HYDROPHILIC POLYMER COMPOUND HAVING ANTICOAGULATION
EFFECT
Technical Field
[0001]
The present invention relates to a hydrophilic polymer compound having
anticoagulation effect (anticoagulant activity).
Background Art
[0002]
The blood coagulation reaction required for coagulating blood is an extremely
complicated reaction in which various blood coagulation factors are involved. It is
thought that the primary hemostasis stage in which platelets are involved and in the
coagulation thrombus formation stage in which blood coagulation factors such as
thrombin are involved to stabilize and strengthen fibrin are particularly important.
[0003]
The blood coagulation reaction is indispensable to the hemostasis of bleeding
due to injury or the like. On the other hand, however, in cases where the blood
coagulation reaction proceeds due to contact between the blood and a medical device
or a medical material, such as an extracorporeal circulation circuit in hemodialysis,
there is a risk that the formed coagulation thrombus increases the circulation pressure
or occludes a blood vessel.
[0004]
To decrease these risks, a method of preventing blood coagulation is known
wherein heparin which is an anticoagulant is preliminarily administered to the patient
who is to undergo hemodialysis. However, this method has a number of problems
in that administration of heparin in excess amount causes side effects, the control of
administration dose is complicated, the method cannot be applied to a patient having
bleeding tendency, and so on.
[0005]
Recently, in order to avoid these problems, trials for preventing blood
coagulation during the therapy by immobilizing a compound having an anticoagulant
activity including heparin on the surface of medical devices or medical materials
such as blood circuits and the like, have been reported (Patent Documents 1 to 9).
Prior Art References
Patent Documents
[0006]
Patent Document 1: Japanese Translated PCT Patent Application Laid-open
No. 2003-507082
Patent Document 2: JP 2001-213984A
Patent Document 3: Japanese Translated PCT Patent Application Laid-open
No. 2004-525888
Patent Document 4: JP 2006-291193 A
Patent Document 5: WO 08/032758
Patent Document 6: JP 2009-225824 A
Patent Document 7: JP 2010-082067A
Patent Document 8: JP 2007-181691 A
Patent Document 9: JP 2007-181692 A
Summary of the Invention
Problems to be Solved by the Invention
[0007]
However, at present, as the compound having an anticoagulant activity to be
immobilized on the surface of medical devices or medical materials such as blood
circuit and the like, a specific compound which can inhibit both of the blood
coagulation reactions in the primary hemostasis stage in which platelets are involved
and in the coagulation thrombus formation stage in which blood coagulation factors
are involved has not yet been developed. Even if a conventional compound having
an anticoagulant activity is to be immobilized on the surface of a medical device or
medical material, it is difficult to immobilize the compound retaining a sufficient
anticoagulant activity, and even when the immobilization is successful, there is a
problem in that the immobilized compound is released from the medical device or
the medical material into the blood. Further, in cases where a plurality of
compounds are used for inhibiting both of the blood coagulation reactions in the
primary hemostasis stage in which platelets are involved and in the coagulation
thrombus formation stage in which blood coagulation factors are involved, it is
necessary to control the competitive adsorption between the compounds and to
control the immobilization ratio, which is very complicated.
[0008]
Accordingly, an object of the present invention is to provide a hydrophilic
polymer compound which can inhibit both of the blood coagulation reactions in the
primary hemostasis stage in which platelets are involved and in the coagulation
thrombus formation stage in which blood coagulation factors are involved, which
hydrophilic polymer compound can be firmly immobilized on the surface of medical
devices or medical materials, in the state retaining the anticoagulant activity.
Means for Solving the Problems
[0009]
To solve the above-described problems, the present inventors intensively
studied to discover that a hydrophilic polymer compound comprising a polymer
compound which inhibits platelet adhesion and a compound which inhibits blood
coagulation reaction, bound to the polymer compound, exhibits an outstanding
anticoagulant activity, and can be firmly immobilized on the surface of medical
devices and medical materials.
[0010]
That is, the present invention provides a hydrophilic polymer compound
comprising a polymer compound which inhibits platelet adhesion, and a compound
that inhibits blood coagulation reaction, bound to the above-described polymer
compound.
[0011]
The above-described polymer compound which inhibits platelet adhesion is
preferably a copolymer composed of a hydrophobic polymer and a hydrophilic
polymer, and has an amount of adsorption to poly(methyl methacrylate) of not less
than 0.1 pg/mm2, more preferably, a copolymer of monomers selected from the group
consisting of ethylene glycol, vinyl acetate, vinyl pyrrolidone, propylene glycol, vinyl
alcohol and siloxane, still more preferably, a polyether-modified silicone.
[0012]
The above-described compound which inhibits blood coagulation reaction
preferably has an antithrombin activity, is more preferably a compound represented
by General Formula (I) below, still more preferably (2R,4R)-4-methyl-1-((2 S)-2-
{[(3RS)-3-methyl-l,2,3,4-tetrahydroquinolin-8-yl]sulfonyl}amino-5-
guanidinopentanoyl)piperidin-2-carboxylic acid.

1 9
[wherein R represents a (2R,4R)-4-alkyl-2-carboxypiperidino group; R represents a
phenyl group or a fused polycyclic compound residue, the fused polycyclic
compound residue optionally being substituted with a lower alkyl group, a lower
alkoxy group or an amino group which is substituted with a lower alkyl group].
[0013]
The present invention also provides a surface treatment agent for medical
devices or medical materials, the surface treatment agent comprising the above-
described hydrophilic polymer compound, which has an anticoagulant activity.
[0014]
The present invention further provides a medical device or a medical material
treated with the above-described surface treatment agent.
