CA1244470A - Sulfonated polyethylene amines useful as blood anticoagulants - Google Patents

Abstract

Abstract There is disclosed a blood anticoagulant consisting essentially of a sulfonated polyethyleneimine formed by sulfonating 1 mole % or more of the -NH- and -NH2 groups present in a polyethyleneimine having a molecular weight of 300 or greater. This blood anti-coagulant is suitable for preventing coagulation of blood collected for purpose of hematological examination.

Description

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SPECIFICATION

Title of the Invention Blood Anticoagulant Background of the Invention 1. Field of the Invention This invention relates to blood anticoagulants and, more particularly, to a blood anticoagulant suitable for preventing coagulation of blood colle.cted for purposes of hematological examination.

2. Description of the Prior Art With the progress of clinical medicine, hematological examinat.ion has recently come to play a very important role in the field of preventive medicine or in the judgement of therapeutic effects. Under these circumstances, the development of blood anticoagulants which can prevent coagulation of blood collected for purposes of hematological examination is being ac~ively pursued.
Blood anticoayulants comprising heparin sodium are most widely known. For example, in medical treat-ments using an artifical kidney or a blood oxygenator, they are added to the blood and/or used for the treat-ment of equipment surfaces which are in contact with i~44~70 the blood.
Moreover, blood anticoagulants comprising a metallic salt of e~hylenediaminetetraacetic acid are being used in certain morphological tes~s of blood.
These blood anticoagulants are also being used in coating the internal surfaces of hematocrit tubes for the determination of hematocrit which is an item of hematological examination, and as additives for the separation of blood plasma.
However, since heparin is obtained solely by extraction from animal organs, it cannot be produced as abundantly as æynthetic products and its production cost is far higher. Moreover, it is difficult to obtain heparin preparations having an identical structure and identical properties by extraction from different types of organs.
On the other hand, blood anticoagulants comprising a metallic salt of ethylenediaminetetra-acetic acid can be used in morphological testsiof blood.
However, they are disadvantageous in that they do not allow inorganic ion determinations which are among biochemical tests and they exert an adverse effect on enzyme tests. For these reasons, conventional blood tests have unavoidably involved complicated procedures, i.e., the selection of different blood anticoagulants according to the intended test item and the adoption of 4~0 the serum separation method in which steps must be taken to separate serum from blood prior to measurement.
Accordingly, there is a demand for a blood anticoagulant which is inexpensive, has excellent anti-coagulant properties, and exerts no adverse effect on a wide variety of blood tests. However, no blood anticoagulant meeting this demand has been developed as yet.

Summary of the Invention According to an aspect of the present invention, there is provided a blood anticoagulant consisting essentially of a sulfonated polyethyleneimine formed by sulfonating 1 mole ~ or more of the -NH- and -NH2 groups present in a polyethyleneimine having a molecular weight of 300 or greater.

