Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Crosslinked Polyallylamine or Acid Addition Salt Thereof, and Use Thereof for
Medical Purposes
TECHNICAL FIELD
[0001]
The present invention relates to a cross-linked polyallylamine or an acid
addition salt thereof, and medical use thereof.
BACKGROUND ART
[0002]
Patients with renal function impairment often suffer from hyperphosphatemia
due to decreased phosphate excretion. Hyperphosphatemia causes serious
abnormality in metabolism of calcium and phosphate, leading to decreased serum
calcium, promotion of production and secretion of PTH, ectopic calcification,
and
renal osteodystrophy due to inhibition of vitamin D activation. Even after
starting
dialysis because of renal failure, the above-described disease state continues
unless
the homeostasis of phosphate is maintained. Thus, therapy of hyperphosphatemia
is
indispensable for patients with renal failure who are dialyzed or not
dialyzed. At
present, therapy of hyperphosphatemia is carried out by diet therapy, or
pharmacotherapy by an oral phosphate adsorption agent. However, it is thought
that diet therapy is not sufficient for amelioration of hyperphosphatemia and
that
therapy for these patients requires use of a phosphate adsorption agent.
[0003]
Examples of conventional phosphate adsorption agents which have been
widely used include inorganic salts such as aluminum salts and calcium salts.
Ingested aluminum salts and calcium salts bind to phosphate in the intestine
to form
insoluble phosphates, which inhibit absorption of phosphate. However,
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administration of aluminum salts causes accumulation of aluminum, leading to
brain
diseases, osteomalacia and the like, which is problematic. Further,
administration
of calcium salts causes hypercalcemia, leading to early death of patients by
calcification of the aorta and ectopic calcification, which is problematic.
Recently,
lanthanum carbonate which is one of lanthanum salts has been placed on the
market.
However, since intestinal absorption and accumulation of lanthanum has been
observed, there is a concern about the safety aspect during chronic
administration.
[0004]
Recently, as an oral phosphate adsorption agent, sevelamer hydrochloride, a
polyallylamine which is an organic polymer has been placed on the market.
Since
sevelamer hydrochloride does not cause the side effects observed upon
administration of the above-described inorganic salts, it is widely used for
therapy of
hyperphosphatemia. Sevelamer hydrochloride is a
poly(allylamine/epichlorohydrin) and a production method thereof includes the
steps
of polymerizing allylamine hydrochloride to obtain polyallylamine
hydrochloride
and subsequent cross-linking by reacting the product with epichlorohydrin in
an
aqueous sodium hydroxide solution (Patent Literature 1).
[0005]
However, sevelamer hydrochloride is said to require administration at a high
dose in order to remarkably reduce phosphate absorption. Further, since
sevelamer
hydrochloride absorbs water in the gastrointestinal tract, it causes side
effects such as
constipation, abdominal pain and abdominal distension, which is problematic.
It is
also reported that such side effects may lead to serious side effects such as
intestinal
perforation and intestinal obstruction (Non-patent Literature 1). Further, the
expression frequencies of side effects are known to be dose-dependent (Non-
patent
Literature 2). Since, in sevelamer hydrochloride, there exist these side
effects
caused by swelling, administration thereof at an amount required for
sufficient
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inhibition of phosphate absorption is often difficult, and it needs to be used
in
combination with a calcium formulation at present. Therefore, development of
an
oral phosphate adsorption agent having a lower degree of swelling, which
replaces
sevelamer hydrochloride is demanded (Non-patent Literature 3).
[0006]
Further, the ion-exchange rate of an ion-exchange resin such as a
polyallylamine decreases and the phosphate adsorption capability thereof
decreases
in cases where the degree of swelling is low, so that it is considered to be
difficult to
achieve a low degree of swelling while maintaining a high phosphate adsorption
capacity. Further, in addition to phosphate, there exist acids such as bile
acids in
the intestine, and these are known to compete with each other in terms of
adsorption
to polyallylamine and the like.
[0007]
Since sevelamer hydrochloride has a lower selectivity to phosphate than to
bile acids, there is a possibility that not only its phosphate adsorption
capacity
decreases, but also it causes uptake inhibition of fat-soluble vitamins due to
adsorption of bile acid in the intestine (Non-patent Literature 1). Therefore,
development of an oral phosphate adsorption agent having a higher phosphate
selectivity, which replaces sevelamer hydrochloride is demanded (Non-patent
Literature 3).
[0008]
By the way, reported examples of the production method of a cross-linked
polyallylamine include, other than a method wherein a polyallylamine is
synthesized
first and reacted with a compound which is capable of reacting with amino
groups of
the polyallylamine at multiple sites, as in the method of producing sevelamer
hydrochloride, a method wherein an allylamine salt is copolymerized with a
polyfunctional monomer (Patent Literature 2).
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[0009]
However, in the method described in Patent Literature 2, the amount of the
polyfunctional allylamine derivative to be added is described to be not more
than 2
mol% with respect to the allylamine salt, and the cross-linked polyallylamine
obtained in the Examples was soluble in water, so that it is not appropriate
for use as
an oral phosphate adsorption agent. Further, there is no description on the
degree of
swelling of the obtained cross-linked polyallylamine, and no attempt to
decrease the
degree of swelling was made at all. Further, in the Examples, there are only
examples using an allylamine hydrochloride, and differences in properties of
the
polymers depending on the types of the acids used in the polymerization have
not
been studied. Thus, it is said that the cross-linked polyallylamine described
in
Patent Literature 2 cannot be used as an oral phosphate adsorption agent
having a
low degree of swelling.
[0010]
In Patent Literature 3, there is a report on a production method of a
polyallylamine wherein, in the synthesis of its homopolymer, a phosphate of
allylamine is used as a monomer for polymerization. However, in this report,
there
is no description on synthesis of a cross-linked polyallylamine by cross-
linking
polymerization.
[0011]
In Patent Literature 4, there is a report on a phosphate adsorption agent
produced using the so-called molecular imprinting, wherein the polymerization
is
carried out in the presence of phosphate to obtain a polymer which was given
an
affinity to phosphate ions. In this method, a porous phosphate-imprinted
polymer
was obtained by carrying out polymerization after mixing monomers with
potassium
dihydrogenphosphate in the presence of a diluent such as 2-propanol. However,
in
this report, only methods using as the monomers an acrylic acid derivative are
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disclosed, without disclosing methods using allylamine. Further, the above-
described porous phosphate-imprinted polymer is inferior to sevelamer
hydrochloride in terms of the phosphate adsorption capacity; its selectivities
to other
ions such as bile acids are not investigated; and its volume increases about
tenfold
5 upon swelling; so that it is said that it cannot be used as an oral
phosphate adsorption
agent having a low degree of swelling.
[0012]
In Patent Literature 5, as a method to obtain a polymer having a low degree of
swelling, cross-linking of the surfaces of particles of a cross-linked polymer
is
reported. However, the method described in Patent Literature 5 is a disclosure
about a water-absorbing polymer, and not a disclosure about a polymer which
exerts
the phosphate adsorption capability and, at the same time, has a low degree of
swelling.
[0013]
Patent Literature 1: JP 3113283 B
Patent Literature 2: JP 10-330427 A
Patent Literature 3: JP 2-14364 B
Patent Literature 4: US 2005/0276781 Al
Patent Literature 5: JP 11-302391 A
Non-patent Literature 1: Drug Interview Form: Renagel (registered trademark)
Tablet 250 mg, 8th Revised Edition, 2005, p. 21
,
Non-patent Literature 2: Review Report, Notification No. 3850 of National
Institute
of Health Sciences (Nov. 28, 2002)
Non-patent Literature 3: Hiroyoshi INOUE, "Phosphate excretion
enhancers/absorption inhibitors in the future", Kidney and Dialysis, 2003,
Vol. 55, p.
941-944
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DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0014]
In view of this, the present invention aims to provide a cross-linked
polyallylamine or an acid addition salt thereof, which has both a high
phosphate
adsorption capacity and a low degree of swelling, and a medical use thereof.
MEANS FOR SOLVING THE PROBLEMS
[0015]
The present inventors intensively studied on an oral phosphate adsorption
agent to discover a cross-linked polyallylamine and an acid addition salt
thereof
having a low degree of swelling and a high phosphate adsorption capacity, as a
result
of copolymerization of allylammonium dihydrogen phosphate with an acid
addition
salt of N,N-diallyl-l,3-diaminopropane as a cross-linking agent in an amount
of 5 to
mol% with respect to the amount of the allylammonium dihydrogen phosphate.
[0016]
That is, the present invention provides a cross-linked polyallylamine or an
acid addition salt thereof which is obtained by copolymerization of
allylammonium
20 dihydrogen phosphate with an acid addition salt of N,N-diallyl-1,3-
diaminopropane
in an amount of 5 to 25 mol% with respect to the amount of the allylammonium
dihydrogen phosphate, the cross-linked polyallylamine or an acid addition salt
thereof having:
a phosphate adsorption capacity of 2.7 to 5.0 mmol/g; and
25 a degree of swelling of 2.0 to 5Ø
[0017]
The phosphate adsorption capacity of the above-described cross-linked
polyallylamine or an acid addition salt thereof is preferably 2.7 to 4.8
mmol/g, and
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more preferably 2.7 to 4.5 mmol/g.
[0018]
The above-described cross-linked polyallylamine or an acid addition salt
thereof is preferably given surface cross-linking treatment by reacting a
compound
having 2 or more amino group-reactive functional groups with an amino group.
[0019]
The above-described compound having 2 or more amino group-reactive
functional groups is preferably an acrylic acid ester, methacrylic acid ester,
epihalohydrin, dihalogenated hydrocarbon, diepoxide or dibasic acid chloride,
more
preferably an acrylic acid ester.
[0020]
The acid addition salt of the above-described N,N-diallyl-1,3-
diaminopropane is preferably N,1V'-diallyl-1,3-diaminopropane bis(dihydrogen
phosphate).
[00211
The present invention also provides a pharmaceutical composition comprising
as an effective ingredient the above-described cross-linked polyallylamine or
an acid
addition salt thereof, and further provides a therapeutic or prophylactic
agent for
hyperphosphatemia, comprising as an effective ingredient the above-described
cross-
linked polyallylamine or an acid addition salt thereof. The present invention
further
provides a therapeutic or prophylactic method for hyperphosphatemia,
comprising
administration of an effective amount of the above-described cross-linked
polyallylamine or an acid addition salt thereof to a patient for whom therapy
or
prophylaxis of hyperphosphatemia is desired. The present invention still
further
provides use of the above-described cross-linked polyallylamine or an acid
addition
salt thereof, for the production of a therapeutic or prophylactic agent for
hyperphosphatemia. The present invention still further provides a compound for
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therapy or prophylaxis of hyperphosphatemia, which is the above-described
cross-
linked polyallylamine or an acid addition salt thereof.
EFFECT OF THE INVENTION
[0022]
According to the present invention, a cross-linked polyallylamine or an acid
addition salt thereof, which has both a high phosphate adsorption capability
and a
low degree of swelling can be provided. Further, the cross-linked
polyallylamine or
the acid addition salt thereof of the present invention has a high phosphate
selectivity
and a phosphate adsorption capability not less than that of sevelamer
hydrochloride,
and its degree of swelling is remarkably lower than that of sevelamer
hydrochloride,
so that, in cases where it is used as a pharmaceutical agent, especially as a
therapeutic or prophylactic agent for hyperphosphatemia, administration
thereof at a
high dose which is required for sufficient inhibition of phosphate absorption
is
possible and a therapeutic and prophylactic effect can be observed without
using a
calcium formulation or the like in combination, while reducing side effects
such as
constipation, abdominal pain and abdominal distension.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023]
Fig. 1 shows results of the urinary phosphate excretion test in Example 5.
Fig. 2 shows results of the urinary phosphate excretion test in Example 6.
Fig. 3 shows results of the urinary phosphate excretion test in Example 22.
Fig. 4 shows results of the urinary phosphate excretion test in Example 23.
Fig. 5 shows results of the urinary phosphate excretion test in Example 24.
Fig. 6 shows results of the test for adhesion to the intestinal tract in
Example
25.
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Fig. 7 shows results of the test for adhesion to the intestinal tract in
Example
26.
BEST MODE FOR CARRYING OUT THE INVENTION
[0024]
The cross-linked polyallylamine of the present invention or an acid addition
salt thereof is obtained by copolymerization of allylammonium dihydrogen
phosphate with an acid addition salt ofN,N-diallyl-1,3-diaminopropane in an
amount
of 5 to 25 mol% with respect to the amount of the allylammonium dihydrogen
phosphate, the cross-linked polyallylamine or an acid addition salt thereof
having:
a phosphate adsorption capacity of 2.7 to 5.0 mmol/g; and
a degree of swelling of 2.0 to 5Ø
[0025]
To obtain the above-described cross-linked polyallylamine or an acid addition
salt thereof, allylammonium dihydrogen phosphate is used as a monomer. To
maintain a high phosphate adsorption capacity while suppressing the swelling,
it is
important to use as the monomer allylammonium dihydrogen phosphate which is
the
phosphate of allylamine. This is because, for example, in cases where
allylammonium chloride is used as the monomer, the phosphate adsorption
capacity
of the obtained cross-linked polyallylamine or an acid addition salt thereof
is very
low. Allylammonium dihydrogen phosphate may be prepared in advance from
allylamine and phosphoric acid, or allylamine and phosphoric acid may be mixed
in
a reaction vessel as they are to carry out polymerization reaction.
[0026]
The above-described cross-linking agent means a polyfunctional allylamine
derivative having 2 or more functional groups capable of copolymerizing with
allylammonium dihydrogen phosphate.
[0027]
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To obtain the above-described cross-linked polyallylamine or an acid addition
salt thereof, an acid addition salt of an N,N-diallyl-substituted
alkylenediamine is
used as the cross-linking agent. To obtain a cross-linked polyallylamine or an
acid
addition salt thereof having a low degree of swelling and a high phosphate
adsorption
5 capacity, it is important to use as the cross-linking agent an acid addition
salt of N,N-
diallyl-1,3-diaminopropane. For example, in cases where N,N-diallyl-1,2-
diaminoethane, N,N-diallyl-1,4-diaminobutane or N,N-diallyl-1,5-diaminopentane
is
used, the phosphate adsorption capacity of the obtained polymer is low or the
degree
of swelling thereof is high.
10 [0028]
Examples of the acid added to the above-described N,N-diallyl-l,3-
diaminopropane include inorganic acids such as hydrochloric acid, phosphoric
acid
and sulfuric acid; and organic acids such as formic acid and acetic acid.
Hydrochloric acid, phosphoric acid and sulfuric acid are preferred, and
phosphoric
acid is most preferred. The acid addition salt of N,N-diallyl-1,3-
diaminopropane
may be prepared in advance from N,N-diallyl-1,3-diaminopropane and an acid, or
N,N-diallyl-1,3-diaminopropane and the acid may be mixed in a reaction vessel
as
they are to carry out polymerization reaction.
