Note: Descriptions are shown in the official language in which they were submitted.
12t;~;85S
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BACKGROUND OF THE INVENTION
The present invention relates to a novel
adsorbent and a process for preparing the same, more
particularly, to an adsorbent for removing harmful
substances to be removed from body fluid such as blood or
plasma in extracorporeal circulation treatment.
There has been required a means for selectively
removing harmful substances which appeare in body fluid
and closely relate to a cause or a progress of a disease.
It is known that plasma lipoprotein, especially very low
density lipoprotein (hereinafter referred to as "VLDL")
and/or low density lipoprotein ~hereinafter referred to
as "LDL") contain a large amount of cholesterol and cause
arteriosclerosis. In hyperlipemia such as familial
hyperlipemia or familial hypercholesterolemia, VLDL
and/or LDL show several times higher values than those in
normal condition, and often cause arteriosclerosis such
as coronary arteriosclerosis. Although various types of
treatments such as regimen and medications have been
adopted, they have limitations in effect and a fear of
unfavorable side effects. Particularly in familial
hypercholesterolemia, a plasma exchange therapy which is
composed of plasma removal and compensatory supplement of
exogeneous human plasma protein solutions is probably the
only treatment method being effective nowadays. The
plasma exchange therapy, however, has various defects
such as (1) a need for using expensive fresh plasma or
plasma fractions, (2) a fear of infection by hepatitis
viruses and the like, and (3) loss of all plasma
components containing not only harmful components but
also useful ones, i.e. in case of lipoprotein, not only
VLDL and/or LDL but also high density lipoprotein
(hereinafter referred to as "HDL") which is an important
factor to prevent Arteriosclerosis are lost. For the
purpose of solving the above defects, a selective removal
of harmful components by a membrane and the like has been
adopted. These methods, however, are insufficient in
selectivity and cause a large loss of useful components
3~
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from body fluid. There has been also tried a selective
removal of harmful components by means of adsorption.
For example, a synthetic adsorbent such as active carbon
or Amberlite XAD (a registered trademark, commercially
available from Rohm & Hass Co.) has been utilized for
liver disease. Such an adsorbent however, has many
defects such as poor selectivity and disability for
removing high molecular weight compounds. Furthermore,
for the purpose of increasing selectivity, there has been
adopted an adsorbent based on the principle of affinity
chromatography composed of a carrier on which a material
having an affinity for a substance to be specifically
removed (such material is hereinafter referred to as
"ligand") is immobilized. In that case, however, it is
difficult to obtain a sufficient flow rate for an
extracorporeal treatment because a carrier is a soft gel
such as agarose. Accordingly, a particular modification
in column shape is required in order to obtain a large
flow rate and the risk of an occasional clogging still
remains. Therefore, a stable extracorporeal circulation
cannot be achieved by the above method.
It is an object of the present invention to
provide an adsorbent for selectively removing VLDL and/or
LDL from body fluid such as blood or plasma in
extracorporeal circulation treatment of hyperlipidemia.
' A further object of the present invention is to
provide a process for preparing the adsorbent.
These and other objects of the present
invention will become apparent from the description
hereinafter.
¦ SUMMARY OF THE INVENTION
In accordance with the present invention, there
can be provided an adsorbent for removing LDL and/or VLDL
from body fluid in extracorporeal circulation treatment
comprising a water-insoluble porous hard gel with
exclusion limit of 106 to 109 daltons on which a sulfated
compound is immobilized by a covalent linkage.
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BRIEF DESCRIPTION OF THE DRAWING
Figs. 1 and 2 are graphs, respectively, showing
relations between flow rate and pressure-drop obtained in
Reference Examples 1 and 2, and Fig. 3 is a chart of
polyacrylamide disc gel electrophoresis obtained in
Example 27.
DETAILED DESCRIPTION OF THE INVENTION
It is suitable that carriers used in the
present invention have the following properties:
(1) relatively high mechanical strength,
(2) low pressure-drop and no column clogging in case of
passing body fluid through a column packed with a carrier,
(3) a large number of micro por~s into which LDL and/or
VLDL permeates substantially, and
(4) less change caused by a sterilizing procedure such as
steam sterilization by autoclaving.
Therefore, the most suitable carrier used in
the present invention is a water-insoluble porous polymer
hard gel or a porous inorganic hard gel.
The porous hard gel used in the present
invention is less swelled with a solvent and less
deformed by pressure than a soft gel such as dextran,
agarose or acrylamide.
The term "hard gel" and "soft gel" in the
present invention is explained as follows:
A hard gel is distinguished from a soft gel by
the following method described in Reference Examples 1
and 2. That is, when a relation between flow rate and
pressure-drop is determined by passing water through a
column uniformly packed with a gel, a hard gel shows a
linear relationship while a soft gel shows a non-linear
relationship. In case of a soft gel, a gel is deformed
and consolidated over a certain pressure so that a
flow rate does not increase further. In the present
invention, a gel having the above linear relationship at
least by 0.3 kg/cm2 is referred to as "hard gel".
