Crosslinked anion exchange particles and method for producing the particles

New bile acid sequestrant polymer compositions and a process for preparing the polymers in particulate form, preferably in spherical form, are described. The polymer particles are prepared by crosslinking an amine-containing polymer with an amount of a polyfunctional amine-reactive compound sufficient to crosslink the polymer so that it is essentially water insoluble and has bile acid sequestering efficacy greater than that of cholestyramine, preferably greater than about three times the efficacy of cholestyramine. A preferred polymerization process involves suspension polymerization of water-soluble amine-containing monomers, such as dialkylaminoalkyl (meth)acrylate esters and dialkylaminoalkyl (meth)acrylamides, in the presence of polyfunctional amine-reactive compounds, such as substituted dihaloalkanes. Pharmaceutical compositions containing the bile acid sequestrant polymer particles and a method for lowering blood cholesterol levels using the pharmaceutical compositions are also discribed.

BACKGROUND OF THE INVENTION 
It has been recognized that elevated levels of cholesterol in the blood 
plasma are a major risk factor of coronary heart disease in humans and 
that reducing plasma cholesterol level decreases the risk of coronary 
heart disease. Successful approaches to controlling blood cholesterol 
levels have included dietary modification, e.g., minimizing the intake of 
cholesterol-laden foods and of foods having high fat content, inhibiting 
cholesterol biosynthesis and encouraging an increase in the amount of bile 
acids eliminated by the body. 
Particulate resins, e.g., cholestyramine, described in U.S. Pat. No. 
3383281, and colestipol, described in U.S. Pat. No. 3692895, that are 
capable of sequestering bile acids are known. Such resins, when orally 
administered to a mammalian host, form complexes with bile acid conjugates 
in the intestine and are effective in blocking resorbtion of bile acids 
from the intestine. The resin and sequestered bile acids are subsequently 
excreted from the body in fecal matter thereby increasing the rate at 
which bile acids are eliminated from the body. Other factors being equal, 
an increase in the rate at which bile acids are eliminated from the body 
tends to lower plasma cholesterol level by accelerating the conversion of 
cholesterol to bile acids in order to maintain a constant supply of bile 
acids in the body. A portion of the cholesterol for this increased 
synthesis of bile acids is supplied by removal of cholesterol from the 
blood plasma. 
The bile acid sequestrants may be orally administered in various forms, 
typically as mixtures with food. Although the dosages of known 
sequestrants that are effective in lowering serum cholesterol in humans 
typically fall in the range of 10 to 15 grams/day, dosages of up to about 
50 grams/day may be required. The particulate bile acid sequestrant resins 
can be unpleasant to ingest, particularly when large dosages are required 
and adverse side reactions (bloating, gas formation, constipation, 
diarrhea and the like) are common among patients to whom the resins are 
administered. 
There has been a continuing effort in this field to minimize the unpleasant 
side effects associated with a therapeutically effective bile acid 
sequestrant regimen by developing sequestrants having increased ability to 
sequester bile acids and which are also effective in reducing serum 
cholesterol when administered at lower dosages than presently required 
using cholestyramine and colestipol. 
While new candidate bile acid sequestrants must possess satisfactory bile 
acid sequestering efficacy, they must also be non-toxic to the host 
receiving the treatment. Some bile acid sequestrants may possess 
satisfactory bile acid sequestering efficacy, e.g., water-soluble 
polymers, however, they have been found to be cytotoxic towards the host 
due to sensitivity of living tissue exposed to the water-soluble bile acid 
sequestrant. It is, therefore, desirable to provide a bile acid 
sequestrant that possesses the bile acid sequestering efficacy of such 
water-soluble polymers but without the cytotoxic side effects which occur 
due to intimate contact between the sequestrant used and the living 
tissues exposed to the sequestrant. 
One approach to providing bile acid sequestrants having the proper 
combination of physical properties is to polymerize functionalized 
monomers which are water-soluble due to their functionalized nature and to 
crosslink the polymer to such an extent to render it water-insoluble, thus 
minimizing cytotoxic effects, without hindering accessibility of the 
functionalized sites of the sequestrant to target bile acids to be 
removed. 
It is an object of the present invention to provide a bile acid sequestrant 
with enhanced bile acid sequestering efficacy and low mammalian 
cytotoxicity based on a crosslinked polymer made from functionalized 
water-soluble monomers. Another object of the invention is to provide a 
process for preparing the bile acid sequestrant polymer particles, 
preferably as spherical polymer particles. 
SUMMARY OF THE INVENTION 
The present invention provides essentially water-insoluble bile acid 
sequestrant polymer particles in the form of anion exchange resins and a 
process for preparing the polymer particles comprising (a) polymerizing a 
monomer mixture comprised of amine-containing monomers by free radical 
polymerization and (b) non-free-radical crosslinking with a polyfunctional 
amine-reactive compound, to provide polymer particles that have bile acid 
sequestering efficacy greater than that of cholestyramine. 
In one aspect of the invention the polymerization process comprises 
suspension polymerization of water-soluble amine-containing monomers, 
optionally using a sufficient amount of a dispersant to provide the 
polymer particles in spherical form. Another aspect of the invention 
involves conducting the polymerization wherein crosslinking with 
polyfunctional amine-reactive compounds occurs during formation of the 
polymer particles. 
In another aspect of the invention polymer compositions are provided that 
comprise bile acid sequestrant polymer particles prepared according to the 
aforementioned process, for example, wherein the amine-containing monomer 
is an unsubstituted or substituted aminoalkyl (meth)acrylate ester or an 
unsubstituted or substituted aminoaikyl (meth)acrylamide; and the 
polyfunctional amine-reactive compound is selected from unsubstituted or 
substituted members of the following classes: dihaloalkanes, aralkyl 
dihalides, alkylene diesters, aryl diesters, aralkyl diesters, alkylene 
diacylhalides, aryl diacylhalides, aralkyl diacylhalides, dialdehydes, 
diepoxyalkanes, epihalohydrins and aralkyl diepoxides. 