[0029]
The amount of the above-described acid addition salt of N,N-diallyl-l,3-
diaminopropane to be added is 5 to 25 mol%, more preferably 10 to 25 mol% and
most preferably 15 to 25 mol% with respect to the amount of the monomer,
allylammonium dihydrogen phosphate. With the amount of less than 5 mol%, the
degree of swelling of the obtained polymer is high, so that the obtained
polymer is
not preferably used as an oral phosphate adsorption agent. In cases where the
amount of the cross-linking agent to be added is 5 to 25 mol%, a polymer
having a
degree of swelling of not more than 5.0 is obtained, which degree of swelling
is
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much lower than 6.2, the degree of swelling of sevelamer hydrochloride.
[0030]
The phosphate adsorption capacity of the above-described cross-linked
polyallylamine or an acid addition salt thereof is not less than 2.7 mmol/g,
preferably
not less than 2.8 mmol/g, more preferably not less than 3.0 mmol/g and still
more
preferably not less than 4.0 mmol/g. The upper limit of the phosphate
adsorption
capacity is 5.0 mmol/g, preferably 4.8 mmol/g and more preferably 4.5 mmol/g.
As
indicated in Examples, since the maximum phosphate adsorption capacity of
sevelamer hydrochloride is 2.7 mmol/g, the above-described cross-linked
polyallylamine or an acid addition salt thereof has a phosphate adsorption
capacity
not less than that of sevelamer hydrochloride. The phosphate adsorption
capacity
means the amount of phosphate ions removed by adsorption to a sample to be
measured in a test solution containing phosphate and glycocholate in a molar
ratio of
1:1. More particularly, the measurement is carried out as follows. That is, a
sample to be measured is stirred in 50 mM hydrochloric acid at 37 C for 1
hour, and
each of disodium hydrogen phosphate dodecahydrate and an aqueous sodium
glycocholate solution is added to a final concentration of 10 mM, such that
the final
concentration of the sample to be measured becomes 1 mg/mL in the solution.
After 1 hour of stirring at 37 C, centrifugation (refrigerated centrifuge
5417R
manufactured by Eppendorf, angle rotor FA-45-24-1 1) is carried out at
conditions of
15,000 rpm, 25 C and 15 minutes to remove the sample to be measured, and the
amount of phosphate which was not adsorbed to the sample to be measured is
measured (n=3) using an inorganic phosphate measurement reagent (manufactured
by Wako Pure Chemical Industries; Phosphor C (registered trademark)), to
determine
the amount of the phosphate ions adsorbed to the sample to be measured, that
is, the
phosphate adsorption capacity (a calibration curve was prepared within the
range of
the inorganic phosphate concentration of 0 to 386 mg/mL; the spectrophotometer
is
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Spectra Max Plus manufactured by Molecular Devices). In the assay method
described in the above-described Patent Literature 1(JP 3113283 A), the
phosphate
adsorption capacity was measured in the presence of phosphate alone without
adding
a bile acid such as glycocholate, but, since bile acids represented by
glycocholate are
present in large amounts in the intestine of the body and compete with
phosphate in
terms of adsorption, the assay in the present invention is made based on the
phosphate adsorption capacity under the bile acid-competing condition as
described
above.
[0031]
The cross-linked polyallylamine or the acid addition salt thereof of the
present invention has a degree of swelling of 2.0 to 5Ø In cases where the
degree
of swelling is higher than 5.0, there is a possibility that the risk of side
effects due to
swelling is high. The range of the degree of swelling is preferably 2.0 to
4.5, more
preferably 2.0 to 4.0, still more preferably 2.0 to 3.5. Since the degree of
swelling
of sevelamer hydrochloride is 6.2 as indicated in the Examples, less side
effects due
to swelling are expected in the cross-linked polyallylamine or the acid
addition salt
thereof of the present invention than sevelamer hydrochloride. The degree of
swelling means a value obtained by soaking 200 mg of a sample to be measured,
which was dried under reduced pressure at 40 C for not less than 16 hours, in
50 mL
of distilled water for not less than 24 hours and filtrating the resultant
using a
membrane filter (Omnipore, manufactured by Millipore) having a diameter of 47
mm
and a pore size of 0.45 m under reduced pressure to separate the solid
component,
whose weight is then divided by the dry weight (200 mg).
[0032]
To further decrease the degree of swelling of the cross-linked polyallylamine
or the acid addition salt thereof of the present invention, there is a method
to increase
the crosslink density in the vicinity of the surfaces of particles of the
cross-linked
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polyallylamine or an acid addition salt thereof by a surface cross-linking
treatment
wherein a compound having 2 or more amino group-reactive functional groups
(hereinafter referred to as "surface cross-linking agent") is allowed to react
with
amino groups of the cross-linked polyallylamine or an acid addition salt
thereof.
[0033]
Examples of the surface cross-linking agent used in the present invention
include acrylic acid esters, methacrylic acid esters, epihalohydrins,
dihalogenated
hydrocarbons, diepoxides and dibasic acid chlorides. Acrylic acid esters,
epihalohydrins and dihalogenated hydrocarbons are preferred; acrylic acid
esters and
epihalohydrins are more preferred; and acrylic acid esters are most preferred.
[0034]
Examples of the acrylic acid esters include methyl acrylate, ethyl acrylate,
propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, sec-
butyl
acrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate and glycidyl
acrylate.
Methyl acrylate, ethyl acrylate and 2-hydroxyethyl acrylate are preferred;
methyl
acrylate and 2-hydroxyethyl acrylate are more preferred; and 2-hydroxyethyl
acrylate
is most preferred.
[0035]
Examples of the methacrylic acid esters include methyl methacrylate, ethyl
methacrylate, propyl methacrylate, isopropyl methacrylate, butyl methacrylate,
isobutyl methacrylate, sec-butyl methacrylate, 2-hydroxyethyl methacrylate, 2-
hydroxypropyl methacrylate and glycidyl methacrylate.
[0036]
Examples of the epihalohydrins include epichlorohydrin and epibromohydrin;
and epichlorohydrin is preferred.
[0037]
Examples of the dihalogenated hydrocarbons include 1,2-dichloroethane, 1,2-
dibromoethane, 1,3-dichloropropane, 1,3-dibromopropane, 1,4-dichlorobutane,
1,4-
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dibromobutane, 1,4-dichloro-2-butene and 3,4-dichloro-l-butene.
[0038]
Examples of the diepoxides include 1,2,3,4-diepoxybutane, 1,2-ethanediol
diglycidyl ether and 1,4-butanediol diglycidyl ether.
[0039]
Examples of the dibasic acid chlorides include oxalyl chloride, malonyl
chloride, succinyl chloride, glutaryl chloride and adipoyl chloride.
[0040]
The cross-linked polyallylamine or an acid addition salt thereof obtained in
the present invention by surface cross-linking has a phosphate adsorption
capacity of
not less than 2.7 mmol/g, preferably not less than 2.8 mmol/g, more preferably
not
less than 3.0 mmol/g, still more preferably not less than 4.0 mmol/g. The
upper
limit of the phosphate adsorption capacity is 5.0 mmol/g, preferably 4.8
mmol/g,
more preferably 4.5 mmol/g. As indicated in the Examples, since the phosphate
adsorption capacity of sevelamer hydrochloride is 2.7 mmol/g, the cross-linked
polyallylamine or an acid addition salt thereof obtained in the present
invention by
surface cross-linking has a phosphate adsorption capacity not less than that
of
sevelamer hydrochloride.
[0041]
The cross-linked polyallylamine or an acid addition salt thereof obtained in
the present invention by surface cross-linking has a degree of swelling of 2.0
to 5Ø
In cases where the degree of swelling is higher than 5.0, there is a
possibility that the
risk of side effects due to swelling is high. The range of the degree of
swelling is
preferably 2.0 to 4.5, more preferably 2.0 to 4.0, still more preferably 2.0
to 3.5.
Since the degree of swelling decreases by surface cross-linking, the side
effects are
considered to be reduced.
[0042]
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A higher selectivity to phosphate than to bile acids is one of the
characteristics of the cross-linked polyallylamine or the acid addition salt
thereof of
the present invention. The phosphate selectivity is preferably not less than
1.5,
more preferably not less than 2.0, still more preferably not less than 2.5.
The upper
5 limit is not limited, and a larger value is preferred, but it is not more
than 10 in the
present invention. The phosphate selectivity in the present invention means a
value
obtained by dividing the phosphate adsorption capacity by the bile acid
adsorption
capacity. The bile acid adsorption capacity means the amount of glycocholate
ions
removed by adsorption to a sample to be measured in a test solution containing
10 phosphate and glycocholate in a molar ratio of 1:1. More particularly, the
measurement is carried out as follows. A sample to be measured is stirred in
50
mM hydrochloric acid at 37 C for 1 hour, and each of disodium hydrogen
phosphate
dodecahydrate and an aqueous sodium glycocholate solution is added to a final
concentration of 10 mM, such that the final concentration of the sample to be
15 measured becomes 1 mg/mL in the solution. After 1 hour of stirring at 37 C,
centrifugation (refrigerated centrifuge 5417R manufactured by Eppendorf, angle
rotor FA-45-24-11) is carried out at conditions of 15,000 rpm, 25 C and 15
minutes
to remove the sample to be measured, and the amount of glycocholate which was
not
adsorbed to the sample to be measured is measured (n=3) using a bile acid
measurement reagent (manufactured by Wako Pure Chemical Industries; Total Bile
Acids (registered trademark)), to determine from the measured value the amount
of
the phosphate ions adsorbed to the sample to be measured, that is, the
phosphate
adsorption capacity (a calibration curve was prepared within the range of the
inorganic phosphorus concentration of 0 to 386 mg/mL; the spectrophotometer is
Spectra Max Plus manufactured by Molecular Devices).
[0043]
Thus, the cross-linked polyallylamine or the acid addition salt thereof of the
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present invention has excellent characteristics as an oral phosphate
adsorption agent,
which cannot be obtained by the method described in the above-described Patent
Literature 2 (JP 10-330427 A). Further, since it has a higher phosphate
adsorption
capacity and a lower degree of swelling than sevelamer hydrochloride, side
effects
caused by excessive swelling which are problematic in a medical use of
sevelamer
hydrochloride can be reduced. The side effect-reducing effect can be confirmed
by
observing in an animal experiment the moving speed of the content of the
intestinal
tract, excretion speed thereof, and the amount thereof adhered to the
intestinal tract.
[0044]
Further, the cross-linked polyallylamine or the acid addition salt thereof of
the present invention causes less adherence to the intestinal tract than
sevelamer
hydrochloride. Since adherence of a polymer or the like to the intestinal
tract may
be a factor inhibiting the movement of the intestine, it is thought that side
effects of
the gastrointestinal tract such as constipation are smaller in the cross-
linked
polyallylamine or the acid addition salt thereof of the present invention than
in
sevelamer hydrochloride.
[0045]
Examples of a method which can be used for polymerization for production
of the cross-linked polyallylamine or the acid addition salt thereof of the
present
invention include known methods such as solution polymerization, reversed
phase
suspension polymerization, emulsion polymerization and precipitation
polymerization. Examples of the solvent in which monomers are allowed to
dissolve upon the polymerization include water; aqueous solutions of inorganic
acids
such as hydrochloric acid, phosphoric acid and sulfuric acid, and organic
acids such
as formic acid and acetic acid; polar solvents such as methanol, ethanol, 1-
propanol,
2-propanol, N,N-dimethylformamide and dimethylsulfoxide; and mixed solvents
wherein 2 or more of these solvents are arbitrarily mixed, and, among these,
water
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and mixed solvents of water and a polar solvent are preferred. The amount of
the
solvent is preferably 0.2 mL to 3.0 mL, more preferably 0.3 mL to 2.0 mL, most
preferably 0.3 mL to 1.5 mL with respect to 1 g of the total weight of monomer
allylammonium dihydrogen phosphate and the cross-linking agent.
[0046]
In the polymerization condition of a two-phase system such as reversed phase
suspension polymerization or emulsion polymerization, known organic solvents
can
be used as a dispersion medium which disperses the monomer solution, and
examples thereof include hexane, cyclohexane, heptane, octane, decane,
petroleum
ether, liquid paraffin, ethyl acetate, propyl acetate and isopropyl acetate;
and mixed
solvents wherein 2 or more of these solvents are arbitrarily mixed.
Cyclohexane,
heptane and octane are preferred; heptane and octane are more preferred; and
heptane is most preferred.
[0047]
In the polymerization condition of a two-phase system such as reversed phase
suspension polymerization and emulsion polymerization, a surfactant is added
as
required. Examples of the surfactant which may be used include sorbitan
monolaurate, sorbitan monooleate, sorbitan monostearate, sorbitan
monopalmitate,
ethylene glycol monostearate, glyceryl monostearate, polyethylene glycol
monostearate, polyethylene glycol hydrogenated castor oil, polyoxyethylene
sorbitan
monolaurate, polyoxyethylene sorbitan monooleate, polyethylene glycol and
diisooctyl sulfosuccinate. Sorbitan monolaurate, sorbitan monooleate, sorbitan
monostearate and sorbitan monopalmitate are more preferred; and sorbitan
monolaurate is most preferred.
[0048]
To form macropores in the obtained polymer, a diluent such as methanol,
ethanol, 1, 1 -dimethylethanol, octanol, 2-propanol and hexane may be added,
but
CA 02692515 2010-01-04
18
addition of the above-described diluent is not preferred.
[0049]
As the polymerization initiator, an azo radical initiator is used. Examples of
the azo radical initiator which may be used include known azo radical
initiators such
as 2-cyano-2-propylazoformamide, 1,1'-azobis(cyclohexane-l-carbonitrile), 2,2'-
azobis(2-amidinopropane) dihydrochloride, 2,2'-azobis(2-methylbutyronitrile),
2,2'-
azobisisobutyronitrile, 2,2' -azobis(2,4-dimethylvaleronitrile), 2,2' -
azobis(4-
methoxy-2,4-dimethylvaleronitrile), 4,4'-azobis(4-cyanovaleric acid), dimethyl
2,2'-
azobisisobutyrate, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride,
2,2'-
azobis[2-(2-imidazolin-2-yl)propane] disulfate dihydrate, 2,2'-azobis[N-(2-
carboxyethyl)-2-methylpropionamide], 2,2'-azobis {2-[ 1-(2-hydroxyethyl)-2-
imidazolin-2-yl]propane } dihydrochloride, 2,2' -azobis[2-(2-imidazolin-2-
yl)propane], 2,2'-azobis(1-imino-1-pyrrolidino-2-methylpropane)
dihydrochloride,
2,2'-azobis {2-methyl-N-[ 1,1-bis(hydroxymethyl)-2-hydroxyethyl]propionamide
},
2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis(N-butyl-2-
methyipropionamide), 2,2'-azobis(N-cyclohexyl-2-methylpropionamide), dimethyl
1,1' -azobis(1-cyclohexanecarboxylate), 2,2' -azobis[N-(2-propenyl)-2-
methylpropionamide] and 2,2'-azobis(2,4,4-trimethylpentane). 2,2'-azobis(2-
amidinopropane)dihydrochloride, 4,4'-azobis(4-cyanovaleric acid), 2,2'-
azobis[2-(2-
2 0 imidazolin-2-yl)propane] dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-
yl)propane]
disulfate dihydrate, 2,2'-azobis{2-[1-(2-hydroxyethyl)-2-imidazolin-2-
yl]propane}
dihydrochloride and 2,2' -azobis(1-imino-1-pyrrolidino-2-methylpropane)
dihydrochloride are preferred; 2,2'-azobis(2-amidinopropane) dihydrochloride,
2,2'-
azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride and 2,2'-azobis[2-(2-
2 5 imidazolin-2-yl)propane] disulfate dihydrate are more preferred; and 2,2'-
azobis(2-
amidinopropane) dihydrochloride and 2,2'-azobis[2-(2-imidazolin-2-yl)propane]
dihydrochloride are most preferred.