A pore size of the porous hard gel is selected
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lZ~6~355
depending on molecular weight, shape, or size of a
substance to be removed, and the most suitable pore size
may be selected in each case. For measuring the pore
size, there are various kinds of methods such as mercury
porosimetry and observation by an electron microscope as
a direct measuring method. With respect to
water-containing particles, however, the above methods
sometimes cannot be applied. In such a case, an
exclusion limit may be adopted as a measure of pore size.
The term "exclusion limitl' in the present invention means
the minimum molecular weight of a molecular which cannot
permeate into a pore in a gel permeation chromatography
~cf. Hiroyuki ~atano and Toshihiko Hanai: Zikken Kosoku
Ekitai Chromatography (Experimental High-Pressure Li~uid
Chromatography)~ published by Kagaku Dojin).
Phenomenally, a molecule having a molecular weight of
more than exclusion limit is eluted near the void volume.
Therefore, an exclusion limit can be determined by
studying the relations between molecular weights and
elution volumes using substances of various molecular
weights in a gel permeation chromatography. An exclusion
limit varies with a kind of substances to be excluded.
In the present invention, an exclusion limit of the
porous hard gel is measured by using globular proteins
and/or viruses, and the preferable exclusion limit is
1 x 106 to 1 x 109. When the exclusion limit is more
than 1 x 109, the adsorbing amount of LDL and VLDL
decreases with a decrease of amount of immobilized
ligand and a decrease of surface area, and further a
mechanical strength of gel is reduced.
Removing VLDL and/or LDL being giant molecules
having a molecular weight of more than 1 x 106, a porous
hard gel having an exclusion limit of less than 1 x
106 is not practically available. On the other hand, a
porous hard gel having an exclusion limit of from 1 x
106 to several million which is near a molecular weight
of VLDL or LDL per se may be practically available to a
certain extent. A preferable exclusion limit for removal
12~685S
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of VLDL and/or LDL is 1 x 106 to 1 x 109, more preferably
1 x 106 to 1 x lO8.
With respect to a porous structure of the
porous hard gel used in the present invention, a
structure uniformly having pores at any part of the gel
~hereinafter referred to as "uniform structure") is more
preferable than a structure having pores only on the
surface of the gel. It is preferred that a porosity of
the gel is not less than 20 ~. The carrier may be
selected from suitable shapes such as particle, fiber,
sheet and hollow fiber. In case of using a carrier in
the shape of particle, although a particle having a
smaller size generally shows an excellent adsorbing
capacity, the pressure-drop increases with an extremely
small size. Therefore, a particle having a size of 1 ~m
to 5000 ~m in diameter is preferred. Furthermore, it is
preferred that a carrier has functional groups to be
utilized for the immobilization of ligand or groups to be
easily activated. Examples of the group are, for
instance, amino, carboxyl, hydroxyl, thiol, acid
anhydride, succinylimide, chlorine, aldehyde, aminde,
epoxy group, and the like.
Representative examples of the water-insoluble
porous hard gel used in the present invention are, for
instance, a porous hard gel of a synthetic or a natural
polymer such as stylene-divinylbenzene copolymer,
cross-linked polyvinyl alcohol, cross-linked
polyacrylate, crosslinked vinyl ether-maleic anhydride
copolymer, cross-linked stylene-maleic anhydride
copolymer or cross-linked polyamide, a porous cellulose
gel, an inorganic porous hard gel such as silica gel,
porous glass, porous alumina, porous silica alumina,
porous hydroxyapatite, porous calcium silicate, porous
zirconia or porous zeolite, and the like. Of course it
is to be understood that the porous hard gels used in the
present invention are not limited to those set forth as
; examples above. The surface of the above-mentioned
porous hard gel may be coated with polysaccharides,
.,
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synthetic polymers, and the like. These porous hard gels
may be employed alone or in an admixture thereof.
In the above representative examples, some of
the porous polymer hard gels composed of synthetic
polymers have a fear of toxicity due to unreacted
monomers and a less adsorbing capacity than that of a
soft gel.
Therefore, in the above representative
examples, a porous cellulose gel is one of the
particularly preferable carriers for the present
invention, and it satisfies the above all four points
required for the carrier. In addition, the porous
cellulose gel has various excellent advantages such as
hydrophilicity due to being composed of cellulose, a
large number of hydroxyl group to be utilized for
immobilization, less nonspecific adsorption, and
sufficient adsorbing capacity not inferior to that of a
soft gel due to its relatively high strength even with a
large porisity. Therefore, the porous cellulose gel on
zo which a ligand is immobilized provides a nearly ideal
adsorbent.