Effects of the Invention
[0015]
By the present invention, both of the blood coagulation reactions in the
primary hemostasis stage in which platelets are involved and in the coagulation
thrombus formation stage in which blood coagulation factors are involved can be
prominently inhibited, and the hydrophilic polymer compound can be firmly
immobilized on the surface of medical devices or medical materials, in the state
retaining the anticoagulant activity. Further, the hydrophilic polymer compound of
the present invention can be used as a surface treatment agent for giving
anticoagulant activity to medical devices or medical materials.
Brief Description of the Drawings
[0016]
Fig. 1 is a schematic view showing the mini-module prepared in an example.
Fig. 2 is a schematic view showing the closed circuit used in an in vitro blood
circulation test.
Fig. 3 is a schematic view showing the human blood plasma circulation
circuit used in the measurement of the amount of eluted hydrophilic polymer
compound.
Mode for Carrying out the Invention
[0017]
Unless otherwise specified, the terms used herein have the following
definitions:
[0018]
The "hydrophilic polymer compound" of the present invention is
characterized in that a polymer compound which inhibits platelet adhesion and a
compound which inhibits blood coagulation reaction are bound.
The term "hydrophilic" herein means that the compound is water-soluble, or
even if the compound is not water-soluble, the compound interacts with water
molecules by electrostatic interaction or hydrogen bond. The "hydrophilic polymer
compound" includes, for example, the hydrophilic polymer compounds wherein a
copolymer of the monomers selected from the group consisting of ethylene glycol,
vinyl acetate, vinyl pyrrolidone, propylene glycol, vinyl alcohol and siloxane, is
bound with a compound represented by the following General Formula (I):

[wherein R1 represents a (2R,4R)-4-alkyl-2-carboxypiperidino group; R2 represents a
phenyl group or a fused polycyclic compound residue, the fused polycyclic
compound residue optionally being substituted with a lower alkyl group, a lower
alkoxy group or an amino group which is substituted with a lower alkyl group].
[0019]
The term "polymer compound which inhibits platelet adhesion" herein means
a polymer compound having a number average molecular weight of not less than
1000, which has a blood compatibility and can inhibit platelet adhesion to the surface
of a substrate or material by making the polymer compound exist on the surface of a
medical device or a medical material.
[0020]
Examples of the "polymer compound which inhibits platelet adhesion"
include poly(vinyl alcohol), polyvinyl pyrrolidone), poly(ethylene glycol),
poly(propylene glycol), polymer compounds composed of polyether and polysiloxane,
polyethyleneimine, polyallylamine, polyvinylamine, polyvinyl acetate), poly(acrylic
acid), polyacrylamide, poly(hydroxyethyl methacrylate), or a copolymer or graft of
monomer of these polymer compounds and other monomer, which may preferably
have amino group, carboxyl group, hydroxyl group, epoxy group or mercapto group
to bind with a compound which inhibits blood coagulation, more preferably a
copolymer composed of a hydrophobic polymer and a hydrophilic polymer for
adsorption to the surface of medical devices or medical materials, still more
preferably a polymer compound composed of polyether and polysiloxane which are
highly hydrophilic, or a partially-saponified poly(vinyl alcohol) or a copolymer of
vinyl pyrrolidone and vinyl acetate.
[0021]
Examples of the "polymer compound composed of polyether and
polysiloxane" include copolymers, polymer complexes or polymer blends of
polyether and polysiloxane. The copolymer of polyether and polysiloxane is
composed of polyether units and polysiloxane units, and the copolymer form thereof
may be any of random copolymer, block copolymer or graft copolymer. Among
these, polyether-modified silicone which is highly hydrophilic is preferred.
[0022]
Examples of "polyether" include poly(ethylene oxide) and structures
originated from polyethylene oxide. "Polyether" herein means the structure
represented by General Formula (II) (R3 represents an alkyl group having not more
than 6 carbon atoms), and "structure originated from polypropylene glycol" which is
one example of polyether means the structure represented by General Formula (III).

[0023]
"Polyether-modified silicone" means silicone wherein polyether units bind to
side chains of silicone chains, and polyether-modified silicone which is amino-
modifled or carboxy-modificd may also be employed.
[0024]
When the polymer compound which inhibits platelet adhesion is partially-
saponified polyvinyl alcohol), the degree of saponification is preferably 50 to less
than 100 mol%, more preferably 74 to 99.9 mol%, still more preferably 78 to 95
mol% from the viewpoint of attaining a preferred ease of handling or hydrophilicity.
"degree of saponification" herein means a value calculated according to the Equation
1.
Saponification level = m/(n+m)xl00......Equation 1
m: the number of structures represented by General Formula (IV) in polyvinyl
alcohol)
n: the number of structures represented by General Formula (V) in polyvinyl
alcohol)

[0025]
When the polymer compound which inhibits platelet adhesion is a copolymer
of vinyl pyrrolidone and vinyl acetate, vinyl pyrrolidone units are preferably not less
than 50 unit mol%, more preferably not less than 60 unit mol%, from the viewpoint
of attaining a preferred ease of handling or hydrophilicity. On the other hand, from
the viewpoint of attaining a preferred amount of adsorption to base material, vinyl
pyrrolidone units are preferably less than 100 unit mol%. The percentage of vinyl
pyrrolidone units in a copolymer of vinyl pyrrolidone and vinyl acetate (unit mol%)
is calculated by 1H-NMR measurement (solvent: CDCl3) of the copolymer.
[0026]
The amount of adsorption of the polymer compound which inhibits platelet
adhesion, to base materials such as medical device, medical material or the like, is
preferably not less than 0.1 pg/mm2, more preferably not less than 1 pg/mm2, still
more preferably not less than 10 pg/mm2.