CA 02692515 2010-01-04
19
[0050]
The amount of the initiator to be added is preferably 0.1 mol% to 10 mol%,
more preferably 0.5 mol% to 8 mol%, most preferably 0.7 mol% to 5 mol% with
respect to the total number of moles of the monomers and the cross-linking
agent.
[0051]
The polymerization temperature is preferably 30 C to 100 C, more preferably
40 C to 80 C, most preferably 40 C to 70 C. The polymerization time varies
depending on the polymerization temperature and the like, and not more than
100
hours is usually sufficient.
[0052]
By allowing an acid addition salt ofN,N-diallyl-1,3-diaminopropane having
two terminal double bonds to coexist when the radical polymerization of
allylammonium dihydrogen phosphate is carried out, the polymer strand is
elongated
at these both termini to form a cross-linking structure. During this, in a
part of the
cross-linking agent, one of the double bonds may not react and remain as the
double
bond without being involved in cross-linking.
[0053]
When the production is carried out as described above, the cross-linked
polyallylamine or the acid addition salt thereof of the present invention
having a
phosphate adsorption capacity and a degree of swelling within the above-
described
ranges can be obtained. However, depending on reaction conditions
(concentration
of the reactant, reaction temperature, reaction time and the like) used,
properties
(phosphate adsorption capacity, degree of swelling and phosphate selectivity)
of the
obtained cross-linked polyallylamine polymer may not fall within the above-
described ranges. In this case, the properties can be adjusted to fall within
the
above-described ranges by changing reaction conditions as appropriate.
Further,
even in cases where the properties of the obtained cross-linked polyallylamine
polymer are within the above-described ranges, reaction conditions may be
changed
in order to obtain a cross-linked polyallylamine polymer having better
properties.
The relationship between reaction conditions and properties of the obtained
cross-
CA 02692515 2010-01-04
linked polymer is shown in Example 1 below. Concrete examples of the method
for
changing reaction conditions for adjustment of the properties include, but are
not
limited to: (1) increase of the concentration of the reactant during
polymerization
reaction (decrease of the solvent volume); (2) lowering of the polymerization
5 temperature and extension of the reaction time; (3) use of a larger amount
of the
radical initiator. In cases where (1) is carried out, the phosphate adsorption
capacity
of the obtained cross-linked polyallylamine polymer slightly decreases, but
the
degree of swelling decreases, which is preferred. In cases where (2) is
carried out,
the phosphate adsorption capacity slightly decreases, but the phosphate
selectivity is
10 improved and the degree of swelling decreases, which are preferred. In
cases where
(3) is carried out, the phosphate adsorption capacity slightly decreases, but
the degree
of swelling decreases, which is preferred. Further, in cases where the
purities of the
monomers and the initiator are increased, the radical termination reaction is
suppressed, so that the degree of swelling decreases. Further, in cases where
the
15 diameter of the obtained cross-linked polyallylamine or an acid addition
salt thereof
is decreased, the phosphate adsorption capacity is improved.
[0054]
Since the cross-linked polyallylamine obtained by the polymerization reaction
is a phosphate, it needs to be converted to the free form or another acid
addition salt
20 in order to use it as a phosphate adsorption agent. The conversion to the
free form
is carried out by washing with water or neutralization. Examples of the alkali
used
for the neutralization include inorganic bases such as sodium hydroxide,
potassium
hydroxide, lithium hydroxide, barium hydroxide, sodium hydrogen carbonate,
potassium carbonate and ammonia; and metal alkoxides such as sodium methoxide,
sodium ethoxide and potassium tert-butoxide.
[0055]
In cases where the free-form cross-linked polyallylamine is converted to an
acid addition salt, a pharmaceutically acceptable acid other than phosphoric
acid can
be used, and examples thereof include hydrochloric acid, hydrobromic acid,
hydroiodic acid, sulfuric acid, nitric acid, carbonic acid, acetic acid,
benzoic acid,
CA 02692515 2010-01-04
21
succinic acid, tartaric acid and citric acid. Hydrochloric acid, carbonic acid
and
acetic acid are more preferred; and hydrochloric acid and acetic acid are most
preferred. The amount of the acid may be an arbitrary amount not more than 1
equivalent with respect to the amino group of the cross-linked polyallylamine,
and
0.1 equivalent to 0.8 equivalent is more preferred, and 0.1 equivalent to 0.5
equivalent is most preferred. Further, by allowing an excess amount of the
acid to
act on the phosphate of the cross-linked polyallylamine obtained by the
polymerization, it can be converted to the acid addition salt of interest
without
conversion to the free form. Conversion to the free form is preferably carried
out
by a method wherein the phosphate of the polymer is allowed to react with an
aqueous sodium hydroxide solution and the phosphate is removed by filtration,
followed by stirring in water, washing and filtration. The cross-linked
polyallylamine may also be used as the free form without conversion to an acid
addition salt.
[0056]
The reaction of the surface cross-linking in the present invention is carried
out with a cross-linked polyallylamine or an acid addition salt thereof
dispersed in
the solvent, and the reaction is preferably carried out with a cross-linked
polyallylamine of the free form.
[0057]
The amount of the surface cross-linking agent to be added is selected as
appropriate within the range of 1.0% by weight to 50% by weight with respect
to the
weight of the cross-linked polyallylamine or an acid addition salt thereof to
be
subjected to surface cross-linking. The amount of 1.5% by weight to 40% by
weight is preferred, 2.5% by weight to 20% by weight is more preferred, and
2.5%
by weight to 10% by weight is most preferred. The larger the amount of the
surface
cross-linking agent to be added, the lower the degree of swelling of the
obtained
CA 02692515 2010-01-04
22
polymer, but addition of an excess amount thereof is not preferred since the
phosphate adsorption capacity decreases thereby.
[0058]
The solvent used for the surface cross-linking is selected as appropriate
depending on the surface cross-linking agent. Examples of the solvent which
may
be used include alcoholic solvents represented by methanol, ethanol, 1-
propanol and
2-propanol; hydrocarbon solvents represented by hexane, heptane, cyclohexane
and
toluene; known solvents such as ethyl acetate, tetrahydrofuran, acetone,
acetonitrile,
N,N-dimethylformamide, dimethylsulfoxide and water; and mixed solvents wherein
2 or more of these solvents are arbitrarily mixed.
[0059]
The temperature for the surface cross-linking is selected as appropriate
depending on the surface cross-linking agent, and preferably 0 C to 80 C, more
preferably 20 C to 60 C, most preferably 20 C to 50 C.
[0060]
The time for the surface cross-linking is selected as appropriate depending on
the surface cross-linking agent and the temperature, and usually preferably 5
minutes
to 10 hours.
[0061]
During the surface cross-linking, the cross-linking reaction occurs by
reaction
of the surface cross-linking agent with two amino groups existing in the
vicinity of
the surface of particles of the cross-linked polyallylamine or an acid
addition salt
thereof. Scheme I shows explanation of an example of a reaction wherein
acrylic
acid ester (2-hydroxyethyl acrylate) is used as the surface cross-linking
agent. First,
Michael addition of an amino group on the surface of a polymer particle to the
acrylic acid ester is allowed to occur (Step 1). Subsequently, an amino group
in the
vicinity on the surface of the particle attacks the carbonyl group of the
ester to allow
CA 02692515 2010-01-04
23
amidation to proceed (Step 2). By this, cross-linking in the vicinity of the
surface
of the polymer particle is strengthened. During this, depending on reaction
conditions, only Step 1 proceeds and cross-linking may not occur in a part of
the
surface cross-linking agent.
[0062]
NH2 NH,,
Polymer NH2 oH Michael addition fv`
particle Iol Step 1 o
2-hydroxyethyl acrylate Amidation
Step 2 _ Ho~~pH
0
HN '-)
NH
Scheme 1
[0063]
After the surface cross-linking, the cross-linked polyallylamine may be
salified to an acid addition salt as required. The conversion to an acid
addition salt
is carried out in the same manner as described above. Examples of the acid
used for
the salt formation include pharmaceutically acceptable acids such as
hydrochloric
acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, carbonic
acid,
acetic acid, benzoic acid, succinic acid, tartaric acid and citric acid; and
hydrochloric
acid, carbonic acid and acetic acid are more preferred; and hydrochloric acid
and
acetic acid are most preferred. The amount of the acid may be an arbitrary
amount
not more than 1 equivalent with respect to the amino group of the polymer, and
0.1
equivalent to 0.8 equivalent is preferred, and 0.1 equivalent to 0.5
equivalent is more
preferred. The polymer obtained in the present invention is subjected to
pulverization as required. The method of pulverization is not limited, and
either dry
CA 02692515 2010-01-04
24
or wet pulverization may be carried out.
[0064]
The cross-linked polyallylamine or the acid addition salt thereof of the
present invention can be used as a pharmaceutical composition because of its
high
phosphate adsorption capacity and low degree of swelling, and can be
preferably
used as especially a therapeutic or prophylactic agent for hyperphosphatemia.
In
this case, by orally administering the cross-linked polyallylamine or the acid
addition
salt thereof of the present invention as it is in the form of powder or as a
medical
composition in an appropriate formulation to a mammal, phosphates in the
intestine
is adsorbed to the cross-linked polyallylamine or an acid addition salt
thereof,
whereby contributing to therapy or prophylaxis of hyperphosphatemia.
[0065]
Examples of the formulation of the pharmaceutical composition containing as
an effective component the cross-linked polyallylamine or the acid addition
salt
thereof of the present invention include tablets, powders, pills, capsules and
granules.
The above-described formulations are produced by known methods and may contain
various carriers normally used in the field of drug formulation. Examples of
the
various carriers include fillers, lubricants, binders and disintegrators. In
addition,
additives such as antiseptics, antioxidants, coloring agents, sweeteners,
adsorbents
and wetting agents may be used as required.
[0066]
Examples of the fillers include lactose, D-mannitol, potato starch, sucrose,
corn starch, crystalline cellulose and light anhydrous silicic acid.
[0067]
Examples of the lubricants include magnesium stearate, calcium stearate, talc
and colloidal silica.
[0068]
CA 02692515 2010-01-04
Examples of the binders include crystalline cellulose, D-mannitol, dextrin,
hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone,
starch, sucrose, methyl cellulose and sodium carboxymethyl cellulose.
[0069]
5 Examples of the disintegrators include starch, carboxymethyl cellulose,
calcium carboxymethyl cellulose, croscarmellose sodium, sodium carboxymethyl
starch and low substituted hydroxypropylcellulose.
[0070]
Examples of the antiseptics include p-oxybenzoic acid esters, chlorobutanol,
10 benzylalcohol, phenetyl alcohol, dehydroacetic acid and sorbic acid.
[0071]
Examples of the antioxidants include sulfurous acid salts and ascorbic acid.
[0072]
The effective dose and the number of doses of the pharmaceutical
15 composition containing as an effective component the cross-linked
polyallylamine or
the acid addition salt thereof of the present invention vary depending on the
dosage
form, and age, body weight and severity of the symptom of the patient, and
usually
0.1 to 15 g, preferably 0.5 to 9 g of the pharmaceutical composition may be
administered to an adult per day before, during or after eating.
20 [0073]
The pharmaceutical composition containing as an effective component the
cross-linked polyallylamine or the acid addition salt thereof of the present
invention
may be administered solely or in combination with oral phosphate adsorption
agents
such as calcium carbonate, calcium lactate, calcium acetate, magnesium oxide
and
25 lanthanum carbonate.
EXAMPLES
[0074]
CA 02692515 2010-01-04
26
The present invention will now be described in more detail by way of
Examples below, but the present invention is not limited to these Examples.
[0075]
(Reference Example 1)
Synthesis of Allylammonium Dihydrogen Phosphate
To a three-necked round bottom flask equipped with a mechanical stirrer and
a thermometer, 46.1 g (0.40 mol) of phosphoric acid (85%) and 500 mL of
ethanol
were placed, and 30.0 mL (0.40 mol) of allylamine was added thereto with ice
cooling. After 30 minutes of stirring at room temperature, precipitated white
crystals were recovered by filtration and washed with ethanol sufficiently.
After
drying the recovered crystals under reduced pressure at 60 C, 59.6 g of
allylammonium dihydrogen phosphate was obtained.
[0076]
(Reference Example 2)
Synthesis of N,N-diallyl-l,3-diaminopropane and Its Bis(dihydrogen phosphate)
To a flask, 38.0 mL (0.40 mol) of 1,3-dichloropropane and 300 mL (4.00
mol) of allylamine were placed, and the resulting mixture was heated with
stirring
under argon atmosphere at 50 to 52 C for 16 hours. About a half amount of the
excessive allylamine was evaporated under reduced pressure, and an aqueous
potassium hydroxide solution (prepared by dissolving 48 g of potassium
hydroxide
into 144 g of water) was added thereto. Precipitated salt was removed by
filtration,
and the filtrate was concentrated under reduced pressure to about a half
volume.
After extraction with diethylether, the organic layer was dried over anhydrous
sodium sulfate and concentrated under reduced pressure, to obtain an oily
crude
product. This crude product was distilled under reduced pressure (42 to 44
C/0.3
kPa) to obtain 38.4 g of N,N-diallyl-1,3-diaminopropane. To a flask, 15.0 g
(130
mmol) of phosphoric acid (85%) and 300 mL of ethanol were placed, and 10.0 g
CA 02692515 2010-01-04
27
(65.0 mmol) of N,N-diallyl-1,3-diaminopropane dissolved in 20 mL of ethanol
with
ice cooling was added thereto. After 30 minutes of stirring at room
temperature,
precipitated white crystals were recovered by filtration and washed with
ethanol.
After drying the recovered crystals under reduced pressure at 60 C, 22.6 g of
N,N-
diallyl-l,3-diaminopropane bis(dihydrogen phosphate) was obtained. The total
amount thereof was suspended in 175 mL of methanol and dissolved by adding 14
mL of water while heating the mixture to reflux. The resulting solution was
stirred
at room temperature, thereby precipitating crystals. The resultant was left to
stand
in a freezer at -10 C for 1 hour and filtered, followed by washing the
precipitate with
ice-cooled methanol/water (100/2 v/v). Subsequently, the precipitate was
washed
with ethanol and dried under reduced pressure at 60 C to obtain 22.0 g of N,N-
diallyl-1,3-diaminopropane bis(dihydrogen phosphate).
