CS265772B1 - A method for removing heparin from blood in vitro - Google Patents

Abstract

The solution relates to a method for removing heparin from blood in vitro. The essence of the method for removing heparin from blood, especially from blood plasma, in vitro consists in adding a preparation, preferably in the form of a tablet, containing 5 to 150 parts by weight of at least one blood-insoluble polysaccharide derivative with bound tertiary or quaternary amino groups with an exchange capacity of 0.3 to 1.3 mmol.g-1 to 0.5 to 3 parts by volume of blood or blood plasma, and allowing the mixture to swell for at least 1 minute, stirring the mixture vigorously or leaving it to stand until the heparin is adsorbed, separating the solid portion and using the blood plasma for testing. The preparation added to blood or blood plasma additionally contains microcrystalline cellulose in an amount of up to 12 parts by weight based on 1 part by weight of the polysaccharide derivative. The polysaccharide derivatives are preferably cellulose derivatives or starch derivatives. The solution has applications in medicine, especially in hematology when examining patients taking heparin for real hemostatic potential. Furthermore, wherever a simple and rapid method of removing heparin from blood or blood plasma is required.

Description

265772 2

The invention relates to a method for removing heparin from blood, in particular blood plasma, in vitro.

When treating thromboembolic diseases with heparin, it is necessary to perform complex haemocoagulation tests in the patient, which characterize the patient's health status and show the success or failure of the treatment process. Heparin present in the collected blood samples has a great deal of distortion. Therefore, a rapid and reliable method is needed to remove it without any side effects on blood plasma. Heparin is commonly neutralized with protamine in clinical practice [J. G. Allen et al., J. Lab. Clin. Copper. 34, 473 (1949); Perkins et al., J. Lab. Clin. Copper. 48, 223 (1956) J, however, the fact that heparin blood levels are not accurately known in the collected blood plasma of patients is difficult to titrate. It follows that the protamine excess in the plasma remains undetected or undetected. unneutralized heparin remains in blood plasma, which negatively affects the results of hemocoagulation tests. J. S. Wright et al. have already pointed out these problems [j. Wright et al., J. Cardiovas. Surg. 5, 244 <1964) 3. To avoid these problems, Thompson used bikes. [_AND. R. Thompson, R. B. Counts: J. Lab. Clin. Copper. 88, 922 (1976) 3 column chromatography to capture heparin and protamine from blood plasma samples. They used medium-base cellulose-based hexane to capture heparin, and protamine was adsorbed on carboxymethylcellulose. Further, a portion of the blood plasma is trapped on the columns of ion exchangers, which is a disadvantage of the prepatric cases where a small amount of plasma is used for testing.