As the porous cellulose gel used in the present
invention, although cellulose per se is preferred, a
cellulose derivative such as an esterified cellulose or
an etherified cellulose, or a mixture of cellulose and
the cellulose derivatives may be employed. Examples of
the cellulose derivative are, for instance, acetyl
cellulose, methyl cellulose, ethyl cellulose,
carboxymethyl cellulose, and the like. It is preferred
that the cellulose gel is in the spherical shape. The
; cellulose gel is prepared, for example, by dissolving or
swelling cellulose and/or a cellulose derivatives with a
solvent, dispersing the resulting mixture into another
solvent being not admixed with the used solvent to make
; 35 beads, and then regenerating the beads. The cellulose
and/or cellulose derivatives may be cross-linked or not.
A porisity of a porous cellulose gel may be a
measure of cellulose content. The cellulose content is
i
lZ~ S5
expressed by the following formula:
w
Cellulose content (%) = x 100
Vt - Vo
wherein w is dry gel weight (g), vt is a volume of column
packed with gel (ml) and vo is a void volume (ml).
It is preferred that the cellulose content of
the porous cellulose gel used in the present invention is
2 % to 20 %. In case of less than 2 %, the mechanical
strength of gel is reduced, and in case of more than 20
%, the pore volume is reduced.
Representative examples of the ligand used in
the present invention are as follows:
As ligands to remove VLDL and/or LDL containing
a large amount of cholesterol and causing
arteriosclerosis, polyanion compounds and/or sulfated
compounds are preferred as a ligand. Representative
examples of the polyanion compounds are polyphosphoric
acid, phosphorus walframic acid, poly acrylic acid and
pOlystyrenesulfonic acid.
As the sulfated compound used for the ligand,
there may be employed a compound obtained by sulfation of
a hydroxy-containing compound. Examples of the sulfated
compounds are, for instance, a sulfated carbohydrate, a
sulfated polyhydric alcohol, polysulfated anethol, a
sulfated hydroxy-containing polymer such as polyvinyl
alcohol or polyhydroxyethyl methacrylate, and the like.
Typical sulfated carbohydrate is a sulfated saccharide.
Examples of the saccharides are, for instance a
polysaccharide, a tetrose such as threose, a pentose such
as arabinose, xylose or ribose, a hexose such as glucose,
galactose, mannose or fructose, a derivative thereof, a
deoxysaccharide such as fucose, an amino-saccharide such
as galactosamine, an acidic saccharide such as uronic
acid, glucronic acid or ascorbic acid. Examples of the
~ polyhydric alcohol are, for instance, a glycol such as
- ethylene glycol, glycerol, sorbite pentaerythritol, and
the like. Examples of the sulfated polysaccharldes are
i855
heparin, dextran sulfate, chondroitin sulfate,
chondroitin poly-sulfate, heparan sulfate, keratan
sulfate, xylan sulfate, caronin sulfate, cellulose
sulfate, chitin sulfate, chitosan sulfate, pectin sulfate,
inulin sulfate, arginine sulfate, glycogen sulfate,
polylactose sulfate, carrageenan sulfate, starch sulfate,
polyglucose sulfate, laminarin sulfate, galactan sulfate,
levan sulfate and mepesulfate. Preferable examples of
the above sulfated compounds are, for instance, sulfated
polysaccharides such as heparin, dextran sulfate,
chondroitin polysulfate, and/or the salts thereof, and
particularly preferable examples are a dextran sulfate
and/or the salt thereof. Examples of the salt of the
above sulfated compound are, for instance, a water-
soluble salt such as sodium salt or potassium salt, andthe like.
Dextran sulfate and/or the salt thereof are
explained in more detail hereinbelow.