In another aspect of the invention polymer compositions are provided that 
comprise bile acid sequestrant polymer particles that have amine 
functionality attached to polymer backbone through a side chain linkage 
group. In yet another aspect the polymer compositions comprise polymer 
particles that are in the form of a pharmaceutically acceptable salt. 
The present invention provides pharmaceutical compositions comprising a 
therapeutically effective amount of the polymer composition of the bile 
acid sequestrant polymer particles and a pharmaceutically acceptable 
carrier. 
The present invention also provides a method for lowering blood cholesterol 
level in a mammal comprising oral administration to the mammal of a 
therapeutically effective amount of the bile acid sequestrant polymer 
particles prepared according to the aforementioned process. 
DETAILED DESCRIPTION OF THE INVENTION 
The anion exchange resins of the present invention may be prepared by 
several variations of the same process. In one variation the polymers may 
be produced by bulk polymerization in which the amine-containing monomer 
is first mixed with a monomer-soluble polyfunctional amine-reactive 
compound; the mixture is then heated, for example on a heated plate, roll 
or sheet, to polymerize the mixture to a solid mass, after which the solid 
polymer is granulated into particles by grinding, flaking or other similar 
means. 
In another variation the polymers may be produced wherein polymerizing a 
monomer mixture comprising amine-containing monomers by free radical 
polymerization is completed to produce an uncrosslinked polymer, followed 
by non-free-radical crosslinking with a polyfunctional amine-reactive 
compound to form the crosslinked polymer particles. Preferably, this type 
of polymer is prepared in aqueous solution and the resultant polymer may 
be further granulated to the desired particle size by grinding and similar 
procedures. 
In yet another variation the polymers of the invention are produced by 
suspension polymerization, preferably in aqueous media. A monomer mixture 
of one or more water-soluble, amine-containing monomers, optionally 
containing one or more additional, copolymerizable monomers, together with 
a monomer-soluble polyfunctional compound having functional groups capable 
of reacting with amine functional groups of the amine-containing monomer, 
is suspended in an aqueous medium and the suspension is polymerized in the 
presence of a monomer-soluble, free-radical initiator to form polymer 
particles which have amine functionality. Preferably suspension aids are 
used to provide polymer particles in spherical form; for example, the 
aqueous phase may contain dissolved inorganic salts and suitable 
dispersants. 
In a preferred embodiment of the invention the process comprises suspension 
polymerizing a monomer mixture comprised of water-soluble amine-containing 
monomers by free radical polymerization using a dispersant to provide the 
polymer particles in spherical form, and non-free-radical crosslinking 
with a polyfunctional amine-reactive compound during formation of the 
particles to provide polymer particles that (1) have bile acid 
sequestering efficacy greater than that of cholestyramine and (2) that 
have amine functionality attached to polymer backbone through a side chain 
linkage group. A more preferred embodiment of the polymer particles is in 
the form of a pharmaceutically acceptable salt having bile acid 
sequestering efficacy at least three times the efficacy of cholestyramine. 
As used herein, the terms "(meth)acrylate" and "(meth)acrylamide" refer to 
either the corresponding acrylate or methacrylate and acrylamide or 
methacrylamide, respectively. Also, as used herein, the term "substituted" 
is used in conjunction with various amine-containing monomers and 
polyfunctional amine-reactive compounds to indicate that one or more 
hydrogens of these compounds has been replaced, for example, with (C.sub.1 
-C.sub.8)alkyl, halogen (e.g., chloro-, bromo-), hydroxyl groups and the 
like, except where such groups may be incompatible with functional groups 
already present. 
Among those amine-containing monomers suitable for use in the present 
invention are those vinyl monomers containing amine functionality that is 
not directly attached to the vinyl group. Such monomers include, for 
example, amide monomers such as dialkylaminoalkyl acrylamides or 
methacrylamides (for example, dimethylaminopropyl methacrylamide), 
N,N-bis-(dimethylaminoalkyl)acrylamides or methacrylamides, 
N-.beta.-aminoethyl acrylamide or methacrylamide, 
N-(methylaminoethyl)acrylamide or methacrylamide, aminoalkylpyrazine 
acrylamides or methacrylamides; acrylic ester monomers such as 
dialkylaminoalkyl acrylates or methacrylates (for example, 
dimethylaminoethyl acrylate or methacrylate), .beta.-aminoethyl acrylate 
or methacrylate, N-(n-butyl)-4-aminobutyl acrylate or methacrylate, 
methacryloxyethoxyethylamine, and acryloxypropoxypropoxypropylamine; vinyl 
monomers such as vinyl pyridines; aminoalkyl vinyl ethers or sulfides such 
as .beta.-aminoethyl vinyl ether, .beta.-aminoethyl vinyl sulfide, 
N-methyl-.beta.-aminoethyl vinyl ether or sulfide, N-ethyl 
.beta.-aminoethyl vinyl ether or sulfide, N-butyl-.beta.-aminoethyl vinyl 
ether or sulfide, and N-methyl-3-aminopropyl Vinyl ether or sulfide; 
N-acryloxyalkyl-oxazolidines and N-acryloxyalkyltetrahydro-l,3-oxazines 
such as oxazolidinylethyl methacrylate, oxazolidinylethyl acrylate, 
3-(.gamma.-methacryloxypropyl)tetrahydro-1,3-oxazine, 
3-(.beta.-methacryloxyethyl)-2,2-pentamethylene-oxazolidine, 
3-(.beta.-methacryloxyethyl)-2-methyl-2-propyloxazolidine, 
N-2-(2-acryloxyethoxy)ethyl-oxazolidine, 
N-2-(2-methacryloxyethoxy)ethyl-5-methyl-oxazolidine, 
3-[2-(2-methacryloxyethoxy)ethyl]-2,2-dimethyloxazolidine, 
N-2-(2-acryloxyethoxy)ethyl-5-methyl-oxazolidine, 
3-[2-(methacryloxyethoxy)ethyl]-2-phenyl-oxazolidine, 
N-2-(2-methacryloxyethoxy)ethyl-oxazolidine, and 
3-[2-(2-methacryloxyethoxy)ethyl]-2,2-pentamethylene-oxazolidine. 