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Detailed Description of the Preferred Embodiments Polyethyleneimines are formed by ring-opening polymerization of ethyleneimine and, in many cases, have a branched structure containing primary, secondary and tertiary amino nitrogen atoms. The polyethylene-imine used in the present invention must have a molecular weight of 300 or greater. Molecular weights less than 300 are not suitable because the resulting product will have unsatisfactory anticoagulant properties. A poly-ethyleneimine which contain primary, secondary andtertiary amino nitrogen atoms in a ratio ranging from approximately 1:1:1 to approximately 1:3O1 may be suitably employed.
Such polyethyleneimines can be obtained by subjecting ethyleneimine to ring-opening polymerization in the presence of a catalyst selected from carbon dioxide, hydrochloric acid, hydrobromic acid, p-toluenesulfonic acid, aluminum chloride, boron tri-fluoride and the like.
In the blood anticoagulant of the present invention, l mole % or more of the -N~- and -NH2 groups present in the aforesaid polyethyleneimine must be sulfonated. If the degree of sulfonation is less than 1 mole %, the sulfonated groups are not enough to exhibit satisfactory anticoagulant properties.
This sulfonated polyethyleneimine can be used ~, lZ9~470 as such, i.e., in sulfonic acid form. However, ik is preferable to neutraliæe a paxt or all of the sulfonic groups with alkali metal, alkaline earth metal, ammonium or like ions. The reason for this is that, if it is used in sulfonic acid form and added to blood as an anticoagulant, the blood tends to undergo coagulation because of an increase in hydrogen ion concentration~
The method of sulfonating polyethyleneimines to form blood anticoagulants in accordance with the present invention will be described hereinbelow.
Typical examples of the method of sulfonating - ~ polyethyleneimines include sulfonation processes involving the reac~ion of a polyethyleneimine with chlorosulfonic acid, fuming sulfuric acid and hot concentrated sulfuric acid, respectively.
The sulfonation process using chlorosulfonic acid is carried out by dissolving a polyethyleneimine in a solvent such as methanol or the like and adding an appropriate amount of chlorosulfonic acid to effect reaction therebetween. Usable solvents include alcohols such as methanol, isopropanol, etc.; ketones such as acetone, methyl ethyl ketone, etc.; and halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloroethane, etc. In this case, the concentration of the polyethyleneimine dissolved in the solvent should be not lower than 0.5% by weight and not higher than ~4470 30% by weight. If the concentration is lower than 0.5%
by weight, the amount of solvent used is so large that is will be difficult to recover the sulfonated product.
On the other hand, if the concentration is higher than 30% by weight, it will be dificult to control the heat of reaction generated during the sulfonation reaction.
Moreover, chlorosulfonic acid should preferably be u~ed in an amount of not less than 10 parts by weight per 100 parts by weight of polyethyleneimine. If the amount is less than 10 parts by weight, the sulfonation reaction will not proceed to a full extent. In this sulfonation process, it is difficult ~o sulfonate both of the two primary amino hydrogen atoms of each-amino group present in the polyethyleneimine, so tha~ only one of them is usually sulfonated.
In the sulfonation process using fuming sul-furic acid, the sulfonation reaction can proceed under substantially the same conditions as described in connection with chlorosulfonic acid.
The sulfonation process using hot concentrated sulfuric acid is carried out by adding concentrated sulfuric acid having a concentration of 96 to 100~ by weight directly to a polyethyleneimine and heating the resulting mixture. In this cas,e, the degree of sulfo-nation is determined by the amount of sulfuric acid to be reacted with the polyethyleneimine, and the heating :.

