Note: Descriptions are shown in the official language in which they were submitted.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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