Dextran sulfate and/or the salt thereof are
sulfuric acid ester of dextran being a polysaccharide
produced by Leuconostoc mesenteroides, etc., and/or the
salt thereof. It has been known that dextran sulfate
and/or the salt thereof form a precipitate with
lipoproteins in the presence of a divalent cation, and
dextran sulfate and/or the salt thereof having a
molecular weight of about 5 x 105 (intrinsic viscosity of
about 0.20 dl/g) are generally employed for this
precipitation. However, as shown in the following
Example 39 of Run Nos. (1) and (2), a porous hard gel on
which some of the above-mentioned dextran sulfate and/or
the salt thereof are immobilized is poor in affinity to
VLDL and/or LDL. As a result of extensive studies to
solve the above problems, it has now been found that
dextran sulfate having an intrinsic viscosity of not more
than 0.12 dl/g, preferably not more than 0.08 dl/g, and a
sulfur content of not less than 15 % by weight has high
affinity and selectivity to VLDL and/or LDL. Furthermore,
the adsorbent of the present invention employing such
~Z~6855
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dextran sulfate and/or the salt thereof as a ligand has
high affinity and selectivity even in the absence of a
divalent cation. Although a toxicity of dextran sulfate
and/or the salt thereof is low, the toxicity increases
S with increasing of molecular weight. From this point of
view, the use of dextran sulfate and/or the salt thereof
having an intrinsic viscosity of not more than 0.12 dl/g,
preferably not more than 0.08 dl/g can prevent a danger
in case that the immobilized dextran sulfate and/or the
salt thereof should be released from a carrier. In
addition, dextran sulfate and/or the salt thereof are
less changed by a sterilizing procedure such as steam
sterilization by autoclaving, because they are linked
mainly by ~ 6)-glycosidic linkage. Although there are
various methods for measuring a molecular weight of
dextran sulfate and/or the salt thereof, a method by
measuring viscosity is general. Dextran sulfate and/or
the salt thereof, however, show different viscosities
depending on various conditions such as ion strength, pH
value, and sulfur content (content of sulfonic acid
group). The term "intrinsic viscosity" used in the
present invention means a viscosity of sodium salt of
dextran sulfate measured in a neutral 1 M ~aCl aqueous
solution, at 25C. The dextran sulfate and/or the salt
thereof used in the present invention may be in the form
of straight-chain or branched-chain.
For coupling a ligand with a carrier, various
methods such as physical adsorption methods, ionic
coupling methods and covalent coupling methods may be
employed. In order to use the adsorbent of the present
invention in extracorporeal circulation treatment, it is
important that the ligand is not released. Therefore, a
covalent coupling method having a strong bond between
` ligand and carrier is preferred. In case of employing
other methods, a modification is necessary to prevent the
release of ligand. If necessary, a spacer may be
introduced between ligand and carrier.
It is preferred that a gel is activated by a
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reagent such as a cyanogen halide, epichlorohydrin, a
polyoxirane compound such as bisepoxide or triazine
halide, and then reacted with a ligand to give the
desired adsorbent. In that case, it is preferred that a
gel having a group to be activated such as hydroxyl group
is employed as a carrier. In the above reagents,
epichlorohydrin or a polyoxirane compound such as
bisepoxide is more preferred, because a ligand is
strongly immobilized on a carrier activated by using such
a reagent and a release of a ligand is reduced.
Epichlorohydrin and a polyoxirane compound,
however, show lower reactivity, particularly lower to
dextran sulfate and/or the salt thereof, because dextran
sulfate and/or the salt thereof have hydroxyl group alone
as a functional group. Therefore, it is not easy to
obtain a sufficient amount of immobilized ligand.
As a result of extensive studies, it has now
been found that the following coupling method is
preferred in case of using dextran sulfate and/or the
salt thereof as a ligand. That is, a porous polymer hard
gel is reacted with epichlorohydrin and/or a polyoxirane
compound to introduce epoxy groups into the gel, and then
dextran sulfate and/or the salt thereof is reacted with
the resulting epoxy-activated gel in a concentration of
not less than 3 % based on the weight of the whole
reaction system excluding the dry weight of the gel, more
preferably not less than 10 ~. This method gives a good
immobilizing efficiency. In that case, a porous
cellulose gel is particularly suitable as a carrier.
On the other hand, when a porous inorganic hard
gel is employed as a carrier, it is preferred that the
gel is activated with a reagent such as an epoxysilane,
e.g. y-glycidoxypropyltrimethoxysilane or an
aminosilane, e.g. y-aminopropyltriethoxysilane, and then
reacted with a ligand to give the desired adsorbent.
The amount of immobilized ligand varies
depending on properties of the ligand used such as shape
and activity. For sufficient removal of VLDL and/or LDL
;1'~`~6S55
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by using a polyanion compound, for instance, it is
preferred that the polyanion compound is immobilized in
an amount of not less than 0.02 mg/ml of an apparent
column volume occupied by an adsorbent (hereinafter
referred to as ~bed volume"), economically 100 mg or
less. The preferable range is 0.5 to 20 mg/ml of bed
volume. Particularly, for removal of VLDL and/or LDL by
using dextran sulfate and/or the salt thereof as a
ligand, it is preferred that the amount of immobilized
ligand is not less than 0.2 mg/ml of bed volume. After
the coupling reaction, the unreacted polyanion compound
may be recovered for reuse by purification, etc.
It is preferred that the remaining unreacted
active groups are blocked by ethanolamine, and the like.
lS In accordance with the present invention, an
adsorbent composed of porous cellulose gel having an
exclusion limit of 106 to 108 and a particle size of 30
to 200 ~m on which sodium salt of dextran sulfate having
an intrinsic viscosity of not more than 0.12 dl/g and a
sulfur content of not less than 15 % by weight is
immobilized, is particularly suitable for removal of VLDL
and/or LDL in extracorporeal circulation treatment of
hypercholesterolemia.