~2~4~70 temperature. Specifically, the amount of sulfuric acid used should be not less than 50 parts by weight per 100 parts by weight of the polyethyleneimine. If the amount is less than 50 parts by weight, sulfonation with sulfuric acid will not proceed effectively.
Concentrated sulfuric acid should preferably be added little by little to the polyethyleneimine, because local addition of a large amount of concentrated sulfuric acid may cause dehydration of the polyethyleneimine prior to sulfonation. After the addition of concentrated sulfuric acid, a reaction temperature equal to or higher than 100C and lower than 200C should suitably be maintained for a period of time ranging from 30 to 120 minutes. At higher temperatures, the sulfona~ion will be completed in a relatively short time, while at lower temperatures, the sulfonation will require a time of the order of 120 minutes. The degree of sulfonation of the polyethyleneimine can be confirmed by Fourier-transform infrared absorption spectroscopic analysis.
Since the sulfonated product of polyethylene-imine synthesized in the above-described manner contains unreacted chlorosulfonic acid, sulfuric acid and the like, such impurities should ~e removed according to a suitable purification procedure. For example, such impurities can be removed by dissolving the sulfonated product of polyethyleneimine in water, adding the ~2~447~;3 resulting aqueous solution dropwise to a poor solvent miscible with water (for example, one selected from alcohols such as methanol, isopropanol, etc.; and ketones such as acetone, methyl ethyl ketone, etc.) to reprecipitate the product, separating the product so formed, and then drying it. In this purification procedure, the amount of water used to dissolve the sulfonated product of polyethyleneimine should preferably be as small as possible. Accordingly, it is preferable that the aqueous solution of the sulfonated product of polyethyleneimine which is added dropwise to a poor solvent for the purpose of reprecipitating the product has a concentration ranging from 10 to 80% by weight and more preferably from 30 to 60g by weight. If the concentration is lower than 10% by weight, only a small amount of the sulfonated product of polyethyleneimine will precipitate upon addition to a poor solvent, resulting in a reduced recovery. On the other hand, if the concentration is higher than 80% by weight, it will be difficult to prepare the aqueous solution of the sulfonated product.
The neutralization of the sulfonated product of polyethyleneimine may be carried out by preparing an aqueous solu$ion of the sulfonated product and adding thereto a predetermined amount of an aqueous alkaline solution. The neutralizing agents which can be used -~ l2~7a _g for this purpose include sodium hydroxide, potassium hydroxide, magnesium hydroxide, barium hydroxide, ammonia, ammonium hydroxide and the like. The sulfonated product which has been neutralized with a metallic salt can also be purified by reprecipitation with a poor solvent selected from alcohols and the like.
In the blood anticoagulant of the present invention, the sulfonated product of polyethyleneimine may be used as such or in the form of an alkali metal, alkaline earth meta~ or ammonium salt or a mixture thereof. Moreover, the blood anticoagulant of the present invention may contain heparin salts, salts of oxalic acid, double salts of oxalic acid, salts of citric acid, and the like, so long as they are present in such low contents as to exert no influence on the results of blood tests.
The blood anticoagulant of the present inven-tion is characterized in that it can be readilysynthesized from polyethyleneimine which is a product obtained in chemical industry, it is inexpensive as compared with conventional blood anticoagulants, and it exhibits satisfactory stable anticoagulant properties without causing any change in the morphology of blood corpuscles, i.e., it can be used without exerting any adverse effect on various types of blood tests.
The present invention is further illustrated by the following examples.
In these examples, the degree of sulfonation ~2~47C~

of the sulfonated polyethyleneimine was determined by Fouriex-transform infrared absorption spectroscopic analysis.

Example 1 5 g of polyethyleneimine having a weight averaye molecular weight of 10,000 and containing primary~ secondary and tertiary amino nitrogen atoms in a ratio of 1:2:1 was dissolved in 50 ml of me~hanol.
Then, 34 g of chlorosulfonic acid was added thereto little by little. After completion of the addition, the resulting mixture was heated to a reaction temperature of 60C with stirring and held at that temperature for 30 minutes to complete the reaction. During this lS reaction, the sulfonated product of polyethyleneimine precipitated as solvent-insoluble matter, which was separated by filtration through a glass filter. Then, 5 ml of water was added to the reaction product and the resulting aqueous solution was slowly added dropwise to 200 ml of stirred methanol to reprecipitate the reaction product. The reaction product so formed was separated and dried under reduced pressure to obtain 4.3 g of a powder. When the degree of sulfonation of this powder was determined, 27~ of the -NH- and -NH2 groups present in the polyethyleneimine were found to be sulfonated.
A portion of this powder was taken and dissolved in iZ~470 water to prepare a 10 wt.~ aqueous solution thereof.
5 ~1, 2 ~ 1 and 0.5 ~1 aliquots of this 10 wt.~
aqueous solution were ~eparately placed in test tubes and about 1 ml o~ human fresh blood was added to each test tube. When these samples were visually examined

3 hours after the addition, none of them exhibited coagulation. Moreover, when these samples were observed under the light microscope to examine the morphology of blood corpuscles, no changes of red blood cells, white blood cells, platelets or the like were noted.