The above drawbacks are substantially altered by a method of removing heparin from blood, in particular from blood plasma, in vitro, according to the invention, which adds a preparation, preferably in the form of 0.5 to 3 parts by volume of blood or blood plasma. tablets containing 5 to 150 wt. parts of at least one blood-insoluble polysaccharide derivative bound by tertiary or quaternary amino groups having an exchange capacity of 0.3-1.3 mmol.g-1, allowed to swell for at least 1 minute, agitated vigorously or not until heparin is adsorbed the solid support is separated and the blood plasma is used in the test. The blood or blood plasma addition preparation contains up to 12 wt.% Of microcrystalline cellulose. parts based on 1 wt. The polysaccharide derivative is preferably cellulose derivatives or starch derivatives. The cellulose derivative is selected from the group consisting of: diethylaminoethyl cellulose, dimethylaminoethyl cellulose, trimethylammonium cellulose, diethylaminopropyl cellulose, triethylammonium propyl cellulose, dimethylamino propyl cellulose, trimethylammonium propyl cellulose, diethylamino-2-hydroxypropyl cellulose, polyethyleneimine cellulose, triethylammonium 2-hydroxypropyl cellulose, dimethylamino- 2-hydroxy-propyl cellulose, cellulose crosslinked with a mixture of epichlorohydrin and diethylamine in alkaline medium, imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) cellulose ether, dimethyl amino-bis- (2) chloride -hydroxy-3-propyl / cellulose ether, benzyldimethylammonium 2-hydroxypropylcellulose. The starch derivative is selected from the group consisting of: diethylaminoethyl cross-linked starch; -2-hydroxypropyl cross-linked starch, trimethylammonium methyl cross-linked starch, diethylaminopropyl-starch starch, epichlorohydrin-diethylamine alkaline starch starch, imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) ether starch ether, dimethyl amino-bis- (2-hydroxy-3-propyl) ether starch ether, benzyldimethylammonium 2-hydroxy-propyl cross-linked starch. The polysaccharide derivative consists of particles having a particle size of 50 to 300 µm, but it is also possible to use a polysaccharide derivative with a smaller or larger dimension. The advantage of the proposed method of removing heparin from the blood of interest Blood Plasma In Vitro is that it utilizes a progressive test tablet method, as a quick and easy way to completely eliminate clinical plasma heparin levels at a determined hemostatic potential and to monitor other coagulation disorders in the patients being examined. A further advantage is that the heparin-adsorbed test preparation is well separated in the plasma, so it cannot affect subsequent hemocoagulation assays in contrast to previous methods. EXAMPLE 1 <0.5 ml of blood plasma in a tube (12x95 mm) is supplemented with a tablet containing 5 mg of chloridimidazolium-N, N'-diyl-bis / 2-hydroxy-3-propyl / cellulose ether with exchange. with a capacity of 1.3 mmol.g 1 and 6 mg mg of microcrystalline cellulose and allowed to swell for 0.5 min at 20 ° C. Thereafter, the tube contents are vigorously mixed on the vibrator until the particles are evenly distributed in the plasma and stirred on the stirrer for 10 minutes at 20 ° C. The heparin-adsorbed tablet substitute is centrifuged in a laboratory centrifuge (1200xg, 5 min) and the respective plasma aliquots are used for hemocoagulation assays. Example 2 To 1 ml of blood plasma in a tube (12x95 mm) is added a tablet containing 20 mg of chloridimidazole-N, N'-diyl-bis (2-hydroxy-3-propyl) cellulose ether with a exchange capacity of 0.7 mmol. 1 and 60 microns of microcrystalline cellulose and allowed to swell for at least 1 min at 22 ° C. Thereafter, the vial contents are vigorously stirred until the tablet is evenly distributed in plasma and stirred for 10 min at 22 ° C on a magnetic stirrer. the heparin-adsorbed substance is centrifuged in a laboratory centrifuge (1200xg, 5 min) and the appropriate plasma aliquots are used for hemocoagulation assays. Example 3 A tablet containing 30 mg of chloridimidazole-N, N'-diyl-bis (2-hydroxy-3-propyl) cellulose ether with a exchange capacity of 1.3 mmol is added to 3 ml of blood plasma in a tube (12x95 mm). 1 and 150 mg microcrystalline cellulose and swelled for 2 min at 22 ° C. The tube is then vigorously mixed on the vibrator until the tablet is evenly distributed in the plasma and allowed to stand at 22 ° C for 15 min. Heparin-adsorbed tablet substitutes are centrifuged in a laboratory centrifuge and appropriate plasma aliquots are used for hemocoagulation assays. Attaches

The procedure is as in Example 3 except that a 150 mg tablet of the chlorimidimidazole-N, N'-diyl-bis (2-hydroxy-3-propyl) cellulose ether with an exchange capacity of 0.3 mmol / g is used. 5

The procedure is as in Example 1 except that a tablet containing 5 mg of diethylaminoethyl cellulose with an exchange capacity of 1 mmol / g and 60 mg of microcrystalline cellulose is added.

Example6

The procedure is as in Example 2, except that a tablet containing 20 mg of trimethylammonium ethyl cellulose with an exchange capacity of 0.7 mmol / g and 60 mg of microcrystalline cellulose is added. Example 7

The procedure is as in Example 3 except that a tablet containing 30 mg of triethylammonium propyl cellulose with an exchange capacity of 0.7 mmol / g and 150 mg of microcrystalline cellulose is added. Example 8

Proceed as in Example 4, except that a 150 mg tablet of diethylamino-2-hydroxypropyl cellulose with an exchange capacity of 0.3 mmol / g is added.