Preferred water-soluble, amine-containing monomers useful in the present 
invention are unsubstituted or substituted aminoalkyl (meth)acrylate 
esters and unsubstituted or substituted aminoalkyl (meth)acrylamides. 
Included among these monomers are: dimethylaminoalkyl acrylamides and 
methacrylamides, N,N-bis-(dimethylaminoalkyl) acrylamides and 
methacrylamides, dimethylaminoalkyl acrylates and methacrylates, or 
mixtures including any of these monomers. Most preferred are the 
dimethylaminoalkyl acrylamides and methacrylamides, dimethylaminoalkyl 
acrylates and methacrylates and mixtures thereof in which the alkyl group 
has from 2 to about 8 carbon atoms, and particularly preferred are 
dimethylaminopropyl methacrylamide, dimethylaminoethyl methacrylate and 
mixtures thereof. The water-soluble monomer is present in the monomer 
mixture as the major component; that is, the water-soluble monomer or 
monomers are present at a level of at least 50 weight percent by weight of 
the total monomers. As used herein, the term "water-soluble," as applied 
to monomers, indicates that the monomer has a solubility of at least about 
1 gram per 100 grams of water, preferably at least about 10 grams per 100 
grams of water, and more preferably at least about 50 grams per 100 grams 
of water. 
Other, non-amine-containing, monomers may optionally be present as minor 
components of the monomer mixture; that is, they may be present in a total 
combined amount of less than about 50% by weight of the total monomer 
mixture. Such non-amine-containing monomers are preferably present at less 
than about 25% by weight of the total monomer mixture. The 
non-amine-containing monomers useful in the present invention include 
those which are copolymerizable with the water-soluble monomer. Examples 
of such other monomers include, but are not limited to, aromatic monomers 
such as styrene and .alpha.-methylstyrene, and aliphatic monomers such as 
methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, 
butyl acrylate, butyl methacrylate, maleic anhydride, vinyl acetate and 
the like, and mixtures thereof. 
In addition to the presence of non-amine-containing monomers, inert 
solvents may also be present in the monomer mixture; that is, they may be 
present at less than about 80%, preferably less than about 50% by weight 
of the total monomer mixture. Such inert solvents are preferably present 
at less than about 25% by weight of the total monomer mixture. Preferred 
inert solvents useful in the present invention include those which are 
themselves water-insoluble, but which are miscible with the water-soluble 
monomer. The inert solvents that combine the properties of 
water-insolubility and monomer-solubility are especially useful for 
enhancing the integrity of the spherical beads formed during the 
suspension polymerization of the water-soluble amine-containing monomers. 
Examples of such other solvents include, but are not limited to, hexane, 
heptane, isooctane, toluene, xylene, ethylbenzene and mixtures thereof. 
Crosslinkers of the general formula B react with amine functionality, 
NRR.sup.1, of the amine-containing polymer (represented in part by 
structure A) or the corresponding amine-containing monomer to produce 
crosslinked polymer (represented in part by structure C) according to 
Equation 1: 
##STR1## 
Z=side chain linkage group; k,m,n=zero or an integer from 1 to 3, and may 
be the same or different; 
R,R.sup.1 =(C.sub.1 -C.sub.8)alkyl groups or hydrogen; or R and R.sup.1 
together with the nitrogen atom to which they are attached may be joined 
to form a saturated ring, optionally containing one or more further 
hetero-atoms, for example oxygen or nitrogen; 
R.sup.2 =(C.sub.1 -C.sub.20)alkylene, aryl, (C.sub.8 
-C.sub.20)aryl-bis-alklene; 
X,X.sup.1 =halogen, tosylate, mesylate, brosylate, nosylate, triflate, 
nonaflate, tresylate, epoxide (X or X.sup.1 is attached to R.sup.2 in C 
as--O.sup.-), and may be the same or different. 
The side chain linkage group, Z, is any chemically stable linkage between 
--NRR.sup.1 and the polymer backbone, i.e., --NRR.sup.1 is not attached 
directly to polymer backbone. By "chemically stable" is meant that Z does 
not substantially decompose or degrade during the polymerization or 
crosslinking reactions. When k is zero the amine functionality is attached 
directly to the polymer backbone. Types of side chain linkage groups 
suitable for use in the present invention include, for example: 
an oxyalkylene group: --O--(CHR).sub.x --, 
a thioalkylene group: --S--(CHR).sub.x --, 
an alkylaminoalkyl group: --(CHR).sub.x --NR--(CHR).sub.x --, 
an alkylene group: --(CHR).sub.x --, 
an arylalkylene group: --C.sub.6 H.sub.4 --(CHR).sub.x --, 
an alkoxyalkyl group: --(CHR).sub.x --O--(CHR).sub.x --, 
an alkylthioalkyl group: --(CHR).sub.x --S--(CHR).sub.x -- (and 
corresponding sulfone and sulfoxide derivatives), 
an amidoalkyl group: --C(=O)NR--(CHR).sub.x --, 
a carboxyalkyl group: --C(=O)O--(CHR).sub.x --, 
where R is as defined above and x is an integer from 1 to 10. When n=m=1, 
the polyfunctional amine-reactive compound is represented by a 
difunctional crosslinker. Sulfur and nitrogen atoms present in the side 
chain linkage may participate in the crosslinking reaction with 
polyfunctional amine-reactive compounds depending on the reactivities of 
the particular materials involved. 
When neither R nor R.sup.1 in Equation 1 is hydrogen, then the crosslinking 
sites in the resultant polymer are represented by the quaternary ammonium 
salt form as illustrated in structure C. When R or R.sup.1 is hydrogen, 
the crosslinking sites in the resultant polymer (represented in part by 
structure C') may be partially or totally in the free base form in the 
presence of excess amine functionality. 
