferably 0.5 to
5 μm is suitable. If the particle size is 0.1 μm or less, spontaneous aggregation of particles occurs, and it becomes necessary to add a chemical substance such as a dispersant, which poses a safety problem when used in medical applications such as catheters. Further, if it exceeds 30 μm, the following problems occur. First, the undulation of the material surface becomes large, and it becomes easy to induce thrombus in terms of blood rheology. Further, at the same addition amount, the distance between the dispersed heparin particles becomes too large, the diffusion of heparin in the matrix is delayed, and the heparin density becomes too small on the material surface,
The initial antithrombotic properties are reduced.

The water-insoluble polymer in the present invention is not particularly limited except that it is water-insoluble and soluble in an organic solvent, but the one having a contact angle on the surface of about 30 to 80 degrees is a heparin-containing polymer. It is preferable in terms of release performance. For example, polyurethane,
Especially polyether urethane, soft vinyl chloride resin, vinyl chloride-vinyl acetate copolymer, vinyl chloride-ethylene-
Vinyl acetate terpolymer, ethylene-vinyl acetate copolymer and the like are suitable.

Next, the method for producing the antithrombotic material in the present invention will be described in detail.

Heparin may be commercially available as a reagent, but if possible, it is preferably clinically widely used.
Recently, low molecular weight heparin (Fragmin ^R , manufactured by Kabi Vitrum, etc.), which has become a hot topic because of few side effects, can be applied according to the purpose. Also, its form is
Powders or solids are preferable, and powders or solids prepared as an aqueous solution are not preferable because it takes time to adjust the concentration again to a required concentration. Water for dissolving heparin is not particularly limited, but distilled water, particularly distilled water for injection, pure water, ultrapure water, etc. can be used.

The heparin precipitant is not particularly limited, but methanol,
Propanol, acetone, dimethylacetamide and the like are preferable. Ethanol, ethylene glycol and the like dissolve heparin depending on the mixing ratio with water, and are not suitable as precipitants for heparin.

The concentration of the aqueous solution of heparin prepared first is 5 to 30 wt%
Is preferred. If it is less than 5 wt%, the recovery rate of low molecular weight heparin will decrease during reprecipitation, and if it exceeds 30 wt%,
The efficiency of reprecipitation is reduced and the heparin particles become too large.

The amount of the precipitating agent is also important and is preferably 3 to 100 times that of the heparin aqueous solution. If it is less than 3 times or more than 100 times, the recovery rate of heparin by precipitation is lowered and the particle size is also unsuitable.

The centrifugation conditions for collecting heparin are preferably 4,000 to 10,000 G, and about 97% or more of heparin can be recovered under these conditions. Below 4,000 G, the recovery rate of heparin is low, 10,000
Even if it exceeds G, there is almost no change in the recovery rate.

When the supernatant is discarded after ultracentrifugation, heparin is recovered in the form of fine particles containing water and a precipitant. If this gel is dried, the particles will agglomerate to form a coarse powder. Therefore, before drying, the organic solvent is rapidly added and stirred. At this time, if the dispersion of heparin is insufficient, heparin can be effectively dispersed without agglomeration by a method such as stirring while irradiating ultrasonic waves in an ultrasonic cleaner or the like.

The organic solvent used here is not particularly limited as long as it can dissolve the above-mentioned water-insoluble polymer, but for example, a general-purpose organic solvent such as tetrahydrofuran, dimethylacetamide, dimethylsulfoxide, acetone, or methylethylketone is used. It is possible.

The solution thus obtained can be easily imparted with antithrombogenicity on the surface of the catheter by applying it to a catheter or the like and drying it, and by using a casting method, it can be applied in the form of a film, a tube, or the like. A thrombosed molded product can be obtained.

The concentrations of the water-insoluble polymer and heparin in the solution can be selected according to the purpose. Basically, first, the amount of heparin added mainly controls the initial sustained release rate of heparin, and the level of antithrombotic property (degree) is determined by defining the heparin density per unit area of the surface.
Dominate. Secondly, the thickness of the coating layer basically controls the sustained release period of heparin.

Furthermore, although depending on the type of water-insoluble polymer of the matrix, the thickness of the coating layer has a minimum thickness necessary for sustained release of heparin. Depending on the amount of heparin and the type of water-insoluble polymer, the minimum thickness is approximately 10
~ 50 μm. The reason why this minimum thickness is required is not clear, but the following can be considered. As a mechanism for sustained release of heparin, it is presumed that water in the blood diffuses and infiltrates into the applied polymer, swells the polymer, dissolves the heparin fine particles in the polymer, and diffuses it out of the tube. In this case, in the vicinity of the base polymer,
It is considered that the swelling of the coated polymer is hindered because the coated polymer adheres to the substrate. Therefore, in order to connect the sustained release of heparin, it is considered that the thickness of the coating polymer must be thick enough not to hinder its swelling.

The antithrombotic material in the present invention needs to be sterilized before use, and ethylene oxide gas sterilization which is widely used can be applied to this. The sterilization conditions are not particularly limited, but it is preferable to sterilize at a temperature as low as possible in order to retain the activity of heparin as much as possible, and it is usually preferable to carry out at 60 ° C or lower.

In the present invention, the antithrombotic material is composed only of heparin and a matrix polymer that disperses it, and does not contain any excess component. Even with such a simple structure, the sustained release of an effective amount of heparin, which was considered to be almost impossible in the past, can be continued for about 1 week or longer. Moreover, there is an advantage that the product can be provided relatively easily and inexpensively, as can be easily estimated from the manufacturing method.

The effects of the present invention will be described below with reference to examples.