[0027]
The amount of adsorption is measured by the following method:
Firstly, untreated sensor chip (Sensor Chip Au; GE Healthcare) is pretreated (distilled
water at 25°C, at a flow rate of 20 µl/min, for 10 minutes) using a surface plasmon
resonance system (hereinafter referred to as "SPR") (BIACORE 3000; GE
Healthcare), and the signal value (RU: resonance unit) is measured.
[0028]
"Base material", i.e., an adsorbent material is dissolved into a solvent to
prepare a 0.5wt% solution of the adsorbent material. One drop of the solution of the
adsorbent material is dropped on to the center of the gold film part and the resultant
is immediately rotated at 3000 rpm for 1 minute at room temperature to coat the
sensor chip with the adsorbent material.
[0029]
After confirming that no droplet exists on the sensor chip, the sensor chip is
washed with distilled water using SPR (at 25°C, at a flow rate of 20 µl/min, for 10
minutes), and further washed three times with 0.025wt% Triton-X100 solution (at
25°C, at a flow rate of 20 ul/min, for 1 minute), and then the signal value at 10
minutes after the end of washing is measured.
[0030]
Among the sensor chips obtained by the method described above, the one
whose signal value difference before and after spin coat was within the range from
3000 to 8000 was selected, then washed with distilled water (at 25°C, at a flow rate
of 20 ul/min, for 10 minutes), and further washed three times with 0.025wt% Triton-
XI00 solution (at 25°C, at a flow rate of 20 ul/min, for 1 minute).
[0031]
Ten minutes after the end of washing, an aqueous solution of a hydrophilic
polymer compound to be adsorbed to the base material (concentration: 100 µg/ml) is
injected (at 25°C, at a flow rate of 20 ul/min, for 1 minute), and then the resultant is
washed with distilled water (at 25°C, at a flow rate of 20 ul/min, for 3 minute). The
difference between the signal value immediately before injection (hereinafter referred
to as "signal value A") and the signal value at 3 minutes after the end of the injection
(hereinafter referred to as "signal value B") is determined, which is then converted as
1 RU = 1 pg/mm2.
[0032]
Next, the sensor chip is washed with distilled water (at 25 °C, at a flow rate of
20 ul/min, for 2 minutes), and further washed three times with 0.025wt% Triton-
X100 solution (at 25°C, at a flow rate of 20 ul/min, for 1 minute), and again the
aqueous solution of the hydrophilic polymer compound to be adsorbed
(concentration: 100 µg/ml) is injected (at 25°C, at a flow rate of 20 ul/min, for 1
minute). Thereafter, the same steps are repeated to determine signal differences 5
times in total, and the mean value is regarded as "the amount of adsorption of
polymer compound which inhibits platelet adhesion to base materials".
[0033]
"Compound which inhibits blood coagulation reaction" means a compound
having an anti-coagulation activity such as an antithrombin activity, and more
specifically means a compound which prolongs the prothrombin time than native
blood by not less than 30% when the compound is added into blood to a
concentration of 10 µg/mL.
[0034]
"Prothrombin time" is measured by the method according to a known
literature (Masamitsu Kanai et al., "Clinical Test Handbook, vol. 30", Kanihara
shuppan, 1993, p416-418). More specifically, 1 part by volume of 3.2% sodium
citrate and 9 parts by volume of blood are mixed, and 0.1 mL aliquot of the sodium
citrate-blood plasma is sampled into a small test tube (inner diameter: 8mm, length:
7.5cm), followed by heating the resultant in a thermostat bath at 37°C for 3 minutes.
Then 0.2 mL of tissue thromboplastin-calcium reagent kept at 37°C is added thereto,
and then the small test tube is lightly shaken, followed by leaving the small test tube
to stand to precipitate fibrin. Here, the time required for the precipitation of fibrin
after addition of the tissue thromboplastin-calcium reagent is measured, which
measured time is defined as "prothrombin time".
[0035]
Examples of the "compound which inhibits blood coagulation reaction"
include heparin, nafamostat mesilate, sodium citrate, sodium oxalate, a 1-antitrypsin,
a2-macroglobulin, C1 inhibitor, thrombomodulin, protein C, compounds having
guanidine structure, prostaglandins, hirudin, Xa inhibitors, tissue factor inhibitor and
antithrombin, and preferred are compounds having an antithrombin activity.
[0036]
"Compound having antithrombin activity" means a compound which has a
high binding affinity to thrombin.
[0037]
An example of the index to evaluate the antithrombin activity of compounds
is the inhibition constant (hereinafter referred to as "Ki") which is calculated from
Lineweaver-Burk plot based on absorbance value of a test solution. Lower Ki
indicates higher binding affinity to thrombin and higher antithrombin activity.
[0038]
Examples of the "compound having antithrombin activity" include
compounds having guanidine structure, and preferred is (2R,4R)-4-methyl-l-((2S)-2-
{[(3RS)-3-methyl-l,2,3,4-tetrahydroquinolin-8-yl]sulfonyl}amino-5-
guanidinopentanoyl)piperidin-2-carboxylic acid(hereinafter referred to as
"argatroban"). Argatroban, which was synthesized in 1978, is a medicinal
compound having a selective antithrombin activity of an arginine derivative.
[0039]
The surface treatment agent of medical device or medical material of the
present invention is characterized by containing the above-described hydrophilic
polymer compound and having an anticoagulant activity.
[0040]
Examples of the "medical device or medical material" include implantable
artificial organs, artificial blood vessels, catheters, stents, blood bags, contact lenses,
intraocular lenses and surgery technical aids; and separation membranes and
adsorbents which are included in modules for biogenic substance separation,
hemocatharsis or the like.