The adsorbent of the present invention may be
employed for various kinds of use. Representative
example of the use is extracorporeal circulation treatment
performed by incorporating a column into extracorporeal
circulation circuit and passing body fluid such as blood
or plasma through the column, the column being packed
with the adsorbent of the present invention. The use of
the adsorbent is not necessarily limited to the above
example.
The adsorbent of the present invention can be
subjected to steam sterilization by autoclaving so long
as the ligand is not largely degenerated, and this
sterilization procedure does not affect on micro pore
structure, particle shape and gel volume of the
adsorbent.
.
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The present invention is more specifically
described and explained by means of the following
Reference Examples and Examples, and it is to be
understood that the present invention is not limited to
the Reference Examples and Examples.
Reference Example 1
Biogel ASm (a commercially available agarose
gel made by Biorad Co., particle size: 50 to 100 mesh) as
a soft gel and Toyopearl HW65 (a commercially available
cross-linked polyacrylate gel made by Toyo Soda
Manufacturing Co., Ltd., particle size: 50 to 100 ym) and
Cellulofine GC-700 (a commercially available porous
cellulose gel made by Chisso Corporation, particle size:
15 45 to 105 ~m) as a hard gel were uniformly packed,
respectively, in a glass column (inner diameter: 9 mm,
height: 150 mm) having filters (pore size: 15 ~m) at
both top and bottom of the column. Water was passed
through the thus obtained column, and a relation between
flow rate and pressure-drop was determined. The results
are shown in Fig. 1. As shwon in Fig. 1, flow rate
increased approximately in proportion to increase of
pressure-drop in the porous polymer hard gels. On the
other hand, the agarose gel was consolidated. As a
result, increasing pressure did not make flow rate
increase.
Reference Example 2
The procedures of Reference Example 1 were
repeated except that FPG 2000 (a commercially available
porous glass made by Wako Pure Chemical Industry Ltd.,
particle size: 80 to 120 mesh) instead of porous polymer
hard gels was employed as a porous inorganic hard gel.
The results are shown in Fig. 2. As shown in Fig. 2,
flow rate increased approximately in proportion to
increase of pressure-drop in the porous glass, while not
in the agarose gel.
* Trade Mark
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Example 1
Toyopearl HW55 (a commercially available
cross-linked polyacrylate gel made by Toyo Soda
Manufacturing Co., Ltd., exclusion limit: 7 x 105,
particle size: 50 to 100 ~m) having a uniform structure
was employed as a carrier.
To 10 ml of the gel were added 6 ml of
saturated NaOH aqueous solution and 15 ml of
epichlorohydrin, and the reaction mixture was subjected
to reaction with stirring at 50C for 2 hours. The gel
was washed successively with alcohol and water to
introduce epoxy groups into the gel. To the resulting
epoxy-activated gel was added 20 ml of concentrated
aqueous ammonia, and the reaction mixture was stirred at
50C for 2 hours to introduce amino groups into the gel.
Three ml portion of the thus obtained
activated-gel containing amino groups was added to 10 ml
of aqueous solution (pH 4.5) containing 200 mg of
heparin. To the resulting reaction mixture was added 200
mg of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide while
maintaining the reaction mixture at pH 4.5, and then the
reaction mixture was shaken at 4C for 24 hours. After
completion of the reaction, the resulting reaction
mixture was washed successively with 2 M NaCl aqueous
solution, 0.5 M NaCl aqueous solution and water to give
the desired gel on which heparin was immobilized
(hereinafter referred to as "heparin-gel"). The amount
of immobilized heparin was 2.2 mg/ml of bed volume.
.
Examples 2 to 4
The procedures of Example 1 were repeated
except that Toyopearl HW60 (exclusion limit: 1 x 106,
particle size: 50 to 100 ~m), Toyopearl HW 65 (exclusion
limit: 5 x 106, particle size: 50 to 10Q ~m) and
35 Toyopearl HW75 (exclusion limit: 5 x 107, particle size:
50 to 100 ~m) instead of Toyopearl HW55 were employed,
respectively, to give each heparin-gel. Toyopearl HW60,
Toyopearl HW65 and Toyopearl HW75 are all commercially
;855
_ 15
available cross-linked polyacrylate gels having a uniform
structure made by Toyo Soda Manufacturing Co., Ltd. The
amounts of immobilized heparin were, respectively, 1.8 mg,
1.4 mg and 0.8 mg/ml of bed volume.
Example 5
Cellulofine GC 700 (a commercially available
porous cellulose gel made by Chisso Corporation,
exclusion limit: 4 x 105, particle size: 45 to 105 ~m)
having a uniform structure was employed as a carrier.