##STR2## 
When the polyfunctional amine-reactive compound is a diester (B', where Y, 
Y.sup.1 =carbalkoxy, represented by --COOR.sup.3) or diacid chloride (B', 
where Y, Y.sup.1 =haloacyl, represented by --COY.sup.2), at least one R or 
R.sup.1 of A=hydrogen, R.sup.3 =(C.sub.1 -C.sub.8)alkyl, and Y.sup.2 
=halogen, then the crosslinking reaction takes place according to Equation 
2. When B' is a diacid chloride some portion of the amine functionality in 
the resultant crosslinked polymer (represented in part by structure D) 
will be in the HY.sup.2 salt form and when B' is a diester the amine 
functionality will be in the free base form with R.sup.3 OH as a byproduct 
of the crosslinking reaction. 
##STR3## 
When at least some of the amine functionality in A is represented by both R 
and R.sup.1 being hydrogen, then dialdehydes may be used to crosslink the 
polymer. In this case, the resultant crosslinked polymer contains imine 
groups, known as Schiff bases when the dialdehyde is an aromatic 
dialdehyde, such as isophthalaldehyde, phthalaldehyde or 
terephthalaldehyde. Glutaraldehyde is an example of a suitable aliphatic 
dialdehyde. 
When R or R.sup.1 is hydrogen, or both R and R.sup.1 are hydrogen, a 
Michael-type reaction (also known as conjugate addition) may be used to 
crosslink the polymer in the absence of free-radical polymerization 
conditions. Examples of crosslinkers suitable for crosslinking the polymer 
in this manner are ethyleneglycol diacrylate, ethyleneglycol 
dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane 
trimethacrylate and the like. 
Crosslinkers useful in practicing the present invention are those compounds 
containing more than one amine-reactive site, i.e., any polyfunctional 
amine-reactive compound. Compounds suitable for use as crosslinkers in the 
present invention (designation B or B' in Equations 1 and 2) include 
unsubstituted and substituted members of the following classes: 
dihaloalkanes, aralkyl dihalides (such as bis(chloromethyl)benzene), 
alkylene diesters, aryl diesters, aralkyl diesters, alkylene diacylhalides 
(such as succinyl chloride), aryl diacylhalides, aralkyl diacylhalides, 
dialdehydes, diepoxyalkanes and aralkyl diepoxides. Polyfunctional 
amine-reactive compounds having mixed functional groups (where X and 
X.sup.1 or Y and Y.sup.1 are different), for example, epihalohydrins such 
as epichlorohydrin or epibromohydrin, are also suitable as crosslinkers. 
In addition, the tosylate(.rho.-toluenesulfonate), 
mesylate(methanesulfonate), brosylate(.rho.-bromobenzenesulfonate), 
nosylate(.rho.-nitrobenzenesulfonate), triflate 
(trifluoromethanesulfonate), nonaflate(nonafluorobutanesulfonate), and 
tresylate (trifluoroethanesulfonate) derivatives of unsubstituted and 
substituted difunctional alkanes and aralkanes are suitable as 
crosslinkers in the present invention. 
Preferred dihaloalkanes are dichloroalkanes and are represented, for 
example, by those selected from the group consisting of 
1,2-dichloroethane, 1,2-dichloropropane, 1,3-dichloropropane, 
1,3-dichloro-2-propanol and 1,4-dichlorobutane. Preferred alkylene 
diesters are dimethyl malonate, dimethyl succinate, diethyl glutarate, 
diethyl adipate, diethyl suberate, diethyl azelate and diethyl sebacate. 
The amount of crosslinking provided by the polyfunctional amine-reactive 
compounds used in the polymers of the present invention may be any amount 
that is effective to render the polymer insoluble in water, e.g., from 
about 0.1 to about 50 mole percent, preferably from about 0.5 to about 20 
mole percent of total monomers, while maintaining efficacy as a bile acid 
sequestrant. When the term "total monomers" is used in this context, 
reference is being made to both the amine-containing monomers and the 
polyfunctional amine-reactive compounds used as crosslinkers. Most 
preferably, the amount of crosslinking approaches the minimum amount 
effective to render the polymer insoluble in water, e.g., from about 2 
mole percent to about 10 mole percent of total monomers present, while 
maintaining high efficacy as a bile acid sequestrant. 
In addition to the polyfunctional amine-reactive crosslinker compounds, the 
polymers of the present invention may also be crosslinked with minor 
amounts of conventional free-radical reactive polyvinyl monomers, i.e., 
less than about 10 mole percent, preferably less than about 2 mole 
percent, and most preferably less than about 0.5 mole percent based on 
total monomers. Conventional polyvinyl monomers, which copolymerize under 
free-radical conditions, include, for example, divinylbenzene, 
trivinylbenzene, divinyltoluene, divinylpyridine, ethyleneglycol 
diacrylate, ethyleneglycol dimethacrylate, trimethylolpropane triacrylate, 
trimethylolpropane trimethacrylate, diethyleneglycol divinyl ether and the 
like. 
While not wishing to be bound by theory, we believe that, in the case of 
the present invention, there is little or no heterogeneity incorporated 
into the polymer backbone since the crosslinker does not act as a 
free-radical reactive comonomer during the polymerization of the 
amine-containing monomer. Instead, the crosslinking reaction takes place 
at sites away from the polymer backbone by nucleophilic displacement 
reaction mechanisms. The process of the present invention provides a 
greater chance for random homogeneous distribution of the crosslinking 
sites in the resultant crosslinked amine-containing polymer particles when 
compared to conventional crosslinked particles prepared by free-radical 
copolymerization (such as cholestyramine and others prepared with 
polyvinyl comonomers) or by crosslinking directly through polymer backbone 
sites (such as colestipol). In addition to the more homogeneous 
distribution of crosslink sites, it is believed that the process of the 
present invention allows for (1) greater control over molecular dimensions 
of the crosslinking moiety and, subsequently, the molecular flexibility of 
the resultant crosslinked structure when compared to conventional 
crosslinked polymers, resulting in (2) bile acid sequestering efficacy 
greater than that of cholestyramine, preferably at least three times, and 
most preferably, at least four times the efficacy of cholestyramine. 