[0077]
(Reference Example 3)
Synthesis of N,N-diallyl-1,2-diaminoethane and Its Bis(dihydrogen phosphate)
To a flask, 7.9 mL (0.10 mol) of 1,2-dichloroethane and 75 mL (1.0 mol) of
allylamine were placed and the resulting mixture was heated with stirring
under
argon atmosphere at 50 to 52 C for 20 hours. About a half amount of the
excessive
allylamine was evaporated under reduced pressure, and an aqueous potassium
hydroxide solution (prepared by dissolving 12 g of potassium hydroxide into 36
g of
water) was added thereto. Precipitated salt was dissolved by addition of
water, and
the resulting solution was extracted with diethylether. The organic layer was
dried
over sodium sulfate and concentrated under reduced pressure to obtain an oily
crude
product. This crude product was distilled under reduced pressure (85 to 87
C/1.2
kPa) to obtain 8.08 g of N,N-diallyl-l,2-diaminoethane.
[0078]
To 300 mL of ethanol, 8.41 g (60.0 mmol) of N,N-diallyl-1,2-diaminoethane
CA 02692515 2010-01-04
28
was dissolved. After cooling the resulting solution to -78 C, 13.8 g (120
mmol) of
phosphoric acid (85%) dissolved to 120 mL of ethanol was slowly added dropwise
thereto. After 30 minutes of stirring, precipitated white crystals were
recovered by
filtration and washed with ethanol. After drying the recovered crystals under
reduced pressure at room temperature, 19.9 g of N,N-diallyl-1,2-diaminoethane
bis(dihydrogen phosphate) was obtained. A 15.0 g aliquot of the obtained N,N-
diallyl-1,2-diaminoethane bis(dihydrogen phosphate) was suspended in 120 mL of
methanol and allowed to dissolve by addition of 12 mL of water while heating
the
mixture to reflux. The resulting solution was stirred at -20 C, thereby
precipitating
crystals. The resulting precipitate was filtered and washed with ice-cooled
methanol/water (100/4 v/v). Subsequently, the precipitate was washed with
ethanol
and dried under reduced pressure at room temperature to obtain 10.7 g of N,N-
diallyl-1,2-diaminoethane bis(dihydrogen phosphate).
[0079]
(Reference Example 4)
Synthesis of N,N-diallyl-1,4-diaminobutane and Its Bis(dihydrogen phosphate)
To 230 mL of acetonitrile, 20.0 g (227 mmol) of 1,4-diaminobutane and 63.3
mL (454 mmol) of triethylamine were dissolved, and 99.0 g (454 mmol) of di-
tert-
butyl dicarbonate was slowly added thereto with ice cooling with stirring. The
resulting mixture was stirred at room temperature for 1 hour and cooled to -20
C.
The precipitated solids were recovered by filtration and dried under reduced
pressure
to obtain 47.1 g of a crude product of tert-butyl butane-1,4-diyldicarbamate.
This
was dissolved to 300 mL of acetonitrile at 60 C and left to stand at room
temperature,
thereby precipitating needle crystals. After cooling at -20 C, the crystals
were
recovered by filtration to obtain 45.6 g of di-tert-butyl butane-1,4-
diyldicarbamate.
[0080]
In 100 mL of N,N-dimethylformamide, 21.0 g (72.9 mmol) of di-tert-butyl
CA 02692515 2010-01-04
29
butane-1,4-diyldicarbamate was suspended. The atmosphere in the flask was
replaced with argon, and 18.9 mL (219 mmol) of allyl bromide was added
thereto.
At an inner temperature of 40 C, 8.74 g (219 mmol) of sodium hydride (60%
dispersion in mineral oil) was added slowly thereto. Thereafter, the resulting
mixture was stirred while adjusting the inner temperature such that a
temperature of
C to 20 C is attained in an ice bath. After confirming that there is no
foaming
and elevation of temperature, 2.91 g (73 mmol) of sodium hydride (60%
dispersion
in mineral oil) and 6.3 mL (73 mmol) of allyl bromide were added to the
mixture,
and the resulting mixture was further stirred at room temperature for 2 hours.
The
10 mixture was cooled on ice, and water was slowly added thereto, followed by
extraction twice with ethyl acetate. After washing the organic layer twice
with
saturated aqueous ammonium chloride solution, 3 times with water and twice
with
saturated brine, it was dried over magnesium sulfate. The resultant was
concentrated under reduced pressure by a rotary evaporator to obtain a crude
product
of di-tert-butyl butane-1,4-diylbis(allylcarbamate) as an oily product. The
total
amount of this oily product was dissolved in 220 mL of ethanol, and 50 mL of 6
M
hydrochloric acid was added thereto, followed by heating the mixture to reflux
for
2.5 hours. After removal of the most part of the oil floating on the liquid,
concentration was carried out under reduced pressure. After repeating the
operation
of adding 100 mL of ethanol to concentrate the solution 3 times, white
crystals were
precipitated. The resultant was cooled on ice and filtered, followed by
washing
with ice-cooled ethanol. The resultant was dried under reduced pressure at
room
temperature to obtain 14.8 g of N,N'-diallyl-1,4-diaminobutane
dihydrochloride. Its
total amount was dissolved in 100 mL of water and 30 mL of 20 w/w% aqueous
sodium hydroxide solution was added thereto, followed by 3 times of extraction
with
diethylether. The organic layer was dried over magnesium sulfate and
concentrated.
The crude product was distilled under reduced pressure (88 C/0.2 kPa) to
obtain 8.65
CA 02692515 2010-01-04
g ofN,N-diallyl-1,4-diaminobutane. To a flask, 8.07 g of phosphoric acid (85%)
was weighed, and 150 mL of ethanol and 75 mL of 2-propanol were added thereto,
followed by cooling with ice. To this, 5.89 g of N,N-diallyl-1,4-diaminobutane
dissolved in 25 mL of ethanol was added, thereby precipitating viscous solids.
The
5 resulting solids were left to stand at room temperature for 3 days to become
solid
masses. These masses were crushed and filtered, followed by washing with
ethanol
and drying under reduced pressure at room temperature, to obtain 12.8 g of N,N-
diallyl-1,4-diaminobutane (dihydrogen phosphate). Its total amount was
suspended
in 200 mL of methanol, and 25 mL of water was added thereto while heating the
10 mixture to reflux. Since a small portion of the solids remained
undissolved, hot
filtration was carried out and the filtrate was left to stand at room
temperature,
thereby precipitating white crystals. The resultant was left to stand in a
freezer at -
10 C and filtered, followed by washing the precipitate with methanol/water
(100/4
v/v). Subsequently, the precipitate was washed with ethanol and dried under
15 reduced pressure at 50 C, to obtain 23.1 g of N,N-diallyl-l,4-diaminobutane
bis(dihydrogen phosphate).
[0081]
(Reference Example 5)
Synthesis of N,N-diallyl-1,5-diaminopentane and Its Bis(dihydrogen phosphate)
20 To 80 mL of acetonitrile, 7.85 g (76.8 mmol) of 1,5-diaminopentane and 21.4
mL (154 mmol) of triethylamine were dissolved, and 33.6 g (154 mmol) of di-
tert-
butyl dicarbonate was slowly added thereto with ice cooling with stirring. The
resulting mixture was stirred at room temperature for 1 hour, and concentrated
to
dryness by a rotary evaporator. To the resultant, 20 mL of ethyl acetate was
added,
25 and it was dissolved therein under heat at 60 C, followed by addition of
100 mL of
hexane and stirring at room temperature, thereby precipitating crystals. The
crystals
were recovered by filtration to obtain 16.1 g of di-tert-butyl pentane-1,5-
CA 02692515 2010-01-04
31
diyldicarbamate.
[0082]
In 200 mL of N,N-dimethylformamide, 45.4 g (150 mmol) of di-tert-butyl
pentane-1,5-diyldicarbamate was suspended. The atmosphere in the flask was
replaced with argon, and 38.9 mL (450 mmol) of allyl bromide was added
thereto.
At an inner temperature of 40 C, 18.0 g (450 mmol) of sodium hydride (60%
dispersion in mineral oil) was added slowly thereto. The resulting mixture was
cooled on ice, and water was slowly added thereto, followed by 3 times of
extraction
with ethyl acetate. After washing the organic layer 3 times with water and
twice
with saturated brine, it was dried over magnesium sulfate. The resultant was
concentrated under reduced pressure by a rotary evaporator, to obtain a crude
product
of di-tert-butyl pentane-1,5-diylbis(allylcarbamate) as an oily product. The
total
amount of this oily product was dissolved in 440 mL of ethanol, and 100 mL of
6M
hydrochloric acid was added thereto, followed by heating the mixture to reflux
for 1
hour. After repeating the operation of adding 200 mL of ethanol to concentrate
the
solution twice, white crystals were precipitated. The resulting precipitate
was
filtered and washed with ethanol. This was dried under reduced pressure at
room
temperature to obtain 24.5 g of N,N-diallyl-1,5-diaminopentane
dihydrochloride.
Its total amount was dissolved in 100 mL of water and 50 g of 20 w/w% aqueous
sodium hydroxide solution was added thereto, followed by extraction with
diethylether twice. The organic layer was dried over magnesium sulfate and
concentrated. The crude product was distilled under reduced pressure (64 C/0.1
kPa) to obtain 7.73 g of N,N-diallyl-1,5-diaminopentane. Into a flask, 9.78 g
of
phosphoric acid (85%) was weighed, and 160 mL of ethanol was added thereto,
followed by cooling with ice. To this, 7.73 g of N,N-diallyl-1,5-
diaminopentane
dissolved in 40 mL of ethanol was added, thereby precipitating white solids.
The
resulting solids were left to stand at room temperature for 3 days to become
solid
CA 02692515 2010-01-04
32
masses. These masses were crushed and filtered, followed by washing with
ethanol
and drying under reduced pressure at 50 C, to obtain 16.0 g of N,N-diallyl-1,5-
diaminopentane dihydrogen phosphate. Its total amount was suspended in 140 mL
of methanol, and 2.8 mL of water was added thereto while heating the mixture
to
reflux. Since a small portion of the solids remained undissolved, hot
filtration was
carried out and the filtrate was left to stand at room temperature, thereby
precipitating white crystals. The resultant was filtered and washed with ice-
cooled
methanol/water (100/4 v/v). Subsequently, the precipitate was washed with
ethanol
and dried under reduced pressure at 50 C to obtain 13.1 g of N,N-diallyl-1,5-
diaminopentane bis(dihydrogen phosphate).
[0083]
(Reference Example 6)
Synthesis of Sevelamer Hydrochloride
To a flask, 173 mL of concentrated hydrochloric acid was added, and 120 g
(2.10 mol) of allylamine was added dropwise thereto at an inner temperature of
5 to
10 C. After completion of the dropping, 90 mL of the liquid was evaporated
under
reduced pressure while being heated in an oil bath at 70 C. After replacing
the
atmosphere in the system with argon 3 times, the mixture was bubbled with
argon for
30 minutes. In 5.4 mL of water, 2.40 g (8.85 mmol) of 2,2'-azobis(2-
amidinopropane) dihydrochloride was suspended, and the resulting suspension
was
added to the above mixture, followed by stirring of the resulting mixture at
an inner
temperature of 50 C for 24 hours. In 5.4 mL of water, 2.40 g (8.85 mmol) of
2,2'-
azobis(2-amidinopropane) dihydrochloride was suspended, and the resulting
suspension was added to the above mixture, followed by stirring of the
resulting
mixture at an inner temperature of 50 C for additional 44 hours. To this
mixture,
48 mL of water was added and the resulting mixture was cooled to room
temperature and poured slowly to 2L of methanol with stirring. The obtained
white
CA 02692515 2010-01-04
33
solids were filtered. The operation wherein the solids were added to 2L of
methanol and the resulting mixture was stirred for 1 hour, followed by
filtration
thereof was repeated twice. The obtained solids were dried in a vacuum oven at
50 C for 24 hours to obtain 111 g of polyallylamine hydrochloride.
[0084]
To a 200 mL beaker, 25.0 g of the obtained polyallylamine hydrochloride was
weighed, and dissolved in 100 mL of water. To the resulting solution, 7.12 g
of
sodium hydroxide was added with stirring with a mechanical stirrer, to achieve
pH
10. Addition of 2.50 mL of epichlorohydrin thereto at an inner temperature of
25 C
and stirring of the resulting mixture caused solidification of the mixture 23
minutes
later. After stopping the stirring, the mixture was left to stand at 25 C for
18 hours.
To this mixture, 75 mL of 2-propaol was added, and the gel was crushed,
followed
by filtration of the resultant. The operation wherein the solids were added to
340
mL of water and the resulting mixture was stirred for 1 hour, followed by
filtration
thereof was repeated 3 times. The resultant was added to 600 mL of 2-propanol
and
the resulting mixture was stirred for 1 hour, followed by filtration thereof.
The
resultant was dried in a vacuum oven at 30 C for 37 hours to obtain 25.7 g of
white
solids. These were freeze-crushed to obtain sevelamer hydrochloride.
[0085]
To confirm that sevelamer hydrochloride having a desired phosphate
adsorption capability was synthesized, the phosphate adsorption capability of
the
sevelamer hydrochloride in the presence of phosphate alone was evaluated
according
to the in vitro assay method described in the above-described Patent
Literature 1.
More concretely, sodium carbonate, sodium chloride and disodium hydrogen
phosphate dodecahydrate were used, and a solution wherein their concentrations
were 30 mM, 80 mM and 12 mM, respectively, was prepared, followed by addition
thereto 1 M hydrochloric acid to adjust the pH to 7 to obtain a test solution.
CA 02692515 2010-01-04
34
Subsequently, 20 mg of the sevelamer hydrochloride was placed in an Erlenmeyer
flask and 10 mL of the above-described test solution was added thereto,
followed by
stirring at 37 C for 3 hours in a water bath. The pH became 8 to 9 after the
stirring,
and the pH of the resulting suspension was adjusted to 7 by 1 M hydrochloric
acid.
A small aliquot of this suspension was taken and sevelamer hydrochloride was
removed therefrom by a centrifuge (refrigerated centrifuge 5417R manufactured
by
Eppendorf, angle rotor FA-45-24-11), followed by measuring (n=3) the amount of
phosphate which was not adsorbed to the sevelamer hydrochloride using an
inorganic
phosphate measurement reagent (manufactured by Wako Pure Chemical Industries;
Phosphor C (registered trademark)), to determine from the measured value the
amount of the phosphate ions adsorbed to the sevelamer hydrochloride, that is,
the
phosphate adsorption capacity (a calibration curve was prepared within the
range of
the inorganic phosphate concentration of 0 to 386 mg/mL; the spectrophotometer
was Spectra Max Plus manufactured by Molecular Devices). As a result, it was
revealed that the phosphate adsorption capacity in the presence of phosphate
alone
was 3.4 0.5 mmol/g, which is equivalent to the phosphate adsorption capacity
described in Patent Literature 1 (3.1 mmol/g).