The gel was filtered with suction, and 4 g of
20 % NaOH and 12 g of heptane were added to 10 g of the
suction-filtered gel. One drop of Tween 20 (nonionic
surfactant) was further added to the reaction mixture
which was stirred for dispersing the gel. After stirring
at 40C for 2 hours, 5 g of epichlorohydrin was added to
the reaction mixture which was further stirred at 40C
for 2 hours. After the reaction mixture was allowed to
stand, the resulting supernatant was discarded, and the
gel was washed with water to introduce epoxy groups into
the gel. To the resulting epoxy-activated gel was added
15 ml of concentratedaqueous ammonia, and the reaction
mixture was stirred at 40C for 1.5 hours, filtered with
suction and washed with water to introduce amino groups
into the gel.
Three ml portion of the thus obtained activated
gel containing amino groups was added to 10 ml of aqueous
solution (pH 4.5) containing 200 mg of heparin. To the
resulting reaction mixture was added 200 mg of 1-ethyl-3-
(dimethylaminopropyl)-carbodiimide while maintaining the
reaction mixture at pH 4.5, and then the reaction mixture
was shaken at 4C for 24 hours. After completion of the
reaction, the resulting reaction mixture was washed
successively with 2 M NaCl aqueous solution, 0.5 M NaCl
aqueous solution and water to give the desired heparin-
Cellulofine A-3. The amount of immobilized heparin was
2.5 mg/ml of bed volume.
1~6~;85S
- 16
Examples 6 to_
The procedures of Example 5 were repeated
except that Cellulofine A-2 (exclusion limit: 7 x 10 ,
particle size: 45 to 105 ~m) and Cellulofine A-3
(exclusion limit: 5 x 107, particle size: 45 to 105 ~m)
instead of Cellulofine GC 700 were employed, respectively,
to give each heparin-gel. Both Cellulofine A-2 and
Cellulofine A-3 are commercially available porous
cellulose gels having a uniform structure made by Chisso
Corporation. The amounts of immobilized heparin were,
respectively, 2.2 mg and 1.8 mg/ml of bed volume.
Example 8
The procedures of Example 5 were repeated
except that Cellulofine A-3 having a particle size of 150
to 200 ~m instead of 45 to 105 ~m was employed. The
amount of immobilized heparin was 1.5 mg/ml of bed volume.
Example 9
The procedures of Example 1 were repeated
except that Toyopearl HW65 instead of Toyopearl HW55 and
chondroitin polysulfate instead of heparin were employed,
to give the desired chondroitin polysulfate-Toyopearl
HW65. The amount of immobilized chondroitin polysulfate
was 1.2 mg/ml of bed volume.
Example 10
To 4 ml of Cellulofine A-3 was added water to
make the volume up to 10 ml, and then 0.5 mole of
NaIO4 was added. After stirring at a room temperature
for one hour, the reaction mixture was washed with water
by filtration to introduce aldehyde groups into the gel.
The thus obtained gel was suspended in 10 ml of phosphate
buffer of pH 8 and stirred at a room temperature for 20
hours after addition of 50 mg of ethylenediamine. The
gel was filtered off and then suspended in 10 ml of 1 ~
NaBH4 solution. After reducing reaction for 15 minutes,
the reaction mixture was filtered and washed with water
855
_ 17
to introduce amino groups into the gel.
In 10 ml of 0.25 M NaIO4 solution was dissolved
300 mg of sodium salt of dextran sulfate. After stirring
at a room temperature for 4 hours, 200 mg of ethylene
glycol was added to the resulting solution and stirred
for one hour. The resulting solution was adjusted to pH
8, and then the above gel containing amino groups was
suspended in the solution and stirred for 24 hours.
After completion of the reaction, the gel was filtered,
washed with water, and then suspended in 10 ml of 1 %
NaBH4 solution. The resulting suspension was subjected
to reducing reaction for 15 minutes and washed with water
by filtration to give the desired sodium salt of dextran
sulfate-Cellulofine A-3. The amount of immobilized
sodium salt of dextran sulfate was 0.5 mg/ml of bed
volume.
Example 11
Cellulofine A-3 was treated in the same manner
as in Example 5 to introduce epoxy groups into the gel.
Two ml of the thus obtained epoxy-activated gel
was added to 2 ml of aqueous solution containing 0.5 g of
sodium salt of dextran sulfate (intrinsic viscosity 0.055
dl/g, average polymerization degree: 40, sulfur content:
19 % by weight), and the reaction mixture was adjusted to
pH 12. The concentration of sodium salt of dextran
sulfate was about 10 % by weight. The resulting reaction
mixture was filtered and washed successively with 2 M
NaCl aqueous solution, 0.5 M ~aCl aqueous solution and
water to give the desired sodium salt of dextran sulfate-
Cellulofine A-3. The remaining unreacted epoxy groups
were blocked with monoethanolamine. The amount of
immobilized sodium salt of dextran sulfate was 1.5 mg/ml
of bed volume.