The mechanism by which polymers of the present invention are crosslinked 
involves reaction between the nucleophilic amine groups of the polymer 
side chains with amine-reactive sites of the crosslinker molecule; these 
reactions may involve quaternization of the side chain amine groups or, in 
the case of primary or secondary amine groups, acylation, alkylation, 
condensation or conjugate addition reactions. The timing of the actual 
crosslinking reaction relative to the formation of polymer may vary 
depending upon the reactivity of the polyfunctional amine-reactive 
compound and the amine-containing monomer. Crosslinking may occur before, 
during or after the actual polymerization of the amine-containing monomer 
or any combination thereof. In the case of aqueous phase suspension 
polymerization, it is preferred that at least some of the crosslinking 
occurs during the polymerization of amine-containing monomer to facilitate 
the formation of water-insoluble spherical particles. Polymers of the 
present invention in the form of spherical particles are preferred because 
of the ease of handling during isolation, cleaning and washing of the 
polymer; however, other forms of the polymers, e.g., precipitation, 
powdered, etc., are equally efficacious regarding bile acid sequestering 
capacity. 
Polymerization initiators useful in the present invention include 
monomer-soluble initiators such as peroxides, hydroperoxides and related 
initiators, as for example benzoyl peroxide, tert-butyl hydroperoxide, 
cumene peroxide, tetralin peroxide, acetyl peroxide, caproyl peroxide, 
tert-butyl peroctoate, tert-butyl perbenzoate, tert-butyl diperphthalate, 
methyl ethyl ketone peroxide and the like. Also useful are azo initiators 
such as azodiisobutyronitrile; azodiisobutyramide, 
2,2'-azo-bis(2,4-dimethylvaleronitrile), 
azo-bis(.alpha.-methylbutyronitrile) and dimethyl-, diethyl- or dibutyl 
azo-bis(methylvalerate). Preferred initiators are the azo initiators, and 
particularly preferred is 2,2'-azo-bis(2,4-dimethylvaleronitrile). 
Preferred use levels of peroxide and azo initiators are from about 0.01% 
to 3% by weight, and from about 0.01% to about 2% by weight, respectively, 
based on the total weight of vinyl monomers. 
Salts useful for reducing solubility of the water-soluble monomer in the 
aqueous phase are water-soluble, non-reactive inorganic salts of a 
monovalent, divalent or aluminum cation and a monovalent or divalent 
anion, including, but not limited to, water-soluble, non-reactive 
inorganic salts of a monovalent, divalent or aluminum cation and a 
monovalent or divalent anion, as for example sodium, potassium, lithium 
and ammonium salts of chloride, bromide, iodide, sulfate, carbonate and 
nitrate and the magnesium and calcium salts of chloride, bromide, iodide 
and nitrate. Preferred salts are sodium chloride, sodium sulfate and 
sodium nitrate. The salt is dissolved in the aqueous medium at levels from 
about 5% by weight, based upon the total aqueous phase weight, to 
saturation of the salt in the aqueous phase. The term "non-reactive," as 
applied to the salts herein, means that the salt does not react chemically 
with water, the monomers or the polymers formed from the monomers. 
The preferred dispersants useful for making the anion exchange resin 
particles of the present invention are nonionic surfactants having a 
hydroxyalkylcellulose backbone, a hydrophobic alkyl side chain containing 
from 1 to about 24 carbon atoms, and an average of from about 1 to about 
8, preferably from about 1 to about 5, ethylene oxide groups substituting 
each repeating unit of the hydroxyalkylcellulose backbone, the alkyl side 
chains being present at a level of from about 0.1 to about 10 alkyl groups 
per 100 repeating units in the hydroxyalkylcellulose backbone. The alkyl 
group in the hydroxyalkylcellulose may contain from 1 to about 24 carbons, 
and may be linear, branched or cyclic. More preferred is a 
hydroxyethylcellulose containing from about 0.1 to about 10 
(C.sub.16)alkyl side chains per 100 anhydroglucose units and from about 
2.5 to about 4 ethylene oxide groups substituting each anhydroglucose 
unit. A particular advantage of these dispersants is that the spherical 
polymer particles of the present invention produced using them are not 
agglomerated, i.e., clumps of particles do not adhere to one another; 
agglomeration occurs when unprotected or poorly protected particles 
collide during the polymerization process. Typical use levels of 
dispersants are from about 0.01 to about 4% by weight, based upon the 
total aqueous-phase weight. 
Other dispersants useful for making the anion exchange resin particles of 
the present invention include finely divided particles such as silica, 
clays, ground ion exchange resins or ground, crosslinked, suspension 
copolymers without ion exchange functionality, and inorganic salts such as 
calcium hydroxyphosphate, particularly in combination with hydroxyapatite. 
The inorganic salts may or may not be fully soluble in water, and where 
they are not fully soluble they may behave similarly to the finely divided 
particles. Still other dispersants useful for making the anion exchange 
resin particles of the present invention are polymers containing 
hydrophilic backbones, which can orient their lipophilic portions to the 
monomer phase and their hydrophilic portions to the aqueous phase at the 
interface of the two phases. These polymeric dispersants include 
celluloses, polyvinyl pyrrolidones, polyvinyl alcohols, starches and the 
like. Mixtures of dispersants may also be used. These other dispersants 
tend to be less preferred, as they tend to produce a somewhat greater 
amount of agglomerated or otherwise undesirable material. 
Bile acid sequestrant polymers of the present invention may be prepared in 
macroporous or macroreticular form according to known methods by 
conducting the polymerization in the presence of precipitants, such as 
those disclosed in Meitzner et al., U.S. Pat. No. 4256840. The precipitant 
may be present in ratios from about 20 parts per 100 parts of monomer, 
i.e., 20% on monomer, to about 600 parts per 100 parts of monomer, i.e., 
600% on monomer, depending on the crosslinking level and precipitant used. 