[0086]
Subsequently, in vitro assays of the degree of swelling and the phosphate
adsorption capability of the obtained sevelamer hydrochloride were carried out
by
the method described below.
[0087]
[Measurement of Degree of Swelling of Sample]
In 50 mL of distilled water, 200 mg of a sample to be measured which was
dried under reduced pressure at 40 C for not less than 16 hours was soaked for
not
less than 24 hours, and the sample was filtered under reduced pressure using a
membrane filter having a diameter of 47 mm and pore size of 0.45 m (Omnipore,
CA 02692515 2010-01-04
manufactured by Millipore) to separate the solid component, whose weight was
then
divided by the dry weight (200 mg).
[0088]
[Measurement of Phosphate Adsorption Capacity and Bile Acid Adsorption
5 Capacity]
After stirring 10 mg of a sample to be measured in 1 mL of 50 mM
hydrochloric acid at 37 C for 1 hour, 9 mL of a mixed solution of 11.1 mM each
of
disodium hydrogen phosphate dodecahydrate and an aqueous sodium glycocholate
solution was added thereto. After stirring the resulting mixture at 37 C for
10 additional 1 hour, the sample to be measured was removed therefrom by a
centrifuge
(refrigerated centrifuge 5417R manufactured by Eppendorf, angle rotor FA-45-24-
11; 15000 rpm, 25 C, 15 minutes), followed by measuring (n=3) the amounts of
phosphate and glycocholate which were not adsorbed to the sample to be
measured
using an inorganic phosphate measurement reagent (manufactured by Wako Pure
15 Chemical Industries; Phosphor C (registered trademark)) and a bile acid
measurement reagent (manufactured by Wako Pure Chemical Industries; Total Bile
Acids (registered trademark)), respectively, to determine from the measured
values
the amount of the phosphate ions adsorbed to the sample to be measured, that
is, the
phosphate adsorption capacity (a calibration curve was prepared within the
range of
20 the inorganic phosphate concentration of 0 to 386 mg/mL; the
spectrophotometer
was Spectra Max Plus manufactured by Molecular Devices). The phosphate
selectivity was indicated as the value calculated by dividing the phosphate
adsorption
capacity by the bile acid adsorption capacity.
[0089]
25 The assay results of sevelamer hydrochloride are shown in Table 1. The
phosphate selectivity was as low as 1.2, and the degree of swelling was 6.2.
[0090]
CA 02692515 2010-01-04
36
[Table 1]
Assay Results of Sevelamer Hydrochloride
Degree of Phosphate Bile acid Phosphate
swelling adsorption adsorption selectivity
capacity capacity
(mmol/g) (mmol/g)
Reference 6.2 2.70 2.30 1.2
Example 6
[0091]
(Example 1)
Cross-linking Copolymerization of Allylammonium Dihydrogen Phosphate under
Various Conditions, Alkali Treatment and Hydrochloride Formation Thereof
Polymerization to obtain the cross-linked polyallylamine or the acid addition
salt thereof of the present invention was carried out under various
conditions. Table
2 shows the amount of the solvent, the amount of the polymerization initiator,
the
polymerization temperature and the polymerization time in each polymerization
condition. Polymerization and hydrochloride formation of the obtained cross-
linked polyallylamine phosphate under each condition were carried out by the
following procedure. To a flask, 6.20 g of allylammonium dihydrogen phosphate,
2.80 g of N,N-diallyl-1,3-diaminopropane bis(dihydrogen phosphate) and a
predetermined amount of a solvent (water) were placed, and the resulting
mixture
was heated in an oil bath at 50 C to be dissolved. After replacing the
atmosphere in
the system with argon 3 times, a predetermined amount of a polymerization
initiator
(2,2'-azobis(2-amidinopropane) dihydrochloride) was added to the above
solution,
and the resulting mixture was heated at a predetermined polymerization
temperature
under stirring for a predetermined polymerization time period. The obtained
solids
were crushed and recovered by filtration, and sufficiently washed with water
and
then ethanol. The solids were then dried under reduced pressure at 60 C to
obtain
white powder of a cross-linked polyallylamine phosphate. The thus obtained
cross-
CA 02692515 2010-01-04
37
linked polyallylamine phosphate was dispersed in water (2.5 mL with respect to
1 g
of the polymer), and 20% sodium hydroxide solution (4 mL with respect to 1 g
of the
polymer) was added thereto with stirring. After 30 minutes of stirring of the
resulting mixture, solids were recovered by filtration. The obtained solids
were
washed with water until the filtrate became neutral and then washed with
ethanol.
This was followed by drying under reduced pressure at 60 C to obtain a free-
form
cross-linked polyallylamine. To a flask, 0.40 g of the obtained free-form
cross-
linked polyallylamine and 8 mL of water were placed, and 4 mL of concentrated
hydrochloric acid was added thereto. After stirring the resulting mixture,
solids
were recovered by filtration. The obtained solids were washed with water until
the
filtrate becomes neutral and then washed with ethanol sufficiently. This was
followed by drying under reduced pressure at 60 C to obtain a cross-linked
polyallylamine hydrochloride.
[0092]
Each of the obtained polyallylamine hydrochloride was assayed in the same
manner as in Reference Example 6. The results are shown in Table 2.
Comparison between the cases of polymerization at 55 C for 15 hours (Reference
Example 7, Example 1-2, Example 1-4) and the cases of polymerization at 45 C
for
61 hours (Example 1-1, Example 1-3, Example 1-5) showed that the cases of
polymerization at 45 C for 61 hours exhibited somewhat lower phosphate
adsorption
capacities, but higher phosphate selectivities and lower degrees of swelling.
When
the amount of the initiator was doubled, the phosphate adsorption capacity
somewhat
decreased but the degree of swelling was found to decrease (Example 1-2,
Example
1-3). Even in cases where the amount of the water used as the solvent was
reduced
by half, the degree of swelling was found to decrease (Example 1-4, Example 1-
5).
By optimizing the reaction temperature, the reaction time, the amount of the
solvent
and the amount of the initiator to be added, a cross-linked polyallylamine
CA 02692515 2010-01-04
38
hydrochloride having a high phosphate adsorption capacity and a low degree of
swelling was obtained.
[0093]
CA 02692515 2010-01-04
39
p~ N h N (V M
~O ~, b^p
U 'i,~-+ = ~-r ,-~i
a: O ~ ~ .-o .-+ o -- O
p ~ =-' o O~ OC)0 r--
p p ~~~ M M M N N N
=-. ,s~
'd G=~ as U ~
p O
O bA
cfj
~ o bu dn
o ~ o 0 0 0, 00 0
to N \O d Cr M M M
y. ~ C~
G~ O
:J N
7~
d)
~
uo
v
^ O
Oo 00 00 00 d- ~r O
~, o M M M M
00 00
U =~ R3 ~
'~ N =~'
vi p bA ~ N N ~
o p~ `' o 0 0 0 0 0
~
~ --~ N M ~ ~ rx"+
-O
~ cn
u ~4 W W W W F+~ F+N W C~
CA 02692515 2010-01-04
[0094]
(Example 2)
Cross-linking Copolymerization of Allylammonium Dihydrogen Phosphate with
Various Amounts of Cross-linking Agent to Be Added and Alkali treatment and
5 Hydrochloride Formation Thereof
Polymerization to obtain the cross-linked polyallylamine or the acid addition
salt thereof of the present invention was carried out with various amounts of
the
added cross-linking agent as described in Table 3. Polymerization and
hydrochloride formation of the obtained cross-linked polyallylamine phosphate
10 under each condition were carried out by the following procedure. To a
flask, 6.20
g of allylammonium dihydrogen phosphate, a predetermined amount of the cross-
linking agent (N,N-diallyl-1,3-diaminopropane bis(dihydrogen phosphate)) and 4
mL of water were placed, and the resulting mixture was heated in an oil bath
at 50 C
to be dissolved. After replacing the atmosphere in the system with argon 3
times,
15 0.11 g of 2,2'-azobis(2-amidinopropane) dihydrochloride was added to the
above
solution, and the resulting mixture was heated at an inner temperature of 45
to 47 C
for 64 hours with stirring. Several hours after the addition of 2,2'-azobis(2-
amidinopropane) dihydrochloride, the reaction system became solidified, and
the
stirring was stopped. The obtained solids were crushed and recovered by
filtration,
20 and sufficiently washed with water and then ethanol. The solids were then
dried
under reduced pressure at room temperature to obtain white powder of a cross-
linked
polyallylamine phosphate. The thus obtained cross-linked polyallylamine
phosphate was dispersed in water (5 mL with respect to 1 g of the polymer),
and 20%
sodium hydroxide solution (4 mL with respect to 1 g of the polymer) was added
25 thereto with stirring. After 1 hour of stirring of the resulting mixture at
room
temperature, solids were recovered by filtration and washed with water until
the
filtrate became neutral. The solids were then stirred in water overnight and
CA 02692515 2010-01-04
41
recovered by filtration, followed by washing thereof with ethanol. After
drying
under reduced pressure, a free-form cross-linked polyallylamine was obtained.
To a
flask, 0.40 g of the obtained free-form cross-linked polyallylamine and 8 mL
of
water were placed, and 2 mL of concentrated hydrochloric acid was added
thereto.
After stirring the resulting mixture for 30 minutes, solids were recovered by
filtration.
The obtained solids were washed with water until the filtrate became neutral
and then
washed with ethanol. This was followed by drying under reduced pressure at
room
temperature to obtain a cross-linked polyallylamine hydrochloride.
[0095]
Each obtained polyallylamine hydrochloride was assayed in the same manner
as in Reference Example 6. The results are shown in Table 3. With the amount
of
the added cross-linking agent of 2 mol%, the degree of swelling was high, so
that the
object of the present invention could not be achieved, but, as the amount of
the
additive increased, the degree of swelling decreased and the phosphate
selectivity
was improved at the same time. With the amount of the added cross-linking
agent
of not less than 5 mol%, the degree of swelling was less than 5Ø However,
the
degree of swelling was found not to have decreased even by increasing the
amount of
the added cross-linking agent from 20 mol% to 30 mol%, so that it was thought
that
the cross-linking did not proceed efficiently.
CA 02692515 2010-01-04
42
[0096]
[Table 3]
Assay Results of Cross-linked Polyallylamine Hydrochloride Synthesized with
Various Amounts of Added Cross-linking Agent
Amount of Degree Phosphate Bile acid Phosphate
added of adsorption adsorption selectivity
cross- swelling capacity capacity
linking (mmol/g) (mmol/g)
agent
(mol%)
Reference 2 7.9 3.06 2.66 1.2
Example 8
Example 2-1 5 4.3 3.42 2.15 1.6
Example 2-2 10 3.5 3.52 1.16 3.0
Example 2-3 15 3.2 3.86 1.29 3.0
Example 2-4 20 3.1 3.21 1.17 2.7
Reference 30 3.2 2.29 1.10 2.1
Example 9
[0097]
(Example 3)
Cross-linking Copolymerization of Allylammonium Dihydrogen Phosphate with
Various Amounts of Cross-linking Agent Added, Alkali treatment and
Hydrochloride Formation Thereof (with Usage of Cross-linking Agent of Product
Purified by Recrystallization)
Polymerization to obtain the cross-linked polyallylamine or the acid addition
salt thereof of the present invention was carried out with various amounts of
the
added cross-linking agent as described in Table 4. Polymerization and
hydrochloride salification of the obtained cross-linked polyallylamine
phosphate
under each condition were carried out in the same manner as in Example 2
except
that the amounts of allylammonium dihydrogen phosphate which is the monomer,
CA 02692515 2010-01-04
43
water which is the solvent, and 2,2'-azobis(2-amidinopropane) dihydrochloride
which is the initiator were changed as appropriate according to the
description in
Table 4 and that N,N-diallyl-1,3-diaminopropane bis(dihydrogen phosphate)
purified
by recrystallization was used as the predetermined amount of the cross-linking
agent.
[0098]
Each obtained polyallylamine hydrochloride was assayed in the same manner
as in Reference Example 6. The results are shown in Table 4. With the amount
of
the added cross-linking agent of 2 mol%, the degree of swelling was high, so
that the
object of the present invention could not be achieved, but, as the amount of
the
additive increased, the degree of swelling decreased and the phosphate
selectivity
was improved at the same time. With the amount of the added cross-linking
agent
of not less than 5 mol%, the degree of swelling was less than 5Ø The degree
of
swelling was found not to have decreased even by increasing the amount of the
added cross-linking agent from 20 mol% to 30 mol%.
[0099]
CA 02692515 2010-01-04
44
...
¾ v .~ ~ N N N N
O U
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CA 02692515 2010-01-04
[0100]
(Comparative Example 1)
Cross-linking Copolymerization of Allylammonium Dihydrogen Phosphate Using
5 Various Cross-linking Agents, Alkali treatment and Hydrochloride Formation
Thereof
Polymerization to obtain the cross-linked polyallylamine or the acid addition
salt thereof of the present invention was carried out with various cross-
linking agents
and various amounts of the added cross-linking agents as described in Table 5.
10 Polymerization and hydrochloride formation of the obtained cross-linked
polyallylamine phosphate under each condition were carried out in the same
manner
as in Example 2 except that the cross-linking agent and the amount of the
added
cross-linking agent were changed as appropriate according to the description
in Table
5.
15 [0101]
Each of the obtained polyallylamine hydrochloride was assayed in the same
manner as in Reference Example 6. The results are shown in Table 5. The data
from Example 3-1 wherein N,N-diallyl-1,3-diaminopropane bis(dihydrogen
phosphate) was used as the cross-linking agent in an amount of 5% are also
shown.
20 In cases where the diphosphate of each of N,N'-diallyl-1,2-diaminoethane,
N,N-
diallyl-1,4-diaminobutane and N,N-diallyl-1,5-diaminopentane was used as the
cross-linking agent, the degree of swelling was high with any amount of the
added
cross-linking agent of 5 mol% to 30 mol%, so that the object of the present
invention
could not be achieved. Thus, it is necessary to use an acid addition salt of
N,N-
25 diallyl-1,3-diaminopropane as the cross-linking agent.
[0102]
CA 02692515 2010-01-04
46
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CA 02692515 2010-01-04
47
[0103]
(Example 4)
Study to Decrease Amount of Hydrochrolic Acid
A cross-linked polyallylamine hydrochloride releases hydrochloric acid upon
adsorption of phosphate thereto. Since excessive release of hydrochloric acid
may
cause hyperchloremic acidosis, the degree of hydrochloride formation is
preferably
as low as possible. Therefore, the free-form cross-linked polyallylamine
obtained
under the conditions of Example 2-4 shown in Table 3 (the cross-linking agent
was
used in an amount of 20 mol%) was used to confirm the effect obtained in cases
where a part of the amino groups were converted to hydrochloride (a cross-
linked
polyallylamine wherein two thirds of the amino groups were subjected to
hydrochloride formation is hereinafter described as a "cross-linked
polyallylamine
2/3 hydrochloride").
