JPS6182760A - Antithrombotic medical material - 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 [Industrial Field of Application] The present invention relates to a UC thrombogenic medical material, specifically, an anti-inflammatory material with thrombomodulin or protein C immobilized thereon.
Concerning pluggable materials.

[Prior Art] When using medical materials such as artificial organs, blood clots caused by contact with blood are one of the major problems. For example, when extracorporeal blood circulation is performed, thrombi occur in blood pumps and artificial N-line circuits.Therefore, it is desired to develop anti-1r1 [pluggable medical materials] that do not cause thrombi and have good blood compatibility. There is.

Currently, when using artificial dialysis or heart-lung machines, anticoagulants (such as Hevarino) are administered, but these have the disadvantage of having side effects.

In recent years, it has been discovered that a protein derived from vascular endothelial cells called thrombomodulin has an anticoagulant effect.

[Object of the Invention] An object of the present invention is to provide an antithrombotic material that has sufficient anticoagulant activity by itself by utilizing thrombomononiline and protein C.

[Structure of the Invention] The gist of the present invention resides in the antithrombotic +4 type comprising thrombomononiline or protein C immobilized on a physiologically acceptable carrier.

Thrombomodulin and protein C used in the present invention are known substances, and the former is, for example, Proc.
, Na11. 8 cad, Sci, USA, Vo
178, No, 4. The latter can be easily collected from the whole surface of a cow (see one example below).

Physiologically acceptable1. The carrier obtained is a substance that does not have an extremely harmful effect on living organisms, and is used as a material for artificial devices, blood pots, or artificial blood circulation circuits, or as a corchinoid for these devices. Refers to substances that are currently used or that will be used in the future. Examples of such substances include polyhydroquinoethyl methacrylate, borihinyl alcohol, Norikono,
Ebokino fat, Boristileno, vinyl chloride, nylon,
Tetoron, cellophane, polyurethane, polytetrafluoroethylene, polyvinylpyrroliton, polypropylene,
1.1 Ethylene, polyvinyl fluoride, polybutylene, neobre/, polyvinylidene chloride, polyethylene terephthalate, polycarbonate, polysulfonate, polyethylene naphthalate, polyvinyl formal, polyvinyl butyrate, acrylic resin, nitrocellulose, gelatin , cellulose, acrylamide, kolaken, acetal, polyester, polyamide, polymethylpenoteno, fibrino, polyvinyl acetate, boriamite, polyacrylonitrile, polyhenoneimidazole, polyglyfuric acid, etc. Mention may be made of molecular or natural materials.

Copolymers consisting of these structural units and pellets of the above materials can also be used.

Depending on the purpose of use, the shape of the carrier may be flat, tubular, particulate, or
It may be fibrous or mesh-like, and its surface may be of any shape, such as smooth, rough, or porous.

As an immobilization method for binding an enzyme and a carrier, a coexistence binding method is preferable from the viewpoint of a fixed lifetime. Examples of the coexistence bonding method include the AND bonding method, the noane bromide activation method, the carboimide activation method, the 0-alkylation bonding method of an amide group, and the noane bromide activation precipitation method is particularly preferred.

[Effect of the invention 1] When the antithrombotic material of the present invention is used, for example, in a blood circulation circuit, a reactor, or other artificial organs that come into direct contact with blood, it is possible to avoid thrombus caused by blood coagulation, and it can be used with peace of mind. It can be done without treatment. This material can also be directly injected into the blood. When using this medical material, no drug is required to prevent blood clots.

In the following, examples of the present invention will be given and explained in more detail.
The present invention is not limited to the examples in any way.

Example] Purified raw persimmons were homogenized with a biomixer and ultracentrifuged (30
0009, 40 minutes), the precipitate was taken, and the surfactant Dryde:/X-100 (lohm & Haas
Thrombomononiline was solubilized using Co., Ltd., USA).

Powdered Sepharose 4F3 (P
Manufactured by hArmacia Fine Chemica1,
Sweden) was added to the Tronohino solution to immobilize it, activated with neuisobropyruph (7 sulfate), and then packed into a column. This column allowed the tJI compatibility of thrombomononiline and Tronohino to be used to immobilize Afsonity Chromatography. 1f Noni with Claffy.

Perseibun purified tronovomononiline fraction 1110. , OIM
NaHCOslo, 5M NaC (added to swollen noane bromide-activated Sepharose 4B at 4°C for 12 min.
Stir gently for ~16 hours. Immobilized Sepharose 4B
Collected twice through a glass filter, and added an O,lM NaHCOs aqueous solution containing 0.5M NaC0, and further 0.5M NaHCOs.
After washing twice with iM acetate buffer (pH 40) containing CQ, the cells were kept in Tris-HCl buffer (pH 7) containing 0.145M NaCQ (for activity measurement, blood was collected from the rabbit ear vein). 25007 citrated whole blood
for 15 minutes, and then /I'I (platelet-poor plasma,
PPP) m, p p p and toa/hino (0,33
U/ffc) and trompomodulino-immobilized Sepharose were mixed, and a fibrometer was used to observe the prolongation of blood coagulation time.For comparison, Sephrose without thrombomononiline immobilized The prolongation of blood clotting time was measured.

The 11th fruit is also shown in the first column.

Table 1 Example 2 0 and 1 M sodium citrate 125a& were added to the total LflIQ at position 'μ, and after centrifugation, 1 M barium chloride 802Q was added to the supernatant, and after further centrifugation, the precipitate was
Suspended in 50v, Q 09% sodium chloride solution.

After centrifugation, the precipitate was dissolved in 0.2 M ethyleneno-amino-tetraacetic acid solution at 150 ml and subjected to ammonium sulfate salting out (40
~67%). The obtained precipitate was dissolved in water and dialyzed against a phosphoric acid solution. Thereafter, it was further purified using DE7\[et al. Sepharose. The eluted protein C fraction was frozen with liquid nitrogen, and then lyophilized and concentrated.

imitation star Immobilization is "N," in Example 1! !　Ute(J-)ta.

Activity measurement Jll, quality is bebutinormedil coumarin amide (B
Using protein C (oc-Leu-Ser-Thr-Arg-MCA), arginino (
The peptide bond between Arg) and methylcoumarinamide (MCA) is cleaved. As a result, the released aminomethylcoumarino emits fluorescence, which is measured using a fluorometer to calculate protein C activity.

In this example, as a control sample, a gel in which triethanolamino was immobilized instead of protein C was used to block unreacted active groups after immobilization.

The results are shown in Table 2.

Table 2

Claims (1)

[Claims] 1. An antithrombotic material comprising thrombomodulin or protein C immobilized on a physiologically acceptable carrier.