Note: Descriptions are shown in the official language in which they were submitted.
1 3351 42
The present invention relates to novel ingestible
covalently cross-linked amine homopolymers which are useful
as adsorbents ~for bile salts. More particularly, the
invention is directed toward the treatment of
hypercholesterolemia by removing through adsorption the bile
; acids and salts from the small intestine, thereby increasing
the catabolism of cholesterol in the liver with a
concomitant decrease in the blood cholesterol level.
Elevation of the blood cholesterol,
hypercholesterolemia, is widely considered as a ma~or risk
factor for the development of atherosclerosis and
cardiovascular diseases. It is presently the leading cause
of death of adults in most of the developed countries. Over
the last few decades, researchers have focussed their
attention on lowering the cholesterol level in the blood to
reduce the risk of cardiovascular diseases. This can be
achieved with limited success by reducing in the cholesterol
intake from food sources and accelerating the elimination of
cholesterol from the human body, although genetic factors
can also be important. In severe cases, the diseases can be
treated clinically by oral drugs, surgery, hemoperfusion or
a combination of these treatments.
Biologically, cholesterol is eliminated from the
human body by conversion to bile acids and excretion
as neutral steroids. Bile acids are synthesized daily
from cholesterol in the liver and enter the bile as glycine
and taurine conjugates. They are released in salt form with
bile during digestion. Bile salts are mostly reabsorbed in
the ileum with only about 1~ loss per cycle, complexed
with proteins and returned to the liver through hepatic
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.,
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1 335 1 42
portal veins. This small loss of bile salts represents a
major route for the elimination of cholesterol from the
body.
The available prescription drugs interrupt
either the biosynthesis of cholesterol in the body or the
enterohcpatic circulation of bile salts. Some of the
inhibitors for the biosynthesis of cholesterol, such as
lovastatin (Mevinolin), are reported to be significantly
effective. Their clinical usefulness is limited however,
due to some of their untoward side effects. Medicines
acting as adsorbents, such as cholestyramine and
colestipol, bind bile salts in the small intestine, thus
preventing the reabsorption of bile salts. The fecal
excretion of bile salts is enhanced under the effect of
cholestyramine and, therefore, the conversion of
cholesterol to bile acids is accelerated to maintain the
bile pool. However, both cholestyramine and colestipol
have major side effects which include the bad taste and
the dryness of cholestyramine, low adsorption capacity of
colestipol, and their poor biological compatibilities.
Cholestyramine, the most widely used adsorbent
for bile salts, is a copolymer of polystyrene and divinyl-
benzene with quaternary ammonium groups as functional
groups. Being a typical strongly basic ion exchanger, its
counterions of the quaternary ammonium, usually chloride
ions, are exchanged with bile salt anions during the
binding. The hydrophobic nature of the polymer backbone
results in its poor biocompatibility. As a consequence,
adverse side effects have been experienced by hypercho-
lesterolemic patients. The drug has to be taken in largedosage and may cause stomach discomfort to patients.
,,,:
-- 2
~ 1 335 1 ~2
It is therefore an object of the present invention
to overcome the above drawbacks and to provide novel bile
salt adsorbents with high adsorption capacity, good
biocompatibility and improved taste.
In accordance with the invention, there is
provided a swellable, covalently cross-linked amine
homopolymers having the formula:
/ [(CH2)oNR]mR
p-[~CH2)nNR]mR or p-(CH2)n ~ N
\ [(CH2)pNR]mR
(Ia) (Ib)
as well as pharmaceutically acceptable salts thereof having
the formulae:
P-[(CH2)nN+(R)2]m R-mX~ (Ic)
and
[(cH2)oN+(R)2]mR mX~
~-(CH2)n - N+ (Id)
X [ (CH2)pN+ (R) 2~mR mX
wherein:
P represents a hydrophilic, covalently cross-
linked and non-digestible homopolymer backbone;
R is a hydrogen atom or a lower alkyl radical;
X~ is a pharmaceutically acceptable anion;
m is an integer varying from 1 to 10 inclusive;
and
n, o and p are, independently, integers varying
from 2 to 12 inclusive.