[0104]
(1) Complete hydrochloride (Example 4-1)
In 60 mL of water, 3.00 g of a free-form cross-linked polyallylamine obtained
under the conditions of Example 2-4 was dispersed, and 30 mL of concentrated
hydrochloric acid was added thereto at room temperature. After stirring the
resulting mixture for 1.5 hours, solids were recovered by filtration. The
obtained
solids were washed with water until the filtrate became neutral and then
washed with
ethanol sufficiently. This was followed by drying under reduced pressure at
room
temperature to obtain 4.84 g of a cross-linked polyallylamine hydrochloride.
[0105]
(2) 2/3 hydrochloride (Example 4-2)
In 4.0 mL of water, 0.35 g of a free-form cross-linked polyallylamine
obtained under the conditions of Example 2-4 was dispersed, and 3.7 mL of 1 M
hydrochloric acid was added thereto at room temperature. After stirring the
resulting mixture for 1 hour, solids were recovered by filtration. The
recovered
CA 02692515 2010-01-04
48
solids were washed with water and then washed sufficiently with ethanol. This
was
followed by drying under reduced pressure at room temperature to obtain 0.53 g
of a
cross-linked polyallylamine 2/3 hydrochloride.
[0106]
The obtained polyallylamine hydrochloride was assayed in the same manner
as in Reference Example 6. The results are shown in Table 6. The degrees of
swelling, phosphate adsorption capacities and phosphate selectivities were
similar
irrespective of the degree of hydrochloride formation, and it was revealed
that it is
possible to reduce the number of equivalence of hydrochloric acid used for the
salt
formation to two thirds. Although not shown in the Table, the phosphate
adsorption
capacity could be increased to 4.79 mmol/g in a case where the diameter of the
polymer particle could be reduced by the operation of stirring the free-form
cross-
linked polyallylamine in water overnight upon alkali treatment (the bile acid
adsorption capacity was 1.21 mmol/g, the phosphate selectivity was 4.0, and
the
degree of swelling was 4.0).
[0107]
[Table 6]
Assay Results of Cross-linked Polyallylamine 2/3 Hydrochloride Synthesized
Equivalence Degree of Phosphate Bile acid Phosphate
of swelling adsorption adsorption selectivity
hydrochloric capacity capacity
acid (mmol/g) (mmol/g)
Example 4-1 1 3.3 3.61 1.42 2.5
Example 4-2 2/3 3.3 3.73 1.16 3.2
[0108]
(Comparative Example 2)
Comparison with Cross-linked Polymer of Allylammonium Chloride
In place of allylammonium dihydrogen phosphate which is the monomer of
the present invention, allylammonium chloride which is the hydrochloride of
CA 02692515 2010-01-04
49
allylamine was used for the polymerization. Polymerization and hydrochloride
formation of the obtained cross-linked polyallylamine phosphate were carried
out in
the same manner as in Example 2 except that 3.74 g (40 mmol) of allylammonium
chloride was used as the monomer and 1.82 g (8 mmol) of N,N-diallyl-1,3-
diaminopropane dihydrochioride was used as the cross-linking agent. The
obtained
cross-linked polyallylamine hydrochloride was assayed in the same manner as in
Reference Example 6. The results are shown in Table 7. Compared to the cross-
linked polyallylamine hydrochloride which differs only in usage of
allylammonium
dihydrogen phosphate as the monomer (Example 2-4), the cross-linked
polyallylamine hydrochloride obtained using allylammonium chloride as the
monomer showed a lower phosphate selectivity and a lower phosphate adsorption
capacity. Thus, it was revealed that it is necessary to use allylammonium
dihydrogen phosphate which is the phosphate of allylamine as the monomer of
the
present invention.
[0109]
[Table 7]
Assay Results of Polymers Obtained by Polymerization with Hydrochloride
Degree of Phosphate Bile acid Phosphate
swelling adsorption adsorption selectivity
capacity capacity
(mmol/g) (mmol/g)
Comparative Example 2 3.5 2.56 1.50 1.7
Example 2-4 3.1 3.21 1.17 2.7
[0110]
(Reference Example 11)
Application of Molecular Imprinting Using Potassium Dihydrogen Phosphate (Part
1)
To confirm whether or not the method of phosphate imprinting described in
the above-described Patent Literature 4 (US 2005/0276781 Al) is applicable to
the
CA 02692515 2010-01-04
allylamine used in the present invention, we attempted synthesis of a
phosphate-
imprinted polymer using allylamine according to the method described in Patent
Literature 4. To a flask, 3.0 mL (40 mmol) of allylamine and 1.23 g (8 mmol)
of
N,N'-diallyl-1,3-diaminopropane were placed, and 2 mL of 2-propanol or water
was
5 added thereto. To the resulting mixture, 0.71 g (5.2 mmol) of potassium
dihydrogen phosphate was added and stirred for 3 hours at room temperature. To
the resulting mixture, 0.108 g (0.4 mmol) of 2,2'-azobis(2-amidinopropane)
dihydrochloride was added, and the atmosphere in the system was replaced with
argon 3 times, followed by heating the mixture at 50 C for 60 hours with
stirring.
10 After cooling the mixture to room temperature, water was added thereto only
to form
a homogeneous solution in either case, so that a water-insoluble polymer could
not
be obtained. From this result, it was revealed that it is impossible to
polymerize
allylamine in the presence of potassium dihydrogen phosphate, and that the
method
of phosphate imprinting described in Patent Literature 4 is not applicable to
the
15 allylamine used in the present invention.
[0111]
(Reference Example 12)
Application of Molecular Imprinting using Potassium Dihydrogen Phosphate (Part
2)
The potassium dihydrogen phosphate used for the molecular imprinting in the
20 above-described Patent Literature 4 was used in an amount of I equivalent
with
respect to the amino groups contained in allylamine and the cross-linking
agent to
convert all the amino groups to hydrochlorides, thereby polymerization was
attempted. To a flask, 3.0 mL (40 mmol) of allylamine and 1.23 g (8 mmol) of
N,N-diallyl-1,3-diaminopropane were placed and 4 mL of water was added
thereto.
25 To the resulting mixture, 7.62 g (56 mmol) of potassium dihydrogen
phosphate was
added, thereby precipitating white solids, and stirring became difficult. The
resultant was left to stand at room temperature for 3 hours, and 4 mL of water
was
CA 02692515 2010-01-04
51
added thereto, followed by heating the mixture at 50 C with stirring to
dissolve the
solids. To the resulting solution, 0.108 g (0.4 mmol) of 2,2'-azobis(2-
amidinopropane) dihydrochloride was added, and the atmosphere in the system
was
replaced with argon 3 times, followed by heating the mixture at 50 C for 110
hours
with stirring. No generation of solids was observed. After cooling the mixture
to
room temperature, water was added thereto only to form a homogeneous solution,
so
that a water-insoluble polymer could not be obtained. According to this
result,
potassium allylammonium hydrogen phosphate obtained by mixing allylamine and
potassium dihydrogen phosphate is inappropriate for obtaining a polymer by
polymerizing the phosphate of allylamine. It was revealed that, to solve the
problem which is to be solved by the present invention, it is indispensable to
use
allylammonium dihydrogen phosphate obtained by mixing allylamine and
phosphoric acid at a ratio of 1:1, and that a desired cross-linked
polyallylamine can
be obtained only in such a case.
[0112]
(Example 5)
Urinary Phosphate Excretion Test in Normal Rat
(Synthesis of Sample for Test and in Vitro Assay)
To a flask, 46.5 g (300 mmol) of allylammonium dihydrogen phosphate, 21.0
g (60 mmol) of N,N-diallyl-1,3-diaminopropane bis(dihydrogen phosphate) and
30.0
mL of water were placed and the atmosphere in the system was replaced with
argon
3 times, followed by heating the mixture in an oil bath at 50 C to dissolve
it. The
atmosphere in the system was replaced once with argon, and the mixture was
bubbled with argon at room temperature for 30 minutes. To this mixture, 0.814
g (3
mmol) of 2,2'-azobis(2-amidinopropane) dihydrochloride was added with
stirring,
and the resulting mixture was heated at an inner temperature of 47 C for 65.5
hours.
Several hours after the beginning of the heating, the mixture became
solidified, and
CA 02692515 2010-01-04
52
stirring was stopped. The obtained solids were crushed and recovered by
filtration,
and sufficiently washed with water and then ethanol. The solids were then
dried
under reduced pressure at 60 C to obtain 51.7 g of white powder of a cross-
linked
polyallylamine phosphate. In 75 mL of water, 51.0 g of the obtained cross-
linked
polyallylamine phosphate was dispersed, and 200 g of 20% aqueous sodium
hydroxide solution was added thereto with stirring. Since salt precipitated,
75 mL
of water was added to the mixture, and the resulting mixture was stirred at
room
temperature for 30 minutes, followed by recovery of solids by filtration and
washing
thereof with water. The solids were transferred to an Erlenmeyer flask, and
500 mL
of water was added thereto, followed by 14 hours of stirring at room
temperature.
The solids were recovered by filtration, and washed with water and then
ethanol.
The solids were then dried at 60 C under reduced pressure to obtain 17.5 g of
a free-
form cross-linked polyallylamine. To a round bottom flask, 4.50 g of the
obtained
free-form cross-linked polyallylamine and 50 mL of water were placed, and 47.8
mL
of I M hydrochloric acid was added to the resulting mixture. After stirring
the
mixture at room temperature for 1 hour, solids were recovered by filtration.
The
recovered solids were washed with water and then ethanol. This was followed by
drying under reduced pressure at 50 C to obtain 6.51 g of a cross-linked
polyallylamine 2/3 hydrochloride. By freeze-crushing 5.00 g of the obtained
cross-
linked polyallylamine 2/3 hydrochloride, 4.94 g of a freeze-crushed product of
the
cross-linked polyallylamine 2/3 hydrochloride was obtained. The obtained
freeze-
crushed product of the cross-linked polyallylamine 2/3 hydrochloride was
assayed in
the same manner as in Reference Example 6. The results are shown in Table 8.
As Comparative Example 3, the assay results of sevelamer hydrochloride in
Reference Example 6 are shown.
CA 02692515 2010-01-04
53
[0113]
[Table 8]
Assay Results of Polymers for in Vivo Test
Degree of Phosphate Bile acid Phosphate
swelling adsorption adsorption selectivity
capacity capacity
(mmol/g) (mmol/g)
Example 5 3.6 3.82 1.09 3.5
Comparative Example 3
(Reference Example 6) 6.2 2.70 2.30 1.2
[0114]
(In Vivo Test)
SD rats (SPF, male, 6 weeks old, JAPAN SLC) were separately kept under a
time-restricted feeding schedule of 8 hours/day with a feeding amount of 10
g/day.
After about I week of habituation, the polymer in Example 5 or sevelamer
hydrochloride (Comparative Example 3 (Reference Example 6)) was mixed with a
feed in an amount of 0.3% by weight or 1% by weight, and the rats were fed
with the
mixture for 3 days. Rats in the control group were fed with only the feed.
During
the 3 days of feeding, urine was collected for 24 hours/day and the daily
urinary
phosphate excretion amounts were measured to obtain the total sum of the
amount
during the 3 days. A decrease in the urinary phosphate excretion amount
indicates
the phosphate adsorption effect by the cross-linked polyallylamine
hydrochloride or
sevelamer hydrochloride.
[0115]
The results are shown in Fig. 1. The cross-linked polyallylamine
hydrochloride in Example 5 showed a dose-dependent and significant decrease in
the
phosphate excretion amount compared to the control group, and sevelamer
hydrochloride also showed the same level of the urinary phosphate excretion-
reducing effect. Therefore, it was shown that, in spite of the fact that the
cross-
linked polyallylamine hydrochloride in Example 5 has a lower degree of
swelling
CA 02692515 2010-01-04
54
than sevelamer hydrochloride, it has the same level of the phosphate
adsorption
effect as sevelamer hydrochloride.
[0116]
(Comparative Example 4)
Synthesis of Polymer Using N,N-diallyl-1,4-diaminobutane Bis(dihydrogen
phosphate) as Cross-linking Agent
The free-form cross-linked polyallylamine obtained during the process of
synthesis of the cross-linked polyallylamine hydrochloride in Comparative
Example
1-8 (N,N-diallyl-1,4-diaminobutane bis(dihydrogen phosphate) was used as the
cross-linking agent in an amount of 5 mol% with respect to the monomers) was
used
for the synthesis. To a round bottom flask, 3.30 g of the free-form cross-
linked
polyallylamine and 37 mL of water were placed and 37.4 mL of IM hydrochloric
acid was added thereto with stirring. After stirring the resulting mixture for
30
minutes at room temperature, solids were recovered by filtration. The
recovered
solids were washed with water and then ethanol. This was followed by drying
under reduced pressure at 50 C to obtain 4.60 g of a cross-linked
polyallylamine 2/3
hydrochloride.
[0117]
(Example 6)
Urinary Phosphate Excretion Test in Normal Rats (Comparison among Cross-
linking
Agents)
(Synthesis of Sample for Test)
The free-form cross-linked polyallylamine obtained during the process of
synthesis of the cross-linked polyallylamine hydrochloride in Example 3-1 (N,N-
2 5 diallyl- 1,3 -diaminopropane bis(dihydrogen phosphate) was used as the
cross-linking
agent in an amount of 5 mol% with respect to the monomers) was used for the
synthesis. To a round bottom flask, 2.70 g of the free-form cross-linked
CA 02692515 2010-01-04
polyallylamine and 30 mL of water were placed, and 30.6 mL of 1 M hydrochloric
acid was added thereto with stirring. After stirring the resulting mixture for
1 hour
at room temperature, solids were recovered by filtration. The recovered solids
were
washed with water and then ethanol. This was followed by drying under reduced
5 pressure at 50 C to obtain 3.89 g of a cross-linked polyallylamine 2/3
hydrochloride.
[0118]
(In Vivo Test)
SD rats (SPF, male, 6 weeks old, JAPAN SLC) were separately kept under a
time-restricted feeding schedule of 8 hours/day with a feeding amount of 10
g/day.
10 After about 1 week of habituation, the cross-linked polyallylamine
hydrochloride in
Example 6, the cross-linked polyallylamine hydrochloride in Comparative
Example
4, or sevelamer hydrochloride (Comparative Example 3 (Reference Example 6))
was
mixed with a feed in an amount of 1% by weight, and the rats were fed with the
mixture for 3 days. Rats in the control group were fed with only the feed.
During
15 the 3 days of feeding, urine was collected for 24 hours/day and the daily
urinary
phosphate excretion amounts were measured to obtain the total sum of the
amount
during the 3 days. A decrease in the urinary phosphate excretion amount
indicates
the phosphate adsorption effect by the cross-linked polyallylamine
hydrochloride or
sevelamer hydrochloride.