Examples 2 to 9 and Comparative-Exam~le 1 The sulfonation of polyethyleneimines was carried out by varying the weight average molecular weight of polyethyleneimine used and the amount of chlorosulfonic acid added. In these polyethyleneimines, the primary, secondary and tertiary amino nitrogen atom ratios were approximately the same as the ratio of the polyethyleneimine used in Example 1. In each case, the starting polyethyleneimine was used in an amount of 5 g and the reaction temperature, reaction time, purification procedure and the like were the same as described in Example 1. The results thus obtained are shown in Table 1.
It can be seen from Comparative Example 1 tha~, when the weight average molecular weight of the 3Lz4g470 starting polyethyleneimine was as low as 250, the sulfonation reaction did not proceed satisfactorily and the resulting product failed to act as an effective blood anticoagulant.

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Example 10 1 g of the sulfonated product of polyethylene-imine synthesized in Example 1 was taken and dissolved in 9 c~c. of water to prepare a 10 wt.% aqueous solution of the reaction product. This aqueous solution was neutralized by adding 2.8 ml of a 4N aqueous solution of sodium hydroxide, and then slowly added dropwise to 400 ml of methanol to reprecipitate the reaction product.
The reaction product 80 formed was separated and dried under reduced pressure to obtain 0.78 g of a powder.
This powder was tested for anticoagulant properties in the same manner as described in Example 2.
Specifically, its anticoagulant properties were evaluated by placing 100 ~1 of a 1.0 wt.~ aqueous solution of the powder in a test tube and adding thereto about 1.0 ml of human blood. No coagulation of the blood was noted even after 5 hours.
When this sample was observed under the micro-scope, no agglutination of red blood cells, white blood cells or platelets was noted.

Example 11 5 g of polyethyleneimine having a weight average molecular weight of 1,800 and containing primary, secondary and tertiary amino nitrogen atoms in a ratio of 1:2:1 was taken, and 5 g of concentrated sulfuric acid ~2~9~47~

having a purity of 98% by weight was slowly added drop-wise thereto and mixed therewith. Then, the resulting sulfuric acid solution was heated to 120C and held at that temperature for 90 minutes. During this period, the sulfuric acid solution was continuously stirred so as to prevent it from being locally heated. After the addition and dissolution of 10 c.c. of water, the reac-tion mixture was added dropwise to 500 ml of methanol to reprecipitate the reaction produck. Any residual ~ulfuric acid was removed by washing with methanol.
The separated reaction product was dried under reduced pressure to obtain 3.6 g of a crystalline powder. The degree of sulfo~ation of this reaction product was determined to be 12%. Its anticoagulant properties were evaluated by preparing a 1.0 wt.~ aqueous solution of the powder, placing 100 ~1 and 50 ~1 aliquots of the solution in test tubes, and adding about 1.0 ml of human blood to each ~est tube. Thus, no coagulation of the blood was noted even 5 hours after its addition. When these samples were observed under the micrvscope, no abnormalities of red blood cells, white blood cells or platelets were noted.
Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention of the scope of the attached claims.

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Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A blood anticoagulant consisting essentially of a sulfonated polyethyleneimine formed by sulfonating 1 mole % or more of the -NH- and -NH2 groups present in a polyethyleneimine having a molecular weight of 300 or greater.

2. The blood anticoagulant of claim 1 wherein at least a part of the sulfonic groups present in the sulfonated polyethyleneimine are neutralized with alkali metal, alkaline earth metal or ammonium ions.

3. A process for preparing a blood anti-coagulant which comprises the step of adding a sulfonat-ing agent selected from the group consisting of chloro-sulfonic acid, concentrated sulfuric acid and fuming sulfuric acid, in the present or absence of a solvent, to a polyethyleneimine having a molecular weight of 300 or greater so as to synthesize a sulfonated polyethylene-imine in which 1 mole % or more of the -NH- and -NH2 groups present therein are sulfonated.

4. The process of claim 3 which further in-cludes the steps of dissolving the sulfonated polyethy-leneimine in water and neutralizing the resulting solu-tion by the addition of an aqueous alkaline solution.

5. The process of claim 3 wherein chloro-sulfonic acid is used as the sulfonating agent in an amount of not less than 10 parts by weight per 100 parts by weight of the polyethyleneimine.