It has been found quite unexpectedly that the
above polymeric amines exhibit increased hydrophilicity and
are highly efficient adsorbents for cholic acid and
glycocholic acid as well as other bile acids, such as
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1 335 1 ~2
chenodeoxycholic acid, lithocholic acid, deoxycholic acid
and taurocholic acid. The significance of the bile acid
adsorption is related to the lowering of serum cholesterol.
As it is known, cholesterol is a major and probably the sole
precursor of bile acids during normal digestion, bile acids
are secreted via the bile from the liver and the gallbladder
into the intestine. Bile acids emulsify the fat and lipid
materials present in the foods, thus facilitating
adsorption. A major portion of bile acids secreted is
reabsorbed from the intestines and returned via the portal
circulation of the liver, thus completing the enterohepatic
cycle. The binding of bile acids in the intestines onto an
insoluble adsorbent that is excreted in the feces results
impartial removal of bile acids from the enterohepatic
circulation, preventing their readsorption. The increased
fecal loss of bile acids leads to an increased oxidation of
cholesterol to bile acids, a decrease in beta lipoprotein or
low density lipoprotein serum levels, and a decrease in
serum cholesterol level. Thus, the compounds of the
invention can be used for reducing hypercholesterolemia in
affected humans.
Accordingly, the present invention also provides,
in a further aspect thereof, a method of treating
hypercholesterolemia in an affected human, which comprises
administering to the affected human an effective amount of a
bile salt adsorbent consisting of a covalently cross-linked
amine homopolymer as defined above.
According to yet another aspect of the invention,
there is provided a pharmaceutical composition for the
treatment of hypercholesterolemia, which comprises as active
ingredient a covalently cross-linked amine homopolymer as
-- 4 --
3~
t 335 1 ~2
defined above, together with a pharmaceutically acceptable
carrier therefor.
The polymer backbone to which the amino groups are
chemically bonded must be hydrophilic so as to swell in an
aqueous medium. This ensures good contact with the medium
and also opens the pores in the polymer so that there is
good access to all of the functional groups. The polymer
backbone must also be cross-linked to prevent the adsorbent
from diffusing from the digestive tract, as well as non-
digestible to prevent the adsorbent from being broken down
and absorbed into the body. It is preferably porous to
permit diffusion of the bile salts which are to be
sequestered, thereby improving adsorption capacity.
A preferred polymer resin for use as backbone to
which the amino groups can be attached is a porous, cross-
linked polymethylacrylate resin. Such a resin is
advantageously prepared by polymerizing methyl acrylate in
the presence of two cross-linking agents used in a ratio of
1:1.
Particularly preferred polymeric amines according
to the invention are the homopolymers functionalized with
linear amines of formula (Ia) and their protonated and
quaternized derivatives of formula (Ic), in which R is a
hydrogen atom or a methyl radical, m is 1, 2 or 3, n is 2 or
3, P represents a polyacrylamide resin and X- is a
ph~rm~ceutically acceptable anion such as Cl-, I- or OH-.
Amongst the homopolymers functionalized with
branched amines of formula (Ib) and their protonated and
quaternized derivatives of formula (Id), the preferred
compounds are those in which R is a hydrogen atom or a
methyl radical, m is 1, n, o and p are each 2, P represents
-- 5
~ 1 335 ~ 42
a polyacrylamide resin and X~ is a pharmaceutically
acceptable anion.
The polymeric amines according to the invention
not only exhibit high adsorption capacity but also high
water swellability, which render them suitable for clinical
application.