[0041]
Methods for treating the surface of the medical devices or medical materials
using the above-described surface treatment agent, i.e., methods for immobilizing the
above-described hydrophilic polymer compound which is active ingredient thereof on
the surface of the medical devices or medical materials include, for example, a
method wherein the above-described surface treatment agent is made to contact the
medical devices or medical materials and then irradiated with a radiation. As the
type of radiation, electron beam or y-ray is preferred.
[0042]
Examples of the material constituting the "medical devices or medical
materials" include cellulose, cellulose acetate, polycarbonates, polysulfones,
poly(ether sulfones), polymethacrylates such as poly(methyl methacrylate)
(hereinafter referred to as "PMMA"), polyacrylates, polyamides, poly(vinylidene
fluoride), poly(vinyl chloride), polyacrylonitrile, polyesters, polyurethanes,
polystyrene, polyethylene, polypropylene, poly(vinylidene fluoride),
polymethylpentene and polyimides.
Examples
[0043]
The present invention will now be described in more detail referring to
examples. However, the present invention is not restricted thereto.
[0044]
(Example 1: Binding between Amino-polyether-modified Silicone and Argatroban)
Argatroban in an amount of 5 mmol was placed in a recovery flask, and 10
mL of anhydrous dimethylformamide (hereinafter referred to as "anhydrous DMF")
was added thereto to dissolve the argatroban. Thereafter, 10 mL of 4N hydrochloric
acid/l,4-dioxane (TOYOKASEI) was added dropwise while cooling the recovery
flask and the resulting mixture was stirred for 1 hour. Then the solvent was
evaporated with a rotary evaporator and the resultant was further dried overnight in a
vacuum dryer, followed by addition of 25 mL of anhydrous DMF to obtain
argatroban hydrochloric acid salt solution in anhydrous DMF.
[0045]
Argatroban hydrochloric acid salt solution in anhydrous DMF in an amount
shown in Table 1 was placed in a two-necked flask, and dicyclohexylcarbodiimide
(hereinafter referred to as "DCC") solution in anhydrous DMF and 4-
hydroxybenzotriazole (hereinafter referred to as " HOBt") solution in anhydrous
DMF were added. Then polyether-modified silicone (X-22-3939A (SHIN-ETSU
CHEMICAL) was further added and the resulting mixture was allowed to react at
room temperature for 3 days. Then the reaction solution was placed in a dialysis
tube (SPECTRAPORE RC, PORE 6, MWCO1000), and dialyzed for 3 days against
distilled water with a volume of more than 10 times while appropriately replacing the
distilled water. The reaction solution after the dialysis was filtered, and the solvent
in the filtrate was evaporated with a rotary evaporator, followed by drying the
resultant overnight in a vacuum dryer to obtain a hydrophilic polymer compound
(hereinafter referred to as "Example 1 Compound").
[0046]
(Measurement of Antithrombin Activity of Example 1 Compound)
ECA-T kit (HAEMOS YS) was used for the measurement. To 100µL of
Example 1 Compound, 900µL of distilled water was added to prepare an aqueous
Example 1 Compound solution. The Example 1 Compound solution in an amount
of 30µL was sampled, and mixed with 100µL of EC A prothrombin buffer and 25
µL of ECA-T substrate. After incubating the resulting mixture at 37°C for 60
seconds, the mixture was set in an apparatus (COATRON Ml(code 80 800 000);
PRODUCTION) and measurement was carried out after adding 50µL of EC A ecarin
reagent.
[0047]
A mixture of 20µL of argatroban solution prepared to an arbitrary
concentration using ethanol/hydrochloric acid (volume ratio: 4/1) mixed solvent and
80µL of human blood plasma, and a mixture of 20µL of blank distilled water and 80
µL of human blood plasma were subjected to the measurement, respectively, in place
of the above-described aqueous Example 1 Compound solution, using the ECA-T kit,
and a calibration curve was prepared from the results thereof. The concentration of
1494.3 ppm by weight in terms of argatroban of the aqueous Example 1 Compound
solution calculated from the calibration curve was defined as the value indicating the
antithrombin activity of Example 1 Compound.
[0048]
(Examples 2 to 13)
Examples 2 to 13 Compounds were obtained, respectively, in the same
manner as in Example 1 except that the molar ratios of DCC, HOBt and polyether-
modified silicone (X-22-3939A) to argatroban hydrochloric acid salt and the volume
ratio of anhydrous DMF to polyether-modified silicone were changed to measure the
antithrombin activity thereof. The molar ratios of DCC, HOBt and polyether-
modified silicone (X-22-3939A) to argatroban and the measurement results of the
antithrombin activity of each Examples 2 to 13 Compounds are shown in Table 1.
[0049]
The antithrombin activity of a polyether-modified silicone (X-22-3939A) was
also measured, but the measured value was the same as that of the blank distilled
water, and it was confirmed that the polyether-modified silicone itself does not have
an antithrombin activity.
[0051]
(Measurement of Thrombin Inhibition Constant of Example 1 Compound)
Aqueous thrombin from bovine plasma solution was prepared by dissolving
10,000 U of thrombin from bovine plasma solution (ITO LIFE SCIENCES) in 1 mL
of physiological saline solution.
[0052]
Aqueous S-2238 stock solution was prepared by dissolving 25 mg of S-2238
stock solution (SEKISUI MEDICAL) in 40 mL of distilled water.
[0053]
The aqueous bovine thrombin solution, aqueous S-2238 stock solution and
the above-described aqueous Example 1 Compound solution were each diluted by
using diluted buffer solution (0.05M Tris, 0.1M NaCl, 1 mg/mL of bovine serum
albumin (BSA), pH 7.4).