20 [0119]
The results are shown in Fig. 2. The cross-linked polyallylamine
hydrochloride in Example 6 showed a significant decrease in the phosphate
excretion
amount compared to the control, and showed a urinary phosphate excretion-
reducing
effect higher than that of sevelamer hydrochloride. On the other hand, while
the
25 cross-linked polyallylamine hydrochloride in Comparative Example 4 showed a
significant decrease in the phosphate excretion amount compared to the
control, its
effect was weaker than that of sevelamer hydrochloride and the cross-linked
CA 02692515 2010-01-04
56
polyallylamine hydrochloride in Example 6. The cross-linked polyallylamine
hydrochloride in Example 6 was shown to have a phosphate adsorption effect not
less than that of sevelamer hydrochloride. On the other hand, the cross-linked
polyallylamine hydrochloride in Comparative Example 4 using as the cross-
linking
agent N,N-diallyl-1,4-diaminobutane diphosphate was shown to have a phosphate
adsorption effect lower than that of the cross-linked polyallylamine
hydrochloride in
Example 6 and sevelamer hydrochloride.
[0120]
(Example 7)
Synthesis of Free-form Cross-linked Polyallylamine and Cross-linked
Polyallylamine
2/3 Hydrochloride for Surface Cross-linking Study and in Vitro Assay of Cross-
linked Polyallylamine 2/3 Hydrochloride
To a flask, 12.4 g (80 mmol) of allylammonium dihydrogen phosphate, 5.60 g
(16 mmol) of N,N-diallyl-1,3-diaminopropane bis(dihydrogen phosphate) and 8.0
mL of water were placed, and the atmosphere in the system was replaced with
argon
3 times, followed by heating the mixture in an oil bath at 50 C to dissolve
it. The
mixture was bubbled with argon for 30 minutes. To this mixture, 0.814 g(3
mmol)
of 2,2'-azobis(2-amidinopropane) dihydrochloride was added at an inner
temperature
of 50 C with stirring, and the resulting mixture was heated at an inner
temperature of
49 to 51 C for 64 hours. Several hours after the beginning of the heating,
the
mixture became solidified, and stirring was stopped. The obtained solids were
crushed and recovered by filtration, and washed with water and then ethanol.
The
solids were then dried under reduced pressure at room temperature to obtain
15.0 g
of white powder of a cross-linked polyallylamine phosphate. In 90 mL of water,
15.0 g of the obtained cross-linked polyallylamine phosphate was dispersed,
and 60
mL of 20% aqueous sodium hydroxide solution was added thereto with stirring.
The resulting mixture was stirred at room temperature for 1 hour, and solids
were
CA 02692515 2010-01-04
57
recovered by filtration, followed by washing thereof with water until the
filtrate
becomes neutral. The solids were transferred to a beaker, and 100 mL of water
was
added thereto, followed by 16 hours of stirring at room temperature. The
solids
were recovered by filtration, and washed with water and then ethanol. This was
followed by drying under reduced pressure at 50 C to obtain 5.12 g of a free-
form
cross-linked polyallylamine.
[0121]
Into a test tube, 0.25 g of the obtained free-form cross-linked polyallylamine
was weighed and dispersed in 5.0 mL of water. To the resulting mixture, 2.65
mL
of 1 M hydrochloric acid was added with ice cooling, while stirring thereof
with a
magnetic stirrer. After 30 minutes of stirring of the resulting mixture,
solids were
recovered by filtration. The obtained solids were washed with water and then
washed with ethanol sufficiently. This was followed by drying under reduced
pressure at room temperature to obtain 0.38 g of a cross-linked polyallylamine
2/3
hydrochloride. The obtained cross-linked polyallylamine 2/3 hydrochloride was
assayed in the same manner as in Reference Example 6. The results are shown in
Table 9.
[0122]
[Table 9]
Assay Results of 2/3 Hydrochloride of the Polymer for Surface Cross-linking
Study
(Sample without Surface Cross-linking)
Degree of Phosphate Bile acid Phosphate
swelling adsorption adsorption selectivity
capacity capacity
(mmol/g) (mmol/g)
Example 6 3.3 4.02 1.16 3.5
[0123]
(Examples 8 to 12)
Study of Reaction Conditions for Surface Cross-linking of Free-form Cross-
linked
CA 02692515 2010-01-04
58
Polyallylamine
The reaction conditions were studied using as the surface cross-linking agent
2-hydroxyethyl acrylate. In 4.0 mL of a solvent shown in Table 10, 0.25 g of
the
free-form cross-linked polyallylamine obtained in Example 7 was dispersed, and
the
resulting mixture was stirred at a temperature shown in Table 10. In 2.0 mL of
the
same solvent, 9.2 mg of 2-hydroxyethyl acrylate (3.7% by weight with respect
to the
free-form cross-linked polyallylamine) was dissolved, and this solution was
added to
the above mixture, followed by stirring the resulting mixture for 1 hour.
Solids
were recovered by filtration and washed with the same solvent as the one used
for the
reaction, followed by drying under reduced pressure at room temperature. The
obtained polymer was dispersed in 5.0 mL of water, and 2.65 mL of 1 M
hydrochloric acid was added thereto with ice cooling and stirring. After 30
minutes
of stirring of the mixture at room temperature, solids were recovered by
filtration.
The recovered solids were washed with water and then ethanol. The solids were
dried under reduced pressure at room temperature to obtain a cross-linked
polyallylamine 2/3 hydrochloride (surface cross-linked product). The obtained
cross-linked polyallylamine 2/3 hydrochloride (surface cross-linked product)
was
assayed in the same manner as in Reference Example 6. The results are shown in
Table 10. Compared to the case where surface cross-linking was not carried out
(Example 7), the degree of swelling decreased with any of the solvents,
although no
large difference was observed for the phosphate adsorption capacity.
CA 02692515 2010-01-04
59
[0124]
[Table 10]
Study Results of Surface Cross-linking Conditions Using 2-Hydroxyethyl
Acrylate
Solvent Temperature Degree Phosphate Bile acid Phosphate
of adsorptio adsorption selectivity
swelling n capacity capacity
(mmol/g) (mmol/g)
Example 7 - - 3.3 4.02 1.16 3.5
Example 8 ethanol 50 C 3.0 3.99 0.95 4.2
Example 9 ethyl 50 C 3.0 3.79 1.04 3.6
acetate
Example 10 heptane 50 C 3.0 3.99 0.92 4.3
Example 11 ethanol 30 C 2.8 3.78 1.03 3.7
Example 12 heptane 30 C 3.0 3.77 1.06 3.6
[0125]
(Examples 13 to 17)
Study of Amount of 2-Hydroxyethyl Acrylate to Be Added in Surface Cross-
linking
of Free-form Cross-linked Polyallylamine
2-Hydroxyethyl acrylate was used as the surface cross-linking agent, and its
amount to be added was studied. In 4.0 mL of ethanol, 0.25 g of the free-form
cross-linked polyallylamine obtained in Example 7 was dispersed, and the
resulting
mixture was stirred at 30 C. In 2.0 mL of ethanol, 2-hydroxyethyl acrylate in
an
amount shown in Table 11 was dissolved, and the resulting solution was added
to the
above mixture, followed by stirring the resulting mixture for 1 hour. Solids
were
recovered by filtration and washed with ethanol, followed by drying under
reduced
pressure at room temperature. The obtained polymer was dispersed in 5.0 mL of
water, and 2.65 mL of 1 M hydrochloric acid was added thereto with ice cooling
and
stirring. After stirring thereof at room temperature for 30 minutes, solids
were
recovered by filtration. The recovered solids were washed with water and then
ethanol. The solids were dried under reduced pressure at room temperature to
obtain a cross-linked polyallylamine 2/3 hydrochloride (surface cross-linked
product).
CA 02692515 2010-01-04
The obtained cross-linked polyallylamine 2/3 hydrochloride (surface cross-
linked
product) was assayed in the same manner as in Reference Example 6. The results
are shown in Table 11. The degree of swelling decreased depending on the
amount
of surface cross-linking agent added. Further, the phosphate adsorption
capacity
5 decreased depending on the amount of surface cross-linking agent added.
[0126]
CA 02692515 2010-01-04
61
¾" U
p U M M M M M
0
a1 o0 l- 00
p--~ N O O Q1
ig,
03 U
u
0
c4
p O O~ ch O~
M M M N
O C1~
N O bA
73
Q N 00 "O
~p ~ M M N N N
0
~
o U
O cd 7a .~bA 'C 'pb
U bA - ~ ..r
s,_ 3 3~ 3 3
cn o~ o 0 0 0 0
~D 73 00 r- oo r--
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~ ~ ~' ~ N t ON1
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CA 02692515 2010-01-04
62
[0127]
(Examples 18 to 21)
Surface Cross-linking Using Methyl Acrylate or Epichlorohydrin
In 4.0 mL of ethanol or heptane, 0.25 g of the free-form cross-linked
polyallylamine obtained in Example 7 was dispersed, and the resulting mixture
was
stirred at a temperature shown in Table 12. In 2.0 mL of the same solvent as
the
one used for dispersion of the above-described free-form cross-linked
polyallylamine,
methyl acrylate or epichlorohydrin was dissolved in an amount shown in Table
12,
and the resulting solution was added to the above mixture, followed by
stirring the
resulting mixture for additional 1 hour. Solids were recovered by filtration
and
washed with the same solvent as the one used for the above-described cross-
linking,
followed by drying under reduced pressure at room temperature. The obtained
polymer was dispersed in 5.0 mL of water, and 2.65 mL of 1 M hydrochloric acid
was added thereto with ice cooling and stirring. After stirring thereof at
room
temperature for 30 minutes, solids were recovered by filtration. The recovered
solids were washed with water and then ethanol sufficiently. The solids were
dried
under reduced pressure at room temperature to obtain a cross-linked
polyallylamine
2/3 hydrochloride (surface cross-linked product). The obtained cross-linked
polyallylamine 2/3 hydrochloride (surface cross-linked product) was assayed in
the
same manner as in Reference Example 6. The results are shown in Table 12. In
all the cases, the degree of swelling decreased compared to that of the case
where
surface cross-linking was not carried out (Example 7).
[0128]
CA 02692515 2010-01-04
63
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Clr v~
=O b~A
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~
b~A
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~~ M a1 01 O d
pp O M N N N N
~
ct
Cd
+~ u
>1
~+ ¾
~
cl ~y o ~" o ~ o =-" o
~." 00 00 o et
"C ~..~
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cC o o ,b o o
vi ~~=.' M V M .Li' ,S~ V~
Cd~
V)
U
a~
~
~
v~ 00 0, o ,.-^ + l~ ^ .- + N N
Cd
u f'tl Ir-1 F+-1 F+I F+1 W
CA 02692515 2010-01-04
64
[0129]
(Example 22)
Urinary Phosphate Excretion Test of Normal Rats Using Surface Cross-Linked
Polymer
(Synthesis of Sample to Be Tested and in Vitro Assay Thereof)
In 90.0 mL of ethanol, 4.50 g of the free-form cross-linked polyallylamine
obtained in Example 7 was dispersed, and the resulting mixture was heated in
an oil
bath with stirring, to achieve an inner temperature of 50 C. In 45 mL of
ethanol,
0.166 g of 2-hydroxyethyl acrylate (3.7% by weight with respect to the free-
form
cross-linked polyallylamine) was dissolved, and the resulting solution was
added to
the above mixture. After 0.5 hours of stirring, solids were recovered by
filtration
and washed with ethanol. This was followed by drying under reduced pressure at
room temperature, and the solids were then dispersed in 50.0 mL of water. To
the
resulting mixture, 47.8 mL of 1 M hydrochloric acid was added with ice cooling
and
stirring. After stirring the resulting mixture at room temperature for 40
minutes,
solids were recovered by filtration. The recovered solids were washed with
water
and then ethanol. The solids were dried under reduced pressure at room
temperature to obtain 6.78 g of a cross-linked polyallylamine 2/3
hydrochloride
(surface cross-linked product). Thereafter, 6.00 g of the obtained cross-
linked
polyallylamine 2/3 hydrochloride (surface cross-linked product) was freeze-
crushed
to obtain 5.99 g of a freeze-crushed product of a cross-linked polyallylamine
2/3
hydrochloride (surface cross-linked product). The obtained freeze-crushed
product
of a cross-linked polyallylamine 2/3 hydrochloride (surface cross-linked
product)
was assayed in the same manner as in Reference Example 6. The results are
shown
in Table 13. The assay results of the sevelamer hydrochloride prepared in
Reference Example 6 are shown as Comparative Example 3.
CA 02692515 2010-01-04
[0130]
[Table 13]
Assay Results of Polymer for in Vivo Assay of Surface Cross-linked Product
Degree Phosphate Bile acid Phosphate
of adsorption adsorption selectivity
swelling capacity capacity
(mmol/g) (mmol/g)
Example 22 3.1 3.97 1.47 2.7
Comparative Example 3 6.2 2.70 2.30 1.2
(Reference Example 6)
5 [0131]
(In Vivo Assay)
SD rats (SPF, male, 6 weeks old, JAPAN SLC) were separately kept under a
time-restricted feeding schedule of 8 hours/day with a feeding amount of 10
g/day.
After about 1 week of habituation, the cross-linked polyallylamine 2/3
hydrochloride
10 (surface cross-linked product) in Example 22 or sevelamer hydrochloride
(Comparative Example 3 (Reference Example 6)) was mixed with a feed in an
amount of 0.3% by weight or 1% by weight, and the rats were fed with the
mixture
for 3 days. Rats in the control group were fed with only the feed. During the
3
days of feeding, urine was collected for 24 hours/day and the daily urinary
phosphate
15 excretion amounts were measured to obtain the total sum of the amount
during the 3
days. A decrease in the urinary phosphate excretion amount indicates the
phosphate
adsorption effect by the cross-linked polyallylamine 2/3 hydrochloride
(surface
cross-linked product) or sevelamer hydrochloride.
[0132]
20 The results are shown in Fig. 3. The cross-linked polyallylamine 2/3
hydrochloride (surface cross-linked product) in Example 22 showed a dose-
dependent and significant decrease in the phosphate excretion amount and the
same
level of the urinary phosphate excretion-reducing effect as sevelamer
hydrochloride.
CA 02692515 2010-01-04
66
Thus, it was shown that, while the cross-linked polyallylamine 2/3
hydrochloride
(surface cross-linked product) in Example 22 has a lower degree of swelling
than
sevelamer hydrochloride, it has the same level of the phosphate adsorption
effect as
sevelamer hydrochloride.
[0133]
(Reference Example 13)
Tablets of Renagel (registered trademark) which is a formulation containing
as an effective component sevelamer hydrochloride (hereinafter referred to as
"Renagel tablets") were pulverized using a grinder (A10, Junke&Kunkel IKA
Labortechnic). The assay results of the pulverized product of Renagel tablets
are
shown in Table 14. The phosphate selectivity was low and the degree of
swelling
was 6.7.