Further features and advantages of the invention
will become more readily apparent from the following non-
limiting examples and the accompanying drawings, in which:
Figs. 1 and 2 show the adsorption isotherms of
compounds according to the in~ention for sodium glycocholate
in 0.0025 M, pH 7.1 Tris buffer, compared with the
adsorption isotherms of cholestyramine and colestipol (used
as reference adsorbents);
Fig. 3 shows bile salt adsorption isotherms in
simulated small intestine contents; and
Fig. 4 shows bile salt adsorption isotherms in an
~ .
extract from pig small intestine.
1. Preparation of Polymer Backbone
A suitable carrier resin was synthesized by
polymerized methyl acrylate in the presence of cross-linking
agents to form a porous, cross-linked polymethylacrylate
(PMA) resin.
The polymerization was carried out in a 1000 ml 3-
necked flask equipped with a mechanical stirrer and a
condenser. Into the flask, 25 grams of NaCl and 480 ml
distilled water were added. The solution was stirred until
all of the NaCl has been dissolved. The temperature of the
~. .
-- 6 --
~;
1 335 1 42
water bath was set at 50 C. 120 ml of 2% polyvinyl alcohol
(PVA) solution was added and the solution was mixed. The
position of the stirring blade was adjusted so that the
top of the blade was at the surface of the water phase.
In a separate beaker, 94 grams of methyl
acrylate, 3.0 grams of each of the cross-linking agents,
divinyl benzene and triallyl-1,3,5-triazine-2,4,6-
(lH,3H,5H)-trione, and 1.4 grams of benzoyl peroxide were
added. The benzoyl peroxide was allowed to dissolve
completely. Then 20-25 grams of butyl ether was added and
the contents were well mixed.
This mixture was then added to the contents of
the 1000 ml flask, and formed an oil phase. Stirring was
then commenced with the speed being controlled so as to
yield the appropriate bead size for the carrier resin.
The temperature of the water bath was then
increased slowly (8C / hr~ until it reached 60C, and
then increased more slowly (4C / hr) to 68C. The system
was maintained at this temperature for 24 hours, following
which the polymerization was continued for 24 hours at
80C and another 24 hours at 95C.
The product was cooled and washed repeatedly
with warm water to remove the PVA. It was then washed by
refluxing methanol for 24 hours to remove butyl ether and
soluble polymer species. The carrier resin thus obtained
was ready for functionalization.
2. Functionalization of Polymer Backbone
A 200 ml 3-necked flask equipped with a
mechanical stirrer, a condenser, a thermometer, CaCO3
drying tube was immersed in an oil bath. 5 grams of PMA
and 100 ml of an alkyldiamine were added into .the flask
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.~
1 335 1 42
and stirred for one hour at room temperature. The temper-
ature was then increased to 50C and was maintained for 3
hours. Thereafter it was increased to 120C, and main-
tained at this temperature for 4 days. The amine-contain-
ing resin thus obtained was repeatedly washed with
methanol, and then with distilled water. It was finally
dried under vacuum.
3. Quaternization of the Amine-Containing
Resins
A 500 ml 3-necked flask equipped with mechani-
cal stirrer, a condenser to which a CaC03 drying tube was
attached, a thermometer was immersed in an oil bath. 5
grams of amine-containing resin prepared above, 25 grams
of KHC03 and 150 ml methanol were added into the flask.
After 2 hours of stirring at 35 C, 80 ml of methyl iodide
was added. The reaction was maintained for 4 days in the
dark. The final product was repeatedly washed with
methanol, with distilled water, with concentrated NaCl
solution, and eventually with water.
4. Protonation of the Amine-Containing Resins
The amine-containing resins were treated with
dilute hydrochloric acid solution at room temperature to
convert the free amine groups to positively charged
organic ammonium groups. This can be done either in a
column where dilute HC1 passes through the column until
the protonation is complete, or simply in a container
where an excess amount of hydrochloric acid is in contact
with the resin ~standing or shaking). Then the excess
hydrochloric acid was washed away with a large amount of
distilled water until the resin is neutral.