[0054]
To a 96-well plate, 100µL of diluted solution of the aqueous S-2238 stock
solution and 50µL of diluted solution of the aqueous Example 1 Compound solution
were dispensed, and the resultant was sealed and then warmed in a thermostat dryer
set at 37°C for 30 minutes. Thereafter, 50µL of the diluted solution of the aqueous
thrombin from bovine plasma solution which was heated at 37°C for 30 minutes was
further dispensed thereto, and absorbance of the resulting mixture was immediately
measured with a microplate reader (measurement wavelength: 405 nm, reference
wavelength: 595nm).
[0055]
Immediately after finishing the first measurement of the absorbance, the
second measurement of the absorbance was carried out. The third or later
measurements of the absorbance were carried out at 4, 6, 8, 10, 12, 14, 16, 18 and 20
minutes, respectively, after the diluted solution of aqueous thrombin from bovine
plasma solution was dispensed. Ki was calculated from each value of the obtained
absorbances by using Lineweaver-Burk plot. The Ki of the Example 1 Compound
was 21 nM.
[0056]
The Ki of the polyether-modified silicone (X-22-3939A) was also calculated
in the same manner, but the Ki of the polyether-modified silicone without an
antithrombin activity was the same as that of the blank, as expected.
[0057]
Further, the Ki of argatroban was also calculated in the same manner, and the
Ki was 42 nM which was not less than twice the value of the Ki of the Example 1
Compound.
[0058]
From these results, it is apparent that the above-described hydrophilic
polymer compound has an extremely high binding affinity to thrombin, and can give
a prominent antithrombin activity much higher than the argatroban which is known to
have an antithrombin activity, to medical devices including hollow fiber dialyzer or
medical material.
[0059]
(Preparation of PMMA Hollow Fiber Membrane Mini-module)
Five parts by weight of isotactic-PMMA and 20 parts by weight of
syndiotactic-PMMA were added to 75 parts by weight of dimethylsulfoxide, and the
resulting mixture was stirred at 110°C for 8 hours to obtain a membrane-forming
liquid. The obtained membrane-forming liquid was extruded from an orifice type
coaxial cylindrical mouthpiece, and after passing 300 mm in the air, the extruded
material was introduced into a coagulation bath containing 100% water, thereby
PMMA hollow fibers having an inner diameter of 0.2 mm and a thickness of a
membrane of 0.03 mm were obtained. As the gas introduced into the inside of the
fiber, dry nitrogen was used.
[0060]
A module case having an inner diameter of 10 mm and a length of 120 mm,
which had two ports communicating to the inside of the hollow fibers (blood port)
and two ports communicating to the outside of the hollow fibers (dialysate port),
respectively, was prepared.
[0061]
Fifty of the above-described PMMA hollow fibers were bundled to form
PMMA hollow fiber membranes, and both ends of the PMMA hollow fiber
membranes were fixed to the above-described module case by using an epoxy potting
material with attention not to clog the hollow portions of the PMMA hollow fiber
membranes. Thereafter, the PMMA hollow fiber membranes and the inner side of
the module case were washed with distilled water to obtain Mini-module 6 as shown
in Fig. 1.
[0062]
(Immobilization of Example 1 Compound to PMMA Hollow Fiber Membranes)
Bis-Tris (DOJINDO LABORATORIES) and sodium chloride were dissolved
inµLtrapure water so as to attain a final concentration of 0.25M and 0.5M,
respectively, and pH of the resulting solution was adjusted to 5 by adding 6N
hydrochloric acid dropwise to prepare Bis-Tris buffer solution having 5 times
concentration.
[0063]
Distilled water remained at the side contacting the blood (the inner side of the
PMMA hollow fiber membranes) and the side not contacting the blood (the outer
side of the PMMA hollow fiber membranes) of the prepared Mini-module 6 was
removed with compressed air. Then aqueous Example 1 Compound solution in a
concentration of 4000 ppm by weight in terms of argatroban, propylene glycol and
Bis-Tris buffer solution having 5 times concentration were mixed in a volume ratio
of 5/3/2 to obtain a filling solution.
[0064]
Only at the side contacting the blood of the Mini-module 6, the above-
described filling solution(400µL) was filled using a syringe. Thereafter, the filling
solution was removed with compressed air, and all of the blood ports la and 1b and
the dialysate ports 2a and 2b were tightly capped to irradiate the Mini-module 6 with
y-ray at a dose of 25 kGy for 3 hours.
[0065]
The PMMA hollow fiber membranes 4 and the inner side of the Mini-module
6 were washed by flowing 0.025% by weight of aqueous poly(oxyethylene octyl
phenyl ether) solution into the PMMA hollow fiber membranes 4 and the inner side
of the Mini-module 6 at a flow rate of 10 mL/min for 8 hours by using a peristaltic
pump 8. Thereafter, distilled water and physiological saline solution were flown at
a flow rate of 10 mL/min for 30 minutes, respectively, to carry out further wash to
obtain a mini-module in which the Example 1 Compound was immobilized
(hereinafter referred to as "Example 1 Mini-module").
[0066]
A mini-module in which polyether-modified silicone was immobilized
(hereinafter referred to as "Comparative Example 1 Mini-module") was obtained by
carrying out the same procedure described above except that polyether-modified
silicone (X-22-3939A) was used in place of aqueous Example 1 Compound solution
in a concentration of 4000 ppm by weight in terms of argatroban.
[0067]
{In vitro Test of Blood Circulation)
The blood supplied by a volunteer and citric acid was mixed in a volume ratio
of 9/1 to obtain blood supplemented with citric acid. Calcicol in an amount of 43.6
µL was added to 1 mL of the blood supplemented with citric acid to obtain a test
blood.