Example 1 500 mg of powdered heparin (manufactured by Wako Pure Chemical Industries, Ltd., special grade heparin sodium) was dissolved in 1.5 ml of pure water, and 100 rpm
The heparin was reprecipitated by pouring it into 50 ml of acetone stirring at a rate of about 1 minute over about 1 minute. With an ultracentrifuge
Heparin was precipitated by centrifugation at 6000 G for 30 minutes. Discard the supernatant, add 45 g of tetrahydrofuran to it, and add 1 at 120 rpm.
The mixture was stirred for 0 minutes to prepare a heparin dispersion liquid. Next, 4.5 g of a soft vinyl chloride resin (1170G-50, manufactured by Sumitomo Bakelite Co., Ltd.) was dissolved in this dispersion. In this way, a solution was prepared in which the amount of heparin in the dry solid content was 10 wt% and the solid content concentration (total of resin and heparin) was 10 wt%.

Vinyl chloride resin (Sumitomo Bakelite Co., Ltd.)
Manufactured by G-540R) (thickness: 200 μm) using a 0.2 mm spacer, and dried under reduced pressure at 50 ° C. for 5 hours.
Prepare the obtained coating sheet for an area of 50 cm ² and
While immersing in l phosphate buffer and incubating at 37 ° C, the amount of heparin that elutes is measured by the absorbance method using toluidine blue (Yoshizawa Zen, "Heparin" (Kodansha Scientific, 1981); Wollin.A). , et al., Throm.Re
s., vol.2,377 (1973)).

The time-dependent change in the sustained release rate of heparin is as shown in FIG. 1, and the material of the present invention has a very simple structure, and is 10 ⁻³ μg / cm ² min or more over a relatively long period of time. It can be seen that heparin is released slowly at a rate. In addition, a longer-term sustained release is possible by considering the prescription. In addition, when the surface and cross section of the coated sheet produced by this method are observed with a scanning electron microscope (Hitachi S-800), the particle size is 0.5 to 10
It was confirmed that heparin particles of about μm were regularly dispersed.

Example 2 Basically the same as in Example 1, except that the solid content concentration was 10 wt%,
A heparin dispersion solution having a heparin concentration of 2 wt% in the solid content (Polyurethane (Tecoflex 80A) and heparin manufactured by Thermedix Co., Ltd.) was prepared. This is a vinyl chloride resin tube (outer diameter 5.0 mm, inner diameter 3.5 mm, length 25
It was applied to the inside of 0 mm, both ends cut at an angle of 45 degrees) and the outside from both sides to 6 cm, and dried under reduced pressure for 7 hours.

The tube thus obtained and the vinyl chloride resin tube not coated with the antithrombotic material of the present invention were sterilized with ethylene oxide gas at 50 ° C. for 5 hours.

The following antithrombotic animal experiments were performed using these tubes.

Under anesthesia with halothane, both jugular veins of a mixed-breed dog (male 20 kg) were exposed, and one tube was inserted into each jugular vein from the upper left to the lower right and from the upper right to the lower left and fixed. A 5-hour blood bypass test was performed while monitoring the blood flow rate with a blood flow meter. About 40 tubes of blood in each tube
There was almost no change, maintaining ml / min. After the 5-hour bypass test, cut off the upstream blood vessel by about 2 cm from the tip of the upstream tube, gently remove the tube from the downstream side, and leave the tube and the upstream blood vessel as one unit. I took it out. After gently rinsing the lumen of the tube with physiological saline, the blood vessel was vertically incised in the blood flow direction, and the state between the tip of the tube and the blood vessel wall and the state of the entire tube were observed. Further, the upstream tip, the central portion, and the downstream tip of the tube were each cut by about 3 cm, fixed with 2% glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd., for electron microscope) for 24 hours, and the scanning electron The inner surface of the tube was observed under a microscope, and the results are shown in Table 1.

(Effect of the Invention) As described above, the antithrombotic material according to the present invention has a considerably excellent antithrombotic property even in a dog experiment, which is said to have a significantly higher activation capacity for platelets and the like than humans. I found out that In addition, the manufacturing method has a feature that it is extremely simple, rich in reproducibility, and can be provided at a low cost as compared with the conventional method.

[Brief description of drawings]

FIG. 1 is a graph showing the change over time in the sustained release rate of heparin of a sheet coated with the antithrombotic material according to the present invention.

Claims (2)

[Claims] 1. A water-insoluble polymer matrix having a particle size of 0.
Heparin particles of 1 to 30 μm are uniformly dispersed in an amount of 5 to 40 wt%, and by contact with blood at 37 ° C., 10
Heparin is gradually released at a rate of ^-1 to 10 ^-3 μg / cm ² min, and 24 to
An antithrombogenic material, which prevents thrombus formation on the surface of the polymer for 150 hours. 2. A heparin aqueous solution having a concentration of 5 wt% or more and 30 wt% or less is poured into a precipitant having a volume of 3 times or more and 100 times or less the volume of the aqueous solution while stirring the precipitating agent at a speed of 50 rpm or more to remove heparin. Re-precipitate, ultracentrifuge at a centrifugal acceleration of 4,000 G or more and 10,000 G or less for 10 to 60 minutes to precipitate heparin, then discard the supernatant, and settle the heparin in an amount equal to that of the above-mentioned precipitant while the heparin is not dried. The organic solvent is poured to disperse the heparin in the form of fine particles, the water-insoluble polymer soluble in the organic solvent is dissolved in the dispersion, and finally the total weight of the water-insoluble polymer and heparin is It is characterized in that the weight of heparin is adjusted to 1 to 40% by weight, and finally, the prepared liquid is molded by a casting method, or by coating on the surface of a substrate by a coating method and drying. Antithrombotic Method of manufacturing a fee.