.,
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1 335 1 42
5. Characterization of the Adsorbents
The products were characterized both qualitati-
vely by infrared spectroscopy and quantitatively by acid-
base back titration. IR results proved that the ester
groups in the former PMA backbone had been converted to
amide groups and the amine-containing small molecules have
been chemically attached to the polymer backbone as
expected. From acid-base back titration, it was found that
all of the products were nitrogen rich materials and that
they had subtitutions (free base or protonated or quater-
nized nitrogen) of about 2-8 mmol/g.
6. Adsorption Studies
(a). Adsorption studies in Tris buffer:
Tris(hydroxymethyl)-aminomethane (Aldrich) and
1.000 N standard HCl solution were used to prepare a
buffered solution with ionlc strength 0.0025 M and pH 7.1.
With this buffer, bile salt solution with concentration
about 50 mg/dl was prepared and was used directly. Into
bottles of different sizes (2-100 ml), about 5-15 mg of
the resin to be tested was weighed. Then different volumes
of bile salt solution (1-50 ml) were added into the
bottles. By changing the volumes of the bile salt solution
added, a whole range of bile salt equilibrium concentra-
tions was easily reached. They were shaken at room temper-
ature (15-25C) for more than 2 hours. Then they were
filtered and the clear solutions were analyzed by High
Performance Liquid Chromatography (HPLC) for which a
perfect linear calibration curve was obtained under the
used experimental conditions.
1 335 1 42
(b). Adsorption Studies in Simulated Intestine
Contents and Extracted Pig Small Intestine:
The simulated small intestine solution with bile
salt concentration about 100 mg/dl was prepared by
dissolving each capsule of COTAZYM*-65B (Organon) in 60 ml
distilled water. Pig small intestine was collected from
freshly killed pigs. The contents of the small intestine
were squeezed into a container and filtered to obtain a
milky fluid for the adsorption studies. The pH of the above
two was measured to be close to 7Ø About 3-5 mg of the
resin was weighed into bottles of different sizes and each
was pre-swollen with one-drop of distilled water. Different
volumes of the solution were added into the resin bottles.
They were sha~en at 37C for 4 hours. 200 ,ul of each fluid
was centrifuged. The upper clear solution was diluted 50
times and was analyzed by fluorescence.
EXAMPLE 1
A hydrophilic amine-contA; n; ng resin was prepared
as described above by grafting onto the cross-linked
polymethylacrylate backbone ethylene diamine and was then
converted to the hydrochloric form by washing with dilute
aqueous HCl. This material, designated 'lunquaternized resin
1", was stirred or shaken with a Na+-glycocholate solution
in Tris buffer at initial concentration of 30-50 mg/dl for
more than 2 hours. The amount of Na+-glycocholate adsorbed
was measured by HPLC as described above. The adsorption
isotherm is shown in Fig. 1. At an equilibrium concentration
of 0.4 mM, this resin adsorbed 4.5 mmol of Na+-glycocholate
per gram of resin.
* Trade Mark
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1 335 1 4 2
EXAMPLE 2
Example 1 was repeated except that diethylene-
triamine instead of ethylene diamine was grafted onto the
polymethylacrylate backbone. The product obtained, desig-
nated "unquaternized resin 2", adsorbed 3.2 mmol of Na -
glycocholate per gram of resin at an equilibrium concen-
tration of 0.4 mM. The adsorption isotherm is shown in
Fig. 2.
EXAMPLE 3
Example 1 was repeated except that 1,4-diamino-
butane instead of ethylene diamine was grafted onto the
polymethylacrylate backbone. The product obtained, desig-
nated "unquaternized resin 3", adsorbed 1.5 mmol of Na -
glycocholate per gram of resin at an equilibrium concen-
tration of 0.4 mM. The adsorption isotherm is shown in
Fig. 2.