[0068]
Silicone tubes 7a and 7b were connected to the Example 1 Mini-module, and
a peristaltic pump 8 was placed in the middle of the silicone tube 7b. The test blood
was flown at a flow rate of 0.9 mL/min for 5 seconds from silicone tube 7a connected
to the blood port 1a, and the test blood discharged from the blood port 1b was
discarded from silicone tube 7b to remove bubbles in the inner side of the PMMA
hollow fiber membranes. Then, the silicone tubes 7a and 7b were connected at
Connecting part 9 to form a closed circuit shown in Fig.2.
[0069]
The circulation of the test blood was started at a flow rate of 0.9 mL/min to
measure the duration of circulation until the silicone tubes 7a or 7b came off the
Connecting part 9 due to an increased inner pressure in the circuit caused by
coagulation thrombus formed in the circuit. The duration of circulation when using
the Example 1 Mini-module was 46 minutes.
[0070]
Mini-module 6 in which no compound was immobilized on the PMMA
hollow fiber membranes (hereinafter referred to as "Comparative Example 2 Mini-
module") was prepared to carry out the same test of blood circulation as described
above. The duration of circulation in this case was 20 minutes which was not more
than half the duration of circulation when using Example 1 Mini-module. From
these results, it is apparent that the above-described hydrophilic polymer compounds
can give a prominent anticoagulant activity to medical devices including hollow fiber
dialyzer or medical materials.
[0071]
When the same test of blood circulation was carried out as described above by
using Comparative Example 1 Mini-module, the duration of circulation was 20
minutes, which was the same as the duration of circulation when using Comparative
Example 2 Mini-module in which no compound was immobilized on the PMMA
hollow fiber membranes.
[0072]
(Measurement of Eluted amount of Example 1 Compound)
Silicone tube 7b having an inner diameter of 0.8 mm and a length of 520 mm
was connected to the blood port 1b of the separately prepared Example 1 Mini-
module, and peristaltic pump 8 was placed in the middle of the silicone tube 7b.
Silicone tube 7a having an inner diameter of 0.8 mm and a length of 160 mm was
connected to the blood port 1a. Thereafter, other ends of the silicone tubes 7a and
7b were each inserted in polystyrene round tube (Code: 352054; BECTON
DICKINSON) 10 containing 5 mL of human plasma to prepare circulating circuit
shown in Fig. 3.
[0073]
After circulation in human plasma at a flow rate of 0.5 mL/min for 4 hours by
using peristaltic pump 8, the concentration of the Example 1 Compound in human
plasma in polystyrene round tube 10 was measured by using ECA-T kit. However,
the concentration of the Example 1 Compound in human plasma after circulation was
below the detection limit of ECA-T kit, and elution of the Example 1 Compound
from Example 1 Mini-module was not confirmed. This result shows that the above-
described hydrophilic polymer compound can be immobilized strongly to medical
devices including hollow fiber dialyzer or medical materials.
[0074]
(Evaluation of Amount of Adsorption of Polymer Compounds having Platelet
Adhesion Inhibitory Activity)
PVP (K-90), VA73, VA64, VA55 and VA37 (any of these were from BASF
Corporation) were provided as a copolymer between vinylpyrrolidone and vinyl
acetate (hereinafter referred to as "VA Copolymer"), which copolymer constitutes the
above-described hydrophilic polymer compounds and was one of polymer
compounds inhibiting platelet adhesion. Similarly, PVA217, PVA417 and
PVA205c (any of these were from KURARAY) were provided as a partially
saponified poly(vinyl alcohol) which was one of the polymer compounds inhibiting
platelet adhesion. Furthermore, as a polyether-modified silicone, Fl 14, F244, F303,
F3031, F348, F350s, F502, F506 and X-22-3939A (any of these were from SHIN-
ETSU SILICONE) were provided. The prepared VA Copolymer, partially
saponified poly(vinyl alcohol) and polyether-modified silicone were each diluted
with distilled water to prepare 10,000 ppm by weight of aqueous solution.
[0075]
On the other hand, as polymer compounds constituting the above-described
hydrophilic polymer compounds, which were not included in the polymer compounds
inhibiting platelet adhesion, PEG2000, PEG4000, PEG6000 and PEG20000 (any of
these were from NACALAI TESQUE), and PEG-methyl ether (PEG-em) and PEG-
dimethyl ether (PEG-dm) (both were from SIGMA-ALDRICH) were provided for
comparison. The prepared polymer compounds were each diluted with distilled
water to prepare 10,000 ppm by weight of aqueous solution.
[0076]
As 0.5% by weight solutions of adsorbent materials adsorbing polymer
compounds which inhibit platelet adhesion, PMMA (weight average molecular
weight: 93000, SIGMA-ALDRICH) solution in toluene, polyurethane solution in
dimethylacetamide, polysulfone (Udel (registered trademark) P-3500 produced by
SOLVAY) solution in dimethylacetamide, poly(vinyl chloride) (weight average
molecular weight: 80000, SIGMA-ALDRICH) solution in tetrahydrofuran,
polystyrene (WAKO) solution in chloroform and polycarbonate (weight average
molecular weight: 20000, TEIJIN) solution in chloroform were each prepared.
[0077]
The amounts of adsorption of various polymer compounds inhibiting platelet
adhesion were measured for each adsorbent material. The results are shown in
Table 2.
[0078]
From the results in Table 2, it is apparent that the polymer compounds
constituting the above-described hydrophilic polymer compounds, which inhibit
platelet adhesion, are not limited to polyether-modiiied silicone (X-22-3939A), and
can be adsorbed strongly to medical devices including hollow fiber dialyzer or
medical materials.