Example 12
To 5 g of suction-filtered Cellulofine A-3 were
added 2.5 ml of 1,4-butanediol diglycidyl ether and 7.5
12668S5
- 18
ml of 0.1 N NaOH aqueous solution, and the reaction
mixture was stirred at a room temperature for 18 hours to
introduce epoxy groups into the gel.
The thus obtained epoxy-activated gel was
reacted with sodium salt of dextran sulfate in the same
manner as in Example 11 to give the desired sodium salt
of dextran sulfate-Cellulofine A-3. The amount of
immobilized sodium salt of dextran sulfate was 1.8 mg/ml
of bed volume.
Example 13
The procedures of Example 11 were repeated
except that Cellulofine A-6 (a commercially available
porous cellulose gel made by Chisso Corporation,
15 exclusion limit: 1 x 108, particle size: 45 to 105 ~m)
having a uniform structure instead of Cellulofine A-3 was
employed to give the desired sodium salt of dextran
sulfate-Cellulofine A-6. The amount of immobilized
sodium salt of dextran sulfate was 1.2 mg/ml of bed
volume.
Example 14
Toyopearl HW65 was treated in the same manner
as in Example 1 to introduce epoxy groups into the gel.
Two ml of the thus obtained epoxy-activated gel
was treated in the same manner as in Example 11 to give
the desired sodium salt of dextran sulfate-Toyopearl
HW65. The amount of immobilized sodium salt of dextran
sulfate was 0.4 mg/ml of bed volume.
Example 15
FPG 2000 (exclusion limit: 1 x 109, particle
size: 80 to 120 mesh, average pore size: 1950 A) was
heated in diluted nitric acid for 3 hours. After washing
and drying, the gel was heated at 500C for 3 hours and
then refluxed in 10 % y-aminopropyltriethoxysilane
solution in toluene for 3 hours. After washing with
methanol, a y-aminopropyl-activated glass was obtained.
~'6~i85X
-- 19
Two g of the thus obtained activated glass was
added to 10 ml of aqueous solution tpH 4.5) containing
200 mg of heparin. The reaction mixture was treated in
the same manner as in Example 1 to give the desired
5 heparin-FPG 2000. The amount of immobilized heparin was
1.2 mg/ml o~ bed volume.
Examples 16 to 18
The procedures of Example 15 were repeated
except that FPG 700 (a commercially available porous
glass made by Wako Pure Chemical Industry Ltd., exclusion
limit: 5 x 107, particle size: 80 to 120 mesh, average
pore size: 70 A), FPG 1000 (a commercially available
porous glass made by Wako Pure Chemical Industry Ltd.,
exclusion limit: 1 x 108, particle size: 80 to 120 mesh,
average pore size: 1091 A) and Lichrospher Si4000 (a
commercially available porous silica gel made by ~erck &
Co. Inc., exclusion limit: 1 x 109, average particle
size: 10 ~m, average pore size: 4000 A) instead of FPG
2000 were employed. The amounts of immobilized heparin
were, respectively, 3.2 mg, 2.2 mg and 0.5 mg/ml of bed
volume.
Example 19
The procedures of Example 15 were repeated
except that chondroitin polysulfate instead of heparin
was employed to give the desired chondroitin poly-
sulfate-FPG 2000. The amount of immobilized chondroitin
polysulfate was 1.0 mg/ml of bed volume.
Example 20
FPG 2000 was treated in the same manner as in
Example 15 to introduce y-aminopropyl groups into the gel.
The thus obtained activated gel was reacted with sodium
salt of dextran sulfate in the same manner as in Example
10 to give the desired sodium salt of dextran sulfate-FPG
2000. The amount of immobilized sodium salt of dextran
- sulfate was 0.5 mg/ml of bed volume.
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- 20
Example 21
FPG 2000 was refluxed in 10 % solution of
y-glycidoxypropyltrimethoxysilane for 3 hours and then
washed with methanol. The thus obtained activated gel
was reacted with sodium salt of dextran sulfate in the
same manner as in Example 11 except that the reaction was
carried out at pH 8.5 to 9 and at 45C to give the
desired sodium salt of dextran sulfate-FPG 2000.
Example 22
The procedures of Example 11 were repeated
except that sodium salt of glucose sulfate instead of
dextran sulfate was employed to give the desired sodium
salt of glucose sulfate-Cellulofine A-3.
The amount of immobilized sodium salt of
glucose was 1.0 mg/ml of bed volume.
Example 23
The procedures of Example 11 were repeated
except that sodium salt of polyvinyl alcohol sulfate
instead of dextran sulfate was employed to give the
desired sodium salt of polyvinyl alcohol
sulfate-Cellulofine A-3.