EXAMPLE 4
Example 1 was repeated except that 1,6-hexane-
diamine instead of ethylene diamine was grafted onto the
polymethylacrylate backbone. The product obtained, desig-
nated "unquaternized resin 4", adsorbed 2.0 mmol of Na+-
glycocholate per gram of resin at an equilibrium concen-
tration of 0.4 mM. The adsorption isotherm is shown in
Fig. 2.
EXAMPLE 5
Example 1 was repeated except that 1,3-diamino-
propane instead of ethylene diamine was grafted onto the
polymethylacrylate backbone. The product obtained, desig-
nated "unquaternized resin 5", adsorbed 4.2 mmol of Na -
~ 335 1 42
glycocholate per gram of resin at an equilibrium concentra-
tion of 0.4 mM. The adsorption isotherm is shown in Fig. 2.
EXAMPLE 6
Example 1 was repeated except that 1,12-diamino-
dodecane instead of ethylene diamine was grafted onto the
polymethylacrylate backbone. The product obtained,
designated "unquaternized resin 6", adsorbed 1.2 mmol of
Na -glycocholate per gram of resin at an equilibrium concen-
tration of 0.4 mM. The adsorption isotherm is shown in Fig.
2.
EXAMPLE 7
~`~~ Example 1 was repeated except that the amine-
containing resin was quaternized with methyl iodide and then
was converted to chloride form by washing with concentrated
sodium chloride solution. The product obtained, designated
"quaternized resin 1", adsorbed 1.7 mmol of Na -glycocholate
per gram of resin at an equilibrium concentration of 0.4 mM.
The adsorption isotherm is shown in Fig. 1.
EXAMPLE 8
Example 2 was repeated except that the amine-
containing resin was quaternized and converted in the manner
described in Example 7. The product obtained, designated
"quaternized resin 2", adsorbed 3.0 mmol of Na -glycocholate
per gram of resin at an equilibrium concentration of 0.4 mM.
The adsorption isotherm is shown in Fig. 1.
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1 33 5 1 4 2
EXAMPLE 9
Example l was repeated except that the tri-
ethylenetetramine instead of ethylene diamine was grafted
onto the polymethylacrylate backbone and that the amine-
containing resin was quaternized and converted the way
mentioned in Example 7. The product obtained, designated
"quaternized resin 7", adsorbed 3.2 mmol of Na -glyco-
cholate per gram of resin at an equilibrium concentration
of 0.4 mM. The adsorption isotherm is shown in Fig. l.
EXAMPLE lO
Example l was repeated except that tris(2-
aminoethyl) amine instead of ethylene diamine was grafted
onto the polymethylacrylate backbone. The product
obtained, designated "unquaternized resin 8", adsorbed 2.2
mmol of Na -glycocholate per gram of resin at an equili-
brium concentration of 0.4 mM. The adsorption isotherm is
shown in Fig. 2.
EXAMPLE ll
The amine-containing resin of Example 2, desig-
nated "unquaternized resin 2", was stirred or shaken witha simulated small intestine solution as defined above for
4 hours at 37C and at Na -glycocholate initial concentra-
tion of about lO0 mg/dl. The amount of Na+glycocholate
adsorbed by this resin, as measured by fluorescence, was
0.7 mmol of Na -glycocholate per gram of resin at an
equilibrium concentration of l.0 mM. The adsorption
isotherm is shown in Fig. 3.
EXAMPLE 12
The amine-containing resin of Example 8, desig-
nated "quaternized resin 2", was stirred or shaken with asimulated small intestine solution as defined above for 4
.,
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1 335 1 42
hours at 37C and at Na -glycocholate initial concentra-
tion of about 100 mg/dl. The amount of Na -glycocholate
adsorbed by this resin, as measured by fluorescence, was
1.8 mmol of Na -glycocholate per gram of resin at an
equilibrium concentration of 1.O mM. The adsorption
isotherm is shown in Fig. 3.