[0080]
(Evaluation of Platelet Adhesion Inhibitory Activity)
The separately prepared module case of Example 1 Mini-module was cut with
µLtrasonic disc cutter to take out the PMMA hollow fiber membranes (hereinafter
referred to as "Example 1 Hollow Fiber Membranes") on which Example 1
Compound was immobilized.
[0081]
A double-stick tape was adhered to one surface of a poly(ethylene
terephthalate) circular film having a diameter of 18 mm, and after fixing the Example
1 Hollow Fiber Membranes thereto, the fixed PMMA hollow fiber membranes were
cut into semicylindrical shape to expose the inner surfaces of the PMMA hollow
fiber membranes. The Example Hollow Fiber Membranes fixed to the circular film
was attached to a Falcon (registered trademark) cylindrical tube cut into cylindrical
shape (diameter 18 mm, NO. 2051), and the gap between the cylindrical tube and the
circular film was sealed with Parafilm. Thereafter, the cylindrical tube was filled
with physiological saline solution.
[0082]
Venous blood right after collection from a volunteer was placed in a blood
collection tube in which heparin was preliminarily collected, and the resulting
mixture was mixed by upside-down mixing to prepare blood supplemented with
heparin. The concentration of the blood supplemented with heparin was set to 50
U/mL.
[0083]
After discarding the physiological saline solution in the above-described
cylindrical tube, 1.0 mL of the blood supplemented with heparin was added thereto,
and the cylindrical tube was shaken at 37°C for 1 hour. Thereafter, Example 1
Hollow Fiber Membranes in the above-described cylindrical tube was washed with
10 mL of physiological saline solution, and then blood components were fixed by
adding physiological saline solution containing 2.5% by volume of glutaraldehyde,
followed by further washing the membranes with distilled water. Then, the circular
film fixing Example 1 Hollow Fiber Membranes was removed from the above-
described cylindrical tube, and the circular film fixing Example Hollow Fiber
Membranes was dried under reduced pressure at normal temperature at an absolute
pressure of 0.5 Torr for 12 hours.
[0084]
The circular film dried under reduced pressure on which Example 1 Hollow
Fiber Membranes was fixed was adhered to the stage of a scanning electron
microscope with a double-stick tape, and platinum/palladium thin film was then
formed on the surface of Example Hollow Fiber Membranes by sputtering. The
inner surfaces in the center portion in the longitudinal direction of the Example
Hollow Fiber Membranes on which surface platinum/palladium thin film was formed
were observed with a field emission scanning electron microscope (S800 produced
by HITACHI) at a magnification of x 1500, and the number of adhered platelets in
one visual field (4.3 x 103 µm2) was counted.
[0085]
The integer value of the mean of the numbers of adhered platelets counted in
different 5 visual fields is defined as the number of adhered platelets (platelets/(4.3 x
10 urn )), and the number of adhered platelets to Example 1 Hollow Fiber
Membranes was one.
[0086]
On the other hand, the separately prepared module case of Comparative
Example 2 Mini-module was cut withµLtrasonic disc cutter, and hollow fiber
membranes in which any compound was not immobilized (hereinafter referred to as
"Comparative Example 2 Hollow Fiber Membranes") were taken out to confirm the
number of adhered platelets as well. As a result, the number of adhered platelets of
Comparative Example 2 Hollow Fiber Membranes was not less than 100.
[0087]
From these results, it is apparent that the above-described hydrophilic
polymer compounds can give a prominent platelet adhesion inhibitory activity to
medical devices including hollow fiber dialyzer or medical materials.
[0088]
(Measurement of Whole Blood Clotting Time)
The blood collected from a volunteer and citric acid were mixed at a volume
ratio of 9/1 to prepare blood supplemented with citric acid.
[0089]
In a cuvette (NON-ACTIVATED CLOTTING TEST KIT), 18µL of
physiological saline solution was placed, and 14.8µL of calcicol was added thereto,
followed by further adding 342µL of blood supplemented with citric acid.
Thereafter, the measurement using Sonoclot Blood Coagulation/Platelet Function
Analyzer (IMI) was carried out to define the obtained ACT ONSET value as whole
blood clotting time. The whole blood clotting time of the blood collected by
volunteer was 545 seconds.
[0090]
The whole blood clotting times were 531, 746 and 849 seconds, respectively,
when the same measurements were carried out using 2, 10 and 20 uM of argatroban
solutions (solvents: methanol/hydrochloric acid (volume ratio: 4/1)), respectively, in
place of physiological saline solution.
[0091]
The whole blood clotting times were 527, 693 and 730 seconds, respectively,
when the same measurements were carried out using 0.3, 1.3 and 2.5 uM of aqueous
Example 1 Compound solutions, respectively, in place of physiological saline
solution.
[0092]
(Example 14: Binding between Vinyl acetate-Vinylpyrrolidone Copolymer and
Argatroban)
In a screw vial, 14.9 g of tetrahydrofuran, 11.5 g of vinyl acetate, 10.8 g of N-
vinylpyrrolidone, 0.028 g of 2-aminoethanethiol and 0.016 g of azobisisobutyronitrile
were placed, and after sealing the screw vial, the resulting mixture was sonicated for
10 minutes. The seal of the screw vial was once taken off, and the mixture was
bubbled with argon gas for 10 minutes, and after sealing the screw vial again, the
screw vial was immersed in a hot water bath at 60°C under stirring for 1 hour, and
further in a hot water bath at 70°C for 6 hours to copolymerize vinyl acetate with
vinylpyrrolidone. To the obtained reaction solution, 80 mL of methanol was added,
and the resulting mixture was added to about 5 times amount of ether, followed by
removing the supernatant. After repeating the washing operation 3 times in which
ether was newly added and the supernatant was removed, the resultant was dried
under reduced pressure to obtain vinyl acetate-vinylpyrrolidone copolymer. The
obtained vinyl acetate-vinylpyrrolidone copolymer was measured by 'H-NMR
(solvent: CDCL3) to obtain 60.6 unit % by mole for vinylpyrrolidone unit.