The amount of immobilized sodium salt of
polyvinyl alcohol sulfate was 1.5 mg/ml of bed volume.
Test Example 1
Each adsorbent obtained in Examples 1 to 23 was
uniformly packed in a column (internal volume: about 3 ml,
inner diameter: 9 mm, height: 47 mm) and 18 ml of plasma
containing 200 U of heparin was passed through the column
at a flow rate of 0.3 ml/minute with varying the plasma
origins depending on the kind of the desired substance to
be removed. That is, human plasma derived from familial
hypercholesterolemia, normal human plasma, normal human
plasma containing about 100 ~g/ml of a commercially
available endotoxin, human plasma derived from
rheumatism, human plasma derived from systemic lupus
126~355
- 21
erythematosus and human plasma derived from myasthenia
gravis were used, respectively, for the tests of removing
VLDL and/or LDL; IgG, Clq or haptoglobin; endotoxin;
rheumatoid factor; anti-DNA antibody or DNA; and
anti-acetylcholine receptor antibody. The pressure-drop
in the column was 15 mmHg or less throughout the test
period and no crogging was observed. In each adsorbent,
LDL, VLDL, HDL, total protein in plasma which was passed
through the column was determined to obtain a removal
efficiency. The results are summarized in Table 1.
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23
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- 24
Example 24
[Effects of intrinsic viscosity and sulfur
content of dextran sulfate and/or the salt thereof]
Cellulofine A-3 was treated in the sa~e manner
s as in Example 5 to introduce epoxy groups into thé gel.
The thus obtained epoxy-activated gel was reacted with
each sodium salt of dextran sulfate having the intrinsic
viscosity and sulfur content shown in the following Table
2 (Run Nos. tl) to (7)) in the same manner as in Example
10 11.
One ml portion of the resulting each adsorbent
was packed in a column, and then 6 ml of human plasma
containing 300 mg/dl of total cholesterol derived from a
familial hypercholesterolemia patient was passed through
the column at a flow rate of 0.3 ml/minute. The removal
efficiency for LDL was determined from the amount of
adsorbed LDL measured by using the total amount of
cholesterol as an indication. That is, the amount of
cholesterol in the human plasma used was mostly derived
from LDL. The results are shown in Table 2.
1266855
-- 25
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- 26
Example 25
[Effect of amount of epoxy group introduced]
Toyopearl 65 was treated in the same manner as
in Example l to introduce epoxy groups into the gel and
CSKA-3 (a cmmercially available porous cellulose gel
made by Chisso Corporation, exclusion limit: 5 x 107,
particle size: 45 to 105 ~m) having a uniform structure
was treated in the same manner as in Example 5 to
introduce epoxy groups into the gel. The amounts of
epoxy groups introduced were, respectively, 250 ~moles
and 30 ~moles/ml of bed volume.
Each gel was reacted with sodium salt of
dextran sulfate ~intrinsic viscosity: 0.027 dl/g, sulfur
content: 17.7 % by weight) in the same manner as in
Example ll except that the conce~tration of sodium salt
of dextran sulfate based on the weight of the whole
reaction system excludig the dry weight of the gel was
charged.
The thus obtained adsorbent was subjected to
the determination of removal efficiency for LDL in the
~ same manner as in Example 24. The results are summarized
: in Table 3.
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~266855
-- 27
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6~355
- 28
xample 26
One ml portion of the adsorbent obtained in
Example 24 of Run No. (3) was uniformly packed in a
column having an internal volume of 1 ml, and 6 ml of
normal human plasma containing LDL and HDL cholesterol in
the ratio of approximately l : l was passed through the
column. LDL in the plasma passed through the column was
greatly reduced, while HDL was scarcely reduced.
Example 27
One ml portion of the adsorbent obtained in
Example 24 of Run No. (3) was uniformly packed in a
column having an internal volume of l ml, and 6 ml of
normal rabbit plasma containing lipoproteins of VLDL, LDL
and HDL was passed through the column. The plasma
obtained before and after the column treatment were,
respectively, examined by polyacrylamide disc gel
electrophoresis. The results are shown in Fig. 3. In
Fig. 3, curves A and B show, respectively, the results
obtained before and after the column treatment. The axis
of ordinates indicates the absorbance at 570 nm and the
axis of abscissas indicates the migration positions at
which bands of VLDL, LDL and HDL were, respectively
appeared.
As shown in Fig. 3, VLDL and LDL were
significantly adsorbed, while HDL was not.
Example 28
The adsorbents obtained in Examples 1 to 7 and
30 11 to 14 were sterilized in an autoclave at 120C for 15
minutes. Each resulting sterilized adsorbent was
subjected to the determination of removal efficiency for
LDL in the same manner as in Test Example 1. As a
result, the removal efficiencies were not inferior to
those obtained without sterilizing by autoclaving. In
addition, pressure-drop was not changed.