EXAMPLE 13
The amine-containing resins of Examples 2 and
8, designated "unquaternized resin 2" and "quaternized
resin 2", respectively, were stirred or shaken with
extracted pig small intestine as defined above for 4 hours
at 37C and at a bile salt initial concentration of about
150 mg/dl. Good adsorption capacities were manifested in
both cases at concentration above 2 mM/L. The adsorption
isotherms are shown in Fig. 4.
The adsorption capacities of the amino-contain-
ing resins prepared in Examples 1 through ~ are summarized
in the following Table:
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1 335 1 ~2
TABLE 1
Ex. ProductStructure Adsorption
No. Designation Capacity (*)
1 Unquater-
nized
Resin 1P (CH ) N+H Cl- 4 5
2 Unquater-
nized +
Resin 2p-[(CH2)2N H2Cl ]2H 3.2
3 Unquater-
nized +
Resin 3P-(CH2)4N H3C1 1.5
4 Unquater-
nized +
Resin 4P-(CH2)6N H3C1 2.0
5 Unquater-
nized
Resin 5 ( 2)3 3C 4.2
6 Unquater-
nized
Resin 6 ( 2)12 3 1.2
7 Quater-
nized +
Resin 1P-(CH2)2N (CH3)3C1 1.7
8 Quater-
nized +
Resin 2P-[(CH2)2N (CH3)2Cl ]2CH3
9 Quater-
nized
Resin 7P-[(CH2)2N (CH3)2Cl ]3C 3 3.2
10 Unquater-/ (CH2)2N H3Cl
nized
Resin 8P-(CH2)2N HCl 2.2
\ (CH2)2N H3Cl
(*) mmol of sodium glycocholate adsorbed per gram of resin
(at an equilibrium concentration of 0.4 mM).
1 335 1 42
As may be seen from Figs 1 and 2, the shapes of
the isotherms are strongly dependent on the structure
(hydrophobicity) of the sorbent. As the ionic strength of
the buffer increases, the adsorption affinity decreases
markedly. The adsorption by the unquaternized sorbents is
strongly dependent on pH and is favoured by an increase in
temperature and smaller particle size. It is also apparent
that the preferred amine-containing resins of the invent-
ion have higher adsorption capacities, in vitro, than the
commonly used cholestyramine and colestipol-
For any medical applications, especially by
oral administration, the water-swellability of the mate-
rial is often considered as a major evaluation parameter
because most of the human fluids have high water contents.
Generally, the more water-swellable the polymer material
is, the more biocompatible it will be.
The measurements of the water-swellability were
done in several 10 ml graduate cylinders. About 0.5 gram
of the resin was weighed and put into the cylinder. After
it was tapped for a while, the initial reading, volume of
the resin, was taken and recorded. Then enough distilled
water was added to the cylinder and let it stand for at
least 24 hours. The final volume was taken. The resin was
separated from the unabsorbed water. The swollen resin was
weighed again. The swellability and the water content
(adsorption capacity of water) were calculated, which were
expressed as percentage of the volume change over the
initial volume.
Swellability = (Vp-VO)/Vo x 100%
Water content = (Wp-WO)/Wo x 100%
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1 335 1 42
where:
VO and WO are the original values
Vp and Wp are the values after swelling.
The results from these measurements are listed
in the following Table:
TAB~E 2
ProductH2O swellability H2O content
Unquaternized
Resin 1 500% 560
Unquaternized
Resin 2 220% 240%
Unquaternized
Resin 4 162% 220%
Quaternized
Resin 1 260% 460%
Quaternized
Resin 2 338% 320%
Quaternized
Resin 4 150% 260%
. . i~ ~- 20 It can be seen that the above materials are
extremely water-swellable. For unquaternized resin 1,
after swelling, its volume is five times larger than
before, and can hold water five times more than its own
weight. Other resins, both quaternized and unquaternized,
have also shown tremendous H2O swellabilities.