[0093]
A vinyl acetate-vinylpyrrolidone copolymer solution in anhydrous DMF was
prepared by dissolving 3.58 g of the obtained vinyl acetate-vinylpyrrolidone
copolymer in 20 mL of anhydrous DMF. The total weight of the prepared vinyl
acetate-vinylpyrrolidone copolymer solution in anhydrous DMF and 0.5 mL of
argatroban hydrochloride solution in anhydrous DMF (0.49 M) were placed in a two-
necked flask, and 0.5 mL of DCC solution in anhydrous DMF (1.04 M) and 0.5 mL
of HOBt solution in anhydrous DMF (1.02 M) were added thereto, respectively, with
ice cooling and stirring, followed by reaction under a nitrogen atmosphere at room
temperature for 3 days. Then, the reaction solution was placed in a dialysis tube
(SPECTRAPORE RC, PORE 6, MWCO=1000), and dialyzed for 3 days against
distilled water with a volume of more than 10 times while appropriately replacing the
distilled water. The reaction solution after the dialysis was filtrated, and the solvent
in the filtrate was evaporated by a rotary evaporator, followed by drying the resultant
overnight in a vacuum drier to obtain a hydrophilic polymer compound (hereinafter
referred to as "Example 14 Compound").
[0094]
(Measurement of Antithrombin Activity of Example 14 Compound)
The antithrombin activity of Example 14 Compound solution in methanol
(concentration: 20% by weight) was measured in the same manner as the
measurement of the antithrombin activity of Example 1 Compound, and the
calculated concentration of 104.1 ppm in terms of argatroban of the Example 14
Compound solution in methanol was defined as the value indicating the antithrombin
activity of the Example 14 Compound solution in methanol.
[0095]
From these results, it is apparent that the above-described hydrophilic
polymer compounds can prolong the whole blood clotting time when compared with
argatroban which is known to have an antithrombin activity, even if the
concentrations of the hydrophilic polymer compounds are very low, and the
hydrophilic polymer compounds can give an excellent anticoagulant activity to
medical devices including hollow fiber dialyzer or medical materials.
(Industrial Applicability)
[0096]
The present invention can be used to give an excellent anticoagulant activity
to medical devices including hollow fiber dialyzer or medical materials.
(Description of Symbols)
[0097]
la, lb Blood port
2a, 2b Dialysate port
3 Module case
4 PMMA hollow fiber membrane
5 Potting material
6 Mini-module
7a, 7b Silicone tube
8 Peristaltic pump
9 Connecting part
10 Polystyrene round tube
We Claim:
1. A hydrophilic polymer compound comprising a polymer compound which
inhibits platelet adhesion, and a compound that inhibits blood coagulation reaction,
bound to said polymer compound.
2. The hydrophilic polymer compound according to claim 1, wherein said
polymer compound which inhibits platelet adhesion is a copolymer composed of a
hydrophobic macromolecule and a hydrophilic macromolecule, and has an amount of
adsorption to poly(methyl methacrylate) of not less than 0.1 pg/mm2.
3. The hydrophilic polymer compound according to claim 1 or 2, wherein said
compound which inhibits blood coagulation reaction is a compound having
antithrombin activity.
4. The hydrophilic polymer compound according to any one of claims 1 to 3,
wherein said compound which inhibits blood coagulation reaction is a compound
represented by General Formula (I):

[wherein R1 represents a (2R,4R)-4-alkyl-2-carboxypiperidino group; R2 represents a
phenyl group or a fused polycyclic compound residue, the fused polycyclic
compound residue optionally being substituted with a lower alkyl group, a lower
alkoxy group or an amino group which is substituted with a lower alkyl group].
5. The hydrophilic polymer compound according to any one of claims 1 to 4,
wherein said copolymer is a copolymer of monomers selected from the group
consisting of ethylene glycol, vinyl acetate, vinyl pyrrolidone, propylene glycol, vinyl
alcohol and siloxane.
6. The hydrophilic polymer compound according to claim 4 or 5, wherein said
copolymer is a polyether-modified silicone.
7. The hydrophilic polymer compound according to any one of claims 4 to 6,
wherein said compound of General Formula (I) is (2R,4R)-4-methyl-l-((2S)-2-
[(3RS)-3-methyl-l,2,3,4-tetrahydroquinolin-8-yl]sulfonyl} amino-5-
guanidinopentanoyl)piperidin-2-carboxylicacid.
8. A surface treatment agent for medical devices or medical materials, the
surface treatment agent comprising said hydrophilic polymer compound according to
any one of claims 1 to 7, which has an anticoagulant activity.
9. A medical device or a medical material treated with said surface treatment
agent according to claim 8.

ABSTRACT

An object of the present invention is to provide a hydrophilic polymer
compound which can inhibit both of the blood coagulation reactions in the primary
hemostasis stage in which platelets are involved and in the coagulation thrombus
formation stage in which blood coagulation factors are involved, which hydrophilic
polymer compound can be firmly immobilized on the surface of medical devices or
medical materials, in the state retaining the anticoagulant activity.
The present invention provides a hydrophilic polymer compound comprising a
polymer compound which inhibits platelet adhesion, and a compound that inhibits
blood coagulation reaction, bound to said polymer compound.