Suitable precipitants for preparing macroporous or macroreticular polymers 
are those materials that are solvents for the monomer and non-solvents for 
the resultant crosslinked polymer. Preferred precipitants include: dialkyl 
ketones, e.g., methyl isobutyl ketone, diisobutyl ketone and the like; 
(C.sub.4 -C.sub.10)alcohols, e.g., t-amyl alcohol, 2-ethylhexanol, 
methylisobutyl carbinol and the like; (C.sub.6 -C.sub.8)alkanes, e.g., 
heptane, isooctane and the like; and (C.sub.7 -C.sub.10)aromatic 
hydrocarbons, e.g., toluene, xylene and the like. 
Uncrosslinked poly(dimethylaminopropylmethacrylamide), while exhibiting 
efficacy as a bile acid sequestrant (relative to cholestyramine), has 
shown evidence of toxicity when orally administered to rats, monkeys and 
dogs. The crosslinked bile acid sequestrants of the present invention 
exhibit reduced toxicity toward mammalian tissue relative to linear, i.e., 
uncrosslinked poly(dimethylaminopropylmethacrylamide). 
Preferably, the bile acid sequestrants of the present invention exhibit 
anion exchange capacities of greater than about 3 milliequivalents per 
gram of dry polymer (meq/g) and, more preferably, greater than about 4 
meq/g. Most preferably, the bile acid sequestrants of the present 
invention exhibit anion exchange capacities of about 5 meq/g to about 6 
meq/g. 
Bile acid sequestrants of the present invention may be used in the form of 
free bases or in the form of pharmaceutically acceptable acid salts, or 
mixtures thereof. Pharmaceutically acceptable acid salts are those whose 
anions, when used in therapeutically effective amounts, are nontoxic to 
the organism to whom the salts are administered. Examples of such salts 
are those derived from mineral acids such as hydrochloric and phosphoric, 
or organic acids such as acetic, citric, lactic and malonic. The various 
salt forms of the present invention may be prepared by dissolving the acid 
in a suitable solvent, e.g., water or a solution of water and an alcohol, 
treating the free base with the solution to form the salt and then 
isolating the insoluble salt from the solution. 
Hydrated, i.e., water-swollen, particles exhibiting a mean particle 
diameter from about 10 microns to about 400 microns, preferably from about 
10 to about 200 microns, are a preferred form of the polymers prepared by 
the process of the present invention for use as bile acid sequestrants. 
In general, bile acid sequestrants of the present invention are used for 
lowering blood cholesterol level in a mammal by oral administration of a 
therapeutically effective amount of the bile acid sequestrant to the 
mammal. The dosage of the sequestrants that will be most suitable for 
reduction of blood cholesterol level will vary with the form of 
administration, the particular embodiment of sequestrant, and the 
physiological characteristics of the host to which the sequestrant is 
administered. In general the amount administered is between about 2 and 
about 125 milligrams per kilogram (mg/kg) of body weight of the mammal per 
day. Based on physiological studies with beagle dogs (as described in 
Example 5), it is expected that the therapeutic dosage in humans will 
generally be from about 2 to about 125 mg/kg of body weight per day. This 
would correspond to a dosage for an 80 kg human host of about 0.2 to about 
10 grams/day. It is expected that more widely used dosages will be from 
about 35 to about 50 mg/kg of body weight per day corresponding to about 
2.5 to about 4 grams/day for an 80 kg host. 
Pharmaceutical compositions of the present invention are prepared by 
combining (1) a therapeutically effective amount of a polymer composition 
containing the bile acid sequestrant polymer particles with (2) a 
pharmaceutically acceptable carrier. Bile acid sequestrants of the present 
invention can be orally administered in any suitable way, including in 
neat form or in the form of pharmaceutical compositions in which the 
sequestrant is combined with pharmaceutically acceptable carriers, for 
example, in the form of tablets, capsules, particles, i.e., granules or 
powders, or as aqueous suspensions. In the case of tablets for oral use, 
commonly used carriers such as lactose and corn starch, and lubricating 
agents such as magnesium stearate, may be added. For oral administration 
in capsule form useful diluents include, e.g., lactose and dried starch. 
When aqueous suspensions are required for oral use the active ingredient 
is combined with emulsifying and suspending agents. If desired, sweetening 
and flavoring agents may be added. Particulate forms of the sequestrant 
may be administered as a mixture with food items such as applesauce, 
stewed fruits, juices and cereals. 
Bile acid sequestrants of the present invention can be used in conjunction 
with other treatments that are designed to lower the level of cholesterol 
in the blood. Preferred pharmaceutical compositions comprise a sequestrant 
of the present invention used in combination with a therapeutically 
effective amount of a material that inhibits cholesterol biosynthesis. 
Examples of such materials would include but are not limited to 
HMG-coenzyme A (HMG-CoA) reductase inhibitors, HMG-CoA synthase 
inhibitors, squalene epoxidase inhibitors and squalene synthase 
inhibitors. More preferred pharmaceutical compositions comprise a HMG-CoA 
reductase inhibitor as the cholesterol biosynthesis-inhibiting material. 
Illustrative of such HMG-CoA reductase inhibitors are lovastatin, 
simvastatin, pravastatin and fluvastatin. Examples of HMG-CoA synthase 
inhibitors are .beta.-lactone derivatives, .beta.-lactam derivatives and 
substituted oxacyclopropane analogues. Other cholesterol level-lowering 
agents that may be administered in conjunction with the sequestrants of 
the present invention include niacin, probucol, the fibric acids 
(clofibrate and gemfibrozil) and LDL-receptor gene inducers.

The following examples are intended to illustrate the invention and not to 
limit it except as it is limited in the claims. All ratios and percentages 
given herein are by weight unless otherwise specified, and all reagents 
used in the examples are of good commercial quality unless otherwise 
specified. 
EXAMPLE 1 
This example illustrates the preparation of spherical crosslinked particles 
of the present invention from water-soluble dimethylaminopropyl 
methacrylamide (DMAPMAM) monomer that has been crosslinked with the 
difunctional amine-reactive compound 1,3-dichloropropane. 