[0134]
[Table 14]
Assay Results of Pulverized Product of Renagel Tablets
Degree of Phosphate Bile acid Phosphate
swelling adsorption adsorption selectivity
capacity capacity
(mmol/g) (mmol/ )
Reference 6.7 3.36 2.51 1.3
Example 13
[0135]
(Example 23)
(Example of Synthesis by Reversed Phase Suspension Polymerization and in Vitro
Assay)
In a 3 L three-necked flask, 17.9 g of sorbitan monolaurate and 1.26 kg of
heptane were placed, and the atmosphere in the flask was replaced with
nitrogen.
The mixture was bubbled with nitrogen for 30 minutes with stirring. In a 200
mL
three-necked flask, 12.9 g (4.00 mmol) of 2,2'-azobis[2-(2-imidazolin-2-
yl)propane]
CA 02692515 2010-01-04
67
dihydrochloride and 25 mL of water were placed, and the atmosphere in the
flask
was replaced with nitrogen with stirring. In a 5 L four-necked flask equipped
with
a mechanical stirrer and a thermometer, 155 g (1.00 mol) of allylammonium
dihydrogen phosphate, 70.1 g (0.200 mol) of N,N-diallyl-1,3-diaminopropane
bis(dihydrogen phosphate) and 75 mL of water were placed, and the resulting
mixture was heated at an inner temperature of 50 C to dissolve the compounds,
followed by replacing the atmosphere in the flask to nitrogen. The aqueous
suspension of the 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride
prepared in advance was added to the resulting solution, and the solution of
sorbitan
monolaurate in heptane prepared in advance was further added thereto. After
stirring the resulting mixture at an inner temperature of 50 to 55 C for 20
hours, the
mixture was cooled to room temperature. To the mixture, 500 mL of ethanol was
added and the resulting mixture was filtered, followed by washing 3 times with
500
mL of ethanol, 7 times with 500 mL of water and twice with 500 mL of ethanol.
The obtained solids were dried under reduced pressure at 50 C to obtain 218 g
of a
cross-linked polyallylamine phosphate. In a 5 L four-necked flask, the
obtained
polyallylamine phosphate and 2 L of water were placed, and 1.2 L of 20%
aqueous
sodium hydroxide solution was added to the resulting mixture with stirring by
a
mechanical stirrer. After 1 hour of stirring at room temperature, the mixture
was
filtered. The solids were washed 8 times with I L of water and 3 times with
250
mL of ethanol. The obtained solids were dried under reduced pressure at 55 C.
A
10-g aliquot thereof was suspended in 200 mL of water and the suspension was
stirred vigorously with a mechanical stirrer for 15 hours, followed by washing
with
water and then ethanol. The solids were dried under reduced pressure at 50 C
to
obtain a free-form cross-linked polyallylamine. The obtained free-form cross-
linked polyallylamine was converted to 20% acetate, 40% acetate and
hydrochloride.
[0136]
CA 02692515 2010-01-04
68
(20% Acetate (Example 23-1))
In 24 mL of water, 3.00 g of the free-form cross-linked polyallylamine was
suspended, and 0.574 g of acetic acid dissolved in 6 mL of water was added to
the
suspension. After stirring the resulting mixture at room temperature for 30
minutes,
the mixture was filtered, and the solids were washed with water and then
ethanol.
The solids were dried under reduced pressure at 40 C to obtain 3.61 g of 20%
acetate
of the cross-linked polyallylamine.
[0137]
(40% Acetate (Example 23-2))
In 24 mL of water, 3.00 g of the free-form cross-linked polyallylamine was
suspended, and 1.15 g of acetic acid dissolved in 6 mL of water was added to
the
suspension. After stirring the resulting mixture at room temperature for 30
minutes,
the mixture was filtered, and the solids were washed with water and then
ethanol.
The solids were dried under reduced pressure at 40 C to obtain 4.18 g of 20%
acetate
of the cross-linked polyallylamine.
[0138]
(Hydrochloride (Example 23-3))
In 25 mL of water, 0.200 g of the free-form cross-linked polyallylamine was
suspended, and 1 mL of concentrated hydrochloric acid was added to the
suspension.
After stirring the resulting mixture at room temperature for 30 minutes, the
mixture
was filtered, and the solids were washed with water and then ethanol. The
solids
were dried under reduced pressure at 50 C to obtain 0.293 g of a cross-linked
polyallylamine hydrochloride.
[0139]
The three obtained samples of the acid addition salts of the cross-linked
polyallylamine were assayed in the same manner as in Reference Example 6. The
results are shown in Table 15. The assay results of the pulverized product of
CA 02692515 2010-01-04
69
Renagel tablets prepared in Reference Example 13 are shown as Comparative
Example 5. All the salts showed a lower degree of swelling, a higher phosphate
adsorption capacity and a higher phosphate selectivity than the pulverized
product of
Renagel tablets.
[0140]
CA 02692515 2010-01-04
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O N '" ~ oo M
r.
= ~ L1~ v~i
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:=d ~ ~ N O O r-+
0i
C/~ ...
cd ~ U
N
v~ y
i-=~
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> V =-\r O Q1 M .~
^O =~ ~ M M ~
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N
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CA 02692515 2010-01-04
71
[0141]
(In Vivo Assay)
SD rats (SPF, male, 6 weeks old, JAPAN SLC) were separately kept under a
time-restricted feeding schedule of 8 hours/day with a feeding amount of 10
g/day.
After about 1 week of habituation, the pulverized product of Renagel tablets
(Reference Example 13) or the cross-linked polyallylamine acetate in Example
23-1
or Example 23-2 was mixed with a feed in an amount shown in Table 16, and the
rats
were fed with the mixture for 3 days. Rats in the control group were fed with
only
the feed. Using sevelamer hydrochloride contained in the pulverized product of
Renagel tablets as the standard, the amount of the polymer was set to attain
the same
weight as the free form. During the 3 days of feeding, urine was collected for
24
hours/day and the daily urinary phosphate excretion amounts were measured to
obtain the total sum of the amount during the 3 days. A decrease in the
urinary
phosphate excretion amount indicates the phosphate adsorption effect by the
polymer.
[0142]
The results are shown in Fig. 4. The polymers in Example 23-1 and
Example 23-2 showed significant decreases in the phosphate excretion amount,
and
showed the same level of the urinary phosphate excretion-reducing effect as
the
pulverized product of Renagel tablets (Reference Example 13). Thus, the
polymers
in Example 23-1 and Example 23-2 were shown to have the same level of the
phosphate adsorption effect as the pulverized product of Renagel tablets,
while
having a lower degree of swelling than that of the pulverized product of
Renagel
tablets.
CA 02692515 2010-01-04
72
[0143]
[Table 16]
Dosage of Samples in Example 23
Dosage (mg)
Control 0
Comparative Example 5 (Reference Example 13) 120
Example 23-1 98
Example 23-2 114
[0144]
(Example 24)
(Example of Synthesis by Precipitation Polymerization and in Vitro Assay)
To a 100 mL four-necked flask equipped with a mechanical stirrer, 9.31 (60
mmol) of allylammonium dihydrogen phosphate, 4.20 g (12 mmol) of N,N-diallyl-
1,3-diaminopropane bis(dihydrogen phosphate), 12 mL of water and 12 mL of
ethanol were placed, and the atmosphere in the system was replaced 3 times
with
argon, followed by addition thereto 0.814 g(3 mmol) of 2,2'-azobis(2-
amidinopropane) dihydrochloride and heating the resulting mixture at a bath
temperature of 60 to 65 C with stirring for 20 hours. The obtained solids were
recovered by filtration and washed with water and then ethanol. The solids
were
dried under reduced pressure at room temperature to obtain 10.3 g of powder of
a
cross-linked polyallylamine phosphate. In 54 mL of water, 9.00 g of the
obtained
cross-linked polyallylamine phosphate was dispersed, and 36 mL of 20% aqueous
sodium hydroxide solution was added thereto with stirring with a magnetic
stirrer.
The resulting mixture was stirred at room temperature for 1 hour, and solids
were
recovered by filtration, followed by washing thereof with water until the
filtrate
becomes neutral. The solids were washed with ethanol and dried under reduced
CA 02692515 2010-01-04
73
pressure at 50 C to obtain 3.5 g of a free-form cross-linked polyallylamine.
[0145]
While stirring 2.26 g of the free-form cross-linked polyallylamine by a
magnetic stirrer, 24 mL of I M hydrochloric acid was added thereto. After 1
hour
of stirring at room temperature, solids were recovered by filtration. The
recovered
solids were washed with water and then ethanol sufficiently. The solids were
dried
under reduced pressure at room temperature to obtain 3.14 g of a cross-linked
polyallylamine 2/3 hydrochloride. The obtained cross-linked polyallylamine 2/3
hydrochloride was assayed in the same manner as in Reference Example 6. The
results are shown in Table 17. The assay results of the pulverized product of
Renagel tablets prepared in Reference Example 13 are shown as Comparative
Example 5. The polymer obtained by precipitation polymerization showed a lower
degree of swelling, a higher phosphate adsorption capacity and a higher
phosphate
selectivity.
[0146]
[Table 17]
Assay Results of Polymer Obtained by Precipitation Polymerization
Degree of Phosphate Bile acid Phosphate
swelling adsorption adsorption selectivity
capacity capacity
(mmol/g) (mmol/g)
Comparative Example 5 6.7 3.36 2.51 1.3
(Reference Example 13)
Example 24 4.0 4.50 0.85 5.3
[0147]
(In Vivo Assay)
SD rats (SPF, male, 6 weeks old, JAPAN SLC) were separately kept under a
time-restricted feeding schedule of 8 hours/day with a feeding amount of 10
g/day.
After about 1 week of habituation, sevelamer hydrochloride contained in the
CA 02692515 2010-01-04
74
pulverized product of Renagel tablets (Reference Example 13) or the polymer in
Example 24 was mixed with a feed in an amount of 1% by weight, and the rats
were
fed with the mixture for 3 days. Rats in the control group were fed with only
the
feed. During the 3 days of feeding, urine was collected for 24 hours/day and
the
daily urinary phosphate excretion amounts were measured to obtain the total
sum of
the amount during the 3 days. A decrease in the urinary phosphate excretion
amount indicates the phosphate adsorption effect by the polymer.
The results are shown in Fig. 5. The polymer in Example 24 showed a
significant decrease in the phosphate excretion amount, and showed the same
level of
the urinary phosphate excretion-reducing effect as the pulverized product of
Renagel
tablets (Reference Example 13). Thus, the polymer in Example 24 was shown to
have the same level of the phosphate adsorption effect as the pulverized
product of
Renagel tablets, while having a lower degree of swelling than that of the
pulverized
product of Renagel tablets.
[0148]
(Example 25)
Into a polypropylene test tube, 500 mg of the allylamine-type polymer
synthesized by the method in Example 15 or the pulverized product of Renagel
tablets prepared in Reference Example 13 was weighed, and a 50 mg/mL
suspension
was prepared with the second fluid of the disintegration test described in
Japanese
Pharmacopoeia 14th revised edition (0.05 mol/L potassium dihydrogen phosphate,
0.0236 mol/L sodium hydroxide, pH about 6.8). After the preparation, the
suspension was rotated on a wave rotor (Thermonics, WR-40) to avoid
precipitation
of the test substance. The colon was isolated from a Sprague Dawley (SD) rat
(SPF,
male, 7 weeks old, JAPAN SLC) under anesthesia with ether and washed with cold
physiological saline, followed by excision of 2 pieces with a length of 5 cm.
The
intestinal tract was reversed using a sonde for mice, which sonde had been
weighed
CA 02692515 2010-01-04
in advance (pre), and the both ends of the intestinal tract were knotted by
surgical
suture to fix the intestinal tract to the sonde, followed by measuring the
weight of the
intestinal tract + the sonde (pre). In each of the polypropylene tubes
containing the
test substance solutions stirred in advance with the wave rotor, the
intestinal tract +
5 the sonde was placed such that it does not move, and the tubes were rotated
on the
wave rotor for 5 minutes. Five minutes later, the intestinal tract + the sound
were
recovered from each polypropylene tube, and the weight of the intestinal tract
+ the
sonde (post) and the weight of the sonde (post) were measured. The amount of
each test substance solution adhered to the intestinal tract was calculated
according
10 to the formula below.
{[the weight of the intestinal tract + the sonde (post)] - [the weight of the
intestinal tract + the sonde (pre)] } - { [the sonde (post)] - [the sonde
(pre)] }
[0149]
The results are shown in Fig. 6. The weights of the polymers synthesized by
15 the method in Example 15 adhered to the colon were significantly lower
compared to
those of the pulverized products of Renagel tablets (Comparative Example 6).
[0150]
(Example 26)
Intestinal tract-adherence tests were carried out on the polymers with and
20 without surface cross-linking, which were obtained by the reversed phase
suspension
polymerization. The polymers to be assayed were synthesized using a free-form
cross-linked polyallylamine synthesized according to the method shown in
Example
23.
[0151]
25 (Synthesis of Polymer without Surface Cross-linking (Example 26-1))
In 80 mL of water, 4.00 g of a free-form cross-linked polyallylamine was
suspended, and 80 mL of 1 M hydrochloric acid was added thereto with stirring.
CA 02692515 2010-01-04
76
After 30 minutes of stirring at room temperature, solids were recovered by
filtration.
The solids were washed with water and then ethanol. The solids were then dried
under reduced pressure at 50 C to obtain 6.45 g of a cross-linked
polyallylamine
hydrochloride.
[0152]
(Synthesis of Polymer with Surface Cross-linking (Example 26-2))
In 80 mL of ethanol, 4.00 g of a free-form cross-linked polyallylamine was
dispersed, and the resulting mixture was stirred at 30 C. To the mixture,
0.370 g of
2-hydroxyethyl acrylate dissolved in 20 mL of ethanol was added, and the
resulting
mixture was stirred for 30 minutes. Solids were recovered by filtration and
washed
with ethanol, followed by drying under reduced pressure at room temperature.
The
obtained polymer was dispersed in 80 mL of water, and 80 mL of 1 M
hydrochloric
acid was added thereto with ice cooling and stirring. After 30 minutes of
stirring at
room temperature, solids were recovered by filtration. The recovered solids
were
washed with water and then ethanol. This was followed by drying under reduced
pressure at room temperature to obtain 6.70 g of a cross-linked polyallylamine
hydrochloride (surface cross-linked product).
[0153]
(Intestinal Tract-adherence Test)
Intestinal tract-adherence tests were carried out by the method shown in
Example 25.
The results are shown in Fig. 7. The cross-linked polyallylamine hydrochloride
(surface cross-linked product) obtained by the reversed phase suspension
polymerization showed significantly lower values of the weight of adherence to
the
colon compared to the pulverized products of Renagel tablets (Comparative
Example
6) irrespective of whether or not the surface cross-linking was carried out.
INDUSTRIAL APPLICABILITY
[0154]
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77
Since the cross-linked polyallylamine or the acid addition salt thereof of the
present invention has a high phosphate adsorption capacity and phosphate
selectivity,
and a remarkably lower degree of swelling than the prior art, it is
appropriate as a
pharmaceutical agent having less side effects such as constipation, abdominal
pain
and abdominal distension, especially as a therapeutic or prophylactic agent
for
hyperphosphatemia.