The procedure is as in Example 1 except that a tablet containing 5 mg of active substance prepared by reaction of cellulose, epichlorohydrin and diethylamine in an alkaline medium with an exchange capacity of 1.3 mmol / l and 60 mg of microcrystalline cellulose is added. Example 9 Example 10

The procedure is as in Example 2 except that a tablet containing 20 mg of polyethyleneimine cellulose with an exchange capacity of 0.6 mmol / l and 60 mg of microcrystalline cellulose is added. Example 11

The procedure is as in Example 3 except that a tablet containing 30 mg of dimethylaminoethyl cellulose with an exchange capacity of 0.7 mmol / l and 150 mg of microcrystalline cellulose is added. Example 12

The procedure is as in Example 4 except that a 150 mg tablet of triethylammonium 2-hydroxypropyl cellulose with an exchange capacity of 0.3 mmol g is added. Example 13 is followed as in Example 1 except that a tablet is added containing 5 mg of trimethylammonium propyl cellulose with an exchange capacity of 0.6 mmol / g and 60 mg of microcrystalline cellulose. Example 14

The procedure is as in Example 2 except that a tablet containing 20 mg of dimethylamino-bis / 2-hydroxy-3-propyl / cellulose ether with an exchange capacity of 0.6 mmol / g and 60 mg of microcrystalline cellulose is added. Example 15

The procedure is as in Example 3 except that a tablet containing 30 mg of benzyldimethylammonium 2-hydroxypropyl cellulose with an exchange capacity of 0.6 mmol / l and 150 mg of microcrystalline cellulose is added. Example 16 I;

Viv

The procedure is as in Example 1, except that a tablet containing 5 mg of imidazolium-N, N'-diyl-bis / 2-hydroxy-3-propyl / ether-starch ether with a capacity of 1.3 mmol is added. g-1 and 60 mg of microcrystalline cellulose. Example 17

The procedure is as in Example 2 except that a tablet containing 10 mg diethylaminoethyl cellulose and 10 mg diethylaminohydroxypropyl cellulose with an exchange capacity of 0.7 mmol / g and 60 mg microcrystalline cellulose is added. Example 18

The procedure is as in Example 3 except that a tablet containing 15 mg of imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) cellulose ether and 15 mg of chloride is added.

Claims (5)

A method of removing heparin from blood, in particular from plasma in vitro, characterized in that 0.5 to 3 vol. a portion of blood or blood plasma is added to the composition, preferably in the form of a tablet containing 5 to 150 wt. parts of at least one blood-insoluble polysaccharide derivative having pendant tertiary amine groups with a quaternary exchange capacity from 0.3 to 1.3 mmol per gram ^1, allowed to swell for at least 1 minute, the mixture was vigorously stirred and allowed to stand until the adsorbed heparin, the solid was collected and blood plasma was used for the tests. 2. A method according to claim 1, wherein the preparation added to the blood or blood plasma contains, in addition, microcrystalline cellulose in an amount of up to 12% by weight. parts per 1 wt. % of the polysaccharide derivative. 3. A method according to claim 1, wherein the polysaccharide derivative is preferably a cellulose derivative or a starch derivative. 4. The method according to claim 3, wherein the cellulose derivatives are selected from the group: diethylaminoethyl cellulose, dimethylaminoethyl cellulose, trimethylammonium methyl cellulose, dimethylaminopropyl cellulose, trimethylammonium propyl cellulose, diethylamino-2-hydroxypropyl cellulose, polyethylenimine cellulose, triethylenimine cellulose, triethylenimine cellulose, triethylenimine cellulose. -hydroxypropyl cellulose, dimethylamino-2-hydroxypropyl cellulose, cellulose Combined with a mixture of epichlorohydrin and diethylamine in an alkaline medium, imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) cellulose ether, dimethylamino-bis chloride (2-hydroxy-3-propyl) cellulose ether, benzyldimethylammonium-2-hydroxypropyl cellulose, diethylaminopropyl cellulose, triethylammonium propyl cellulose. 5. The method according to claim 3, wherein the starch derivative is selected from the group: diethylaminoethyl starch, dimethylaminoethyl starch, triethylammonium propyl starch, dimethylaminopropyl starch, trimethylammonium propyl starch, diethylamino-2-hydroxypropyl starch. polyethyleneimine Cross-linked starch, triethylammonium-2-hydroxypropyl cross-linked starch, dimethylamino-2-hydroxypropyl cross-linked starch, starch cross-linked with a mixture of epichlorohydrin and diethylamine in an alkaline medium, imidazolium · N, N'-diyl-bis (2-hydroxy-3-propyl) cross-linked starch ether, dimethylamino-bis- (2-hydroxy-3-propyl) cross-linked starch ether, benzyldimethylammonium-2-hydroxypropyl cross-linked starch, trimethylammonium ethyl cross-linked starch, diethylaminopropyl cross-linked starch.