The dispersant used was a modified hydroxyethylcellulose which was 
characterized by substitution with about 4.0 moles of ethylene oxide per 
anhydroglucose unit and approximately 0.7-1.0 cetyl groups per 100 
anhydroglucose units, a molecular weight of approximately 300,000 and a 
viscosity in 1% aqueous solution of approximately 400 megaPascals. 
An aqueous solution was prepared by weighing 99.4 g sodium chloride, 
grinding approximately 6 g of this sodium chloride in a mortar with 1.5 g 
dispersant to a homogeneous mixture. The unground sodium chloride was 
added, with stirring, to 274.1 g deionized water at about 55.degree. C. 
The ground sodium chloride-dispersant mixture was added slowly to the 
water, which was then stirred at 55.degree. C. until all the solids had 
dissolved. 
A monomer mixture was made by mixing 67.0 g DMAPMAM, 3.44 g 
1,3-dichloropropane, 56.2 g o-xylene and 0.687 g 
2,2'-azo-bis-(2,4-dimethylvaleronitrile). The difunctional amine-reactive 
compound content, based on the total monomer weight, was 5% (7.3 mole %). 
The aqueous phase was placed in a 1-liter round-bottomed flask equipped 
with 2-blade agitator and stirred at 55.degree. C. The monomer mixture was 
transferred to the reactor vessel and stirred while maintaining a 
temperature of 55.degree. C. for 14 hours, after which the solids were 
drained free of liquid and washed three times with water to remove the 
salt and most of the xylene. 
The washed resin was then dried under vacuum at 60.degree. C. and ground to 
a particle size of less than about 200.mu.. The recovery of dried resin 
was about 80-85%. Electron Spectroscopy for Chemical Analysis (ESCA) 
indicated the presence of charged (quaternary) nitrogen and neutral 
(amide+amine) nitrogen. 
EXAMPLE 2 
This example illustrates the preparation of spherical crosslinked particles 
of the present invention from DMAPMAM monomer that has been crosslinked 
with the difunctional amine-reactive compound 1,3-dichloro-2-propanol. 
The spherical copolymer beads of this example were prepared using the same 
procedure as that of Example 1, except that 121.8 g of DMAPMAM, 6.25 g of 
1,3-dichloro-2-propanol, 1.25 g of 
2,2'-azo-bis-(2,4-dimethylvaleronitrile) and no xylene were used. The 
difunctional amine-reactive compound content, based on the total monomer 
weight, was 5% (6.5 mole %). The recovery of dried resin was 116 g (93%). 
ESCA indicated the presence of charged (quaternary) nitrogen and neutral 
(amide +amine) nitrogen. 
EXAMPLE 3 (comparative) 
In a manner similar to that of Example 1, a sample of crosslinked 
poly(dimethylaminopropylmethacrylamide) in the form of porous, spherical 
beads was prepared by copolymerizing DMAPMAM with a conventional polyvinyl 
crosslinker, divinylbenzene (DVB). 
A monomer mixture was made by mixing DMAPMAM and DVB (55% active (by 
weight), 45% ethylvinylbenzene); no o-xylene was used. A mixed initiator 
solution (30% by weight in acetone) based on 
2,2'-azo-bis-(2,4-dimethylvaleronitrile) and 
2,2'-azo-bis-(2-methylbutanenitrile) was prepared; the 
2,2'-azo-bis-(2,4-dimethylvaleronitrile) initiator was used at 0.7% by 
weight on monomers and the 2,2'-azo-bis-(2-methylbutanenitrile) 
inititiator was used at 0.3% by weight on monomers. 
The aqueous phase containing dispersant (sodium sulfate was used in place 
of sodium chloride as described in Example 1) was placed in a 
round-bottomed flask equipped with agitator. The monomer mixture was 
transferred to the reactor vessel and heated to 72.degree. C. with 
stirring. The inititiator solution was then added and the temperature was 
maintained at 72.degree. C. for 2.5 hours. The temperature was raised to 
90.degree. C. and held for an additional 3 hours and then raised to 
100.degree. C. and held for another 3 hours. The solids were drained free 
of liquid and washed thoroughly to remove salt after cooling the reaction 
mixture. The washed resin was then dried at 60.degree. C. in a convection 
oven and ground to a particle size of less than about 200.mu.. 
In this fashion, 3 different polymers were prepared crosslinked with 
different levels of DVB. Sample 3A contained 1 mole percent DVB, sample 3B 
contained 3 mole percent DVB and sample 3C contained 5 mole percent DVB. 
EXAMPLE 4 (comparative) 
In a manner similar to that of Example 1, a sample of crosslinked 
poly(dimethylaminopropylmethacrylamide) in the form of macroporous, 
spherical beads was prepared by copolymerizing DMAPMAM with conventional 
polyvinyl crosslinkers, divinylbenzene (DVB) and diethyleneglycol divinyl 
ether (DEGDVE). 
A monomer mixture was made by mixing DMAPMAM, DVB (80% active (by weight), 
20% ethylvinylbenzene), DEGDVE, 2,2'-azo-bis-(2,4-dimethylvaleronitrile) 
inititiator (1% by weight of total monomer) and o-xylene (91% by weight on 
monomers). The crosslinker concentration was 4% DVB and 0.5% DEGDVE by 
weight of total monomer (5.7 mole percent total divinyl crosslinker). 
The polymerization and polymer workup was conducted as described in Example 
1, except that residual o-xylene was removed by steam sweep distillation. 
EXAMPLE 5 
The efficacy of the crosslinked copolymer of the present invention as a 
bile acid sequestrant was evaluated in beagle dogs. Beagle dogs weighing 9 
to 11 kg each were fed a semi-synthetic, low cholesterol diet once per day 
in a quantity (200 to 300 grams/dog/day) that stabilized the body weight 
of the respective dogs. The semi-synthetic diet included 32.01% vitamin 
free casein; 43.14% dextrose; 12.42% lard; 2.39% cod liver oil; 2.72% 
calcium phosphate; 4.92% cella flour; and 2.39% hegsted vitamin mix No. 
14. 
Baseline plasma cholesterol levels were assessed for each dog by feeding 
the semi-synthetic diet without a bile acid sequestrant for six months and 
measuring plasma cholesterol levels on blood samples taken twice per week. 
After the baseline serum cholesterol levels were established, 
cholestyramine bile acid sequestrant was mixed with the diet (at dosages 
of 3, 6 and 12 grams/dog/day) plasma cholesterol levels were measured 
twice a week for four weeks to characterize the relationship between 
cholestyramine dosage and serum cholesterol levels for each dog. 
Following derivatization of the dose/response relationship, the dogs were 
maintained on a regimen of 12 grams cholestyramine/dog/day until a 
copolymer of the present invention was substituted for the cholestyramine 
in the diet at a dosage of either 3 grams/dog/day or 6 grams/dog/day. The 
dogs were fed the copolymer of the present invention and the plasma 
cholesterol levels of the dogs were measured daily for four weeks. The 
serum cholesterol level of dog fed a bile acid sequestrant stabilizes at a 
level below to baseline level. The relative efficacy of the crosslinked 
bile acid sequestrant of the present invention and of a control dosage of 
12 grams cholestyramine/day was quantified by calculating an efficacy 
factor CEF") according to Equation 3: 
EQU EF=((N-B)/(N-A))(12/X) [3] 
wherein: 
EF=efficacy factor 
N=serum cholesterol level in milligrams cholesterol/deciliter serum (mg/dl) 
on the semi-synthetic diet without a bile acid sequestrant; 
A=serum cholesterol level (mg/dl) or semi-synthetic diet including 12 grams 
cholestyramine/day; 
X=(grams dosage of bile acid sequestrant of the present invention as bile 
acid sequestrant/day) included in serum synthetic diet; and 
B=serum cholesterol level (mg/dl) on semi-synthetic diet including X grams 
of crosslinked bile acid sequestrant of the present invention. 
The sequestrant of Example 2 and Comparative Examples 3A, 3B, 3C and 4 were 
each tested in beagle dogs according to the above method. A sample of 
uncrosslinked poly(dimethylaminopropylmethacrylamide) prepared by aqueous 
phase solution polymerization and having a number average molecular weight 
of 261,000 and a weight average molecular weight of 588,000 was also 
tested according to this procedure and is listed as sample 5A in the table 
below. Results are set forth below in Table 1 as the EF, calculated 
according to Equation 3 for each of the sequestrants tested, along with 
the dosage administered, expressed as grams sequestrant per dog per day 
(g/dog/day) and a number (Dog No.) identifying the dog to which the dosage 
was administered. 
The results in Table 1 show that the bile acid sequestrant of the present 
invention (Example 2) possesses enhanced efficacy over that of 
sequestrants made with polyvinyl crosslinker (Examples 3A, 3B, 3C and 4) 
or with no crosslinker (Example 5A). 
TABLE 1 
__________________________________________________________________________ 
Mole Percent 
Mole Percent 
Divinyl 
Non-Vinyl Dosage 
Crosslinker 
Crosslinker 
Example No. 
Dog No. 
(g/dog/day) 
EF 
__________________________________________________________________________ 
0 6.5 2 205 3 4 
1.0 0 3A (comparative) 
209 3 1.3 
3.0 0 3B (comparative) 
206 3 1.7 
5.0 0 3C (comparative) 
205 3 2.1 
5.7 0 4 (comparative) 
301 3 2.7 
0 0 5A (uncrosslinked) 
206 6 3.2 
__________________________________________________________________________ 
EXAMPLE 6 
A suspension of particles of the sequestrant in deionized water was 
prepared. The suspension was serially diluted into serumless culture 
medium. The most concentrated suspension tested was 1000 micrograms 
sequestrant per milliliter suspension (.mu.g/ml). 
Exponentially growing Chinese hamster ovary (CHO) cell cultures were 
treated with the sequestrant dilutions for three hours. The cultures were 
gently rocked on a rocker platform during treatment in an attempt to 
maintain a uniform suspension over the cells for the entire treatment 
period. Negative controls, i.e., CHO cell cultures treated with serumless 
culture medium, and solvent controls, i.e., CHO cell cultures treated with 
1% deionized water in serumless culture medium, were included. 
Treatment was terminated by washing the cultures twice with Dulbecco's 
phosphate buffered saline and cells were allowed to recover in McCoy's 5A 
medium containing 10% fetal bovine serum for 0, 5 or 21 hours, i.e., 3, 8 
or 24 hours from the beginning of treatment. 
Cells were harvested at 3 and 24 hours by treating with trypsin-EDTA and 
scraping the cell monolayers from the culture flasks. The harvested cells 
were counted by Coulter counter to determine relative reductions in cell 
numbers. At selected doses, Trypan blue exclusion counts were conducted 
using a hemacytometer to determine cell viability to control for the 
possibility that some dead cells may have been counted with the Coulter 
counter. No cell counts were conducted at 8 hours, but the culture 
monolayers were examined for evidence of toxicity under an inverted 
microscope. 
The sequestrants of Example 2 and comparative Example 5A (uncrosslinked) 
were each tested for cytotoxicity using the procedure set forth above. The 
results of cytotoxicity testing are set forth below in Table 2 as an 
ED.sub.50 value in .mu.g/ml for each sequestrant tested, wherein the 
ED.sub.50 values indicate the minimum dosage of the respective sequestrant 
effective to kill 50% of the cells in the cell culture treated. 
The results in Table 2 show that the bile acid sequestrant of the present 
invention (Example 2) possesses greatly reduced toxicity compared to that 
of a sequestrant made with no crosslinker (Example 5A). Materials with 
EDs0 values of 100 .mu.g/ml or greater are generally considered non-toxic, 
and those with values below 100 .mu.g/ml are considered toxic with the 
degree of toxicity increasing as the value of ED.sub.50 decreases further 
below 100 .mu.g/ml. 
TABLE 2 
______________________________________ 
Example No. ED.sub.50 (.mu.g/ml) 
______________________________________ 
2 &gt;100 
5A 10.0 
______________________________________