Antiviral polymers comprising acid functional groups and hydrophobic groups

The present invention relates to a method of treating a viral infection in an animal, such as a human, by administering to the animal a therapeutically effective amount of a polymer comprising a plurality of pendant hydrophobic groups and a plurality of pendant acid functional groups. The acid functional groups are connected directly to the polymer backbone or via an aliphatic spacer group of 1 to about 20 atoms in length.

BACKGROUND OF THE INVENTION 
One mechanism for infection of a host cell by a microbe, such as a virus, a 
bacterium or a protozoan, proceeds via initial attachment of the microbe 
to the host cell surface. This process is mediated by relatively weak 
attractive interactions between adhesion molecules on the surfaces of the 
microbe and the host cell. In general, microbe-host cell attachment is the 
product of a multiplicity of such interactions, via what has been referred 
to as the polyvalent effect. one well-studied example of such a process is 
the attachment of the influenza A virus to mammalian epithelial cells, 
which results from interaction of terminal N-acetylneuraminic acid groups 
of glycolipids and glycoproteins on the host cell surface with the 
attachment glycoprotein hemagglutinin on the viral surface. 
The scarcity of effective antiviral agents points to the need for new 
approaches to the treatment of viral infections. The attachment step is an 
attractive target for such a treatment, and much activity has focused on 
the development of N-acetylneuraminic acid-containing compounds capable of 
binding to viral hemagglutinin, thus inhibiting viral attachment to host 
cells. Studies have demonstrated that polyvalent compounds, such as 
polymers bearing pendant N-acetylneuraminic acid groups, bind influenza 
virus with association constants which are several orders of magnitude 
higher than those of monomeric N-acetylneuraminic acid derivatives. To 
date, no polyvalent N-acetylneuraminic acid containing compounds are in 
clinical use for treatment or prevention of influenza. Moreover, no data 
demonstrating in vivo efficacy of such compounds have yet been published. 
A disadvantage of N-acetylneuraminic acid-functionalized compounds as 
therapeutic agents for the treatment of infection by influenza A virus 
and, possibly, other viruses, is the great expense of this sugar. In 
addition, the influenza virus has at its surface the enzyme neuramidinase, 
which cleaves N-acetylneuraminic acid moieties from such molecules, 
eventually destroying their ability to bind the virus. There is, thus, a 
need for inhibitors of viral attachment to mammalian cells which have an 
improved efficacy, are readily prepared from inexpensive starting 
materials and have a broad spectrum of activity. 
SUMMARY OF THE INVENTION 
The present invention relates to a method of treating a viral infection in 
an animal, such as a human, by administering to the animal a 
therapeutically effective amount of a polymer having a plurality of 
pendant hydrophobic groups and pendant acid functional groups which are 
directly attached to the polymer backbone or attached to the polymer 
backbone by an aliphatic spacer group. The aliphatic spacer group can have 
a length in the range from 1 to about 20 atoms. 
Suitable acid functional groups include carboxylic acid, sulfonic acid, 
phosphonic acid, hydrosulfate and boronic acid groups. The acid groups can 
also be present in the conjugate base form. Suitable hydrophobic groups 
include normal or branched C.sub.2 -C.sub.20 -alkyl groups, arylalkyl 
groups and aryl groups. 
In one embodiment, the polymer to be administered comprises a monomer or 
repeat unit having an acid functional group and a hydrophobic group. In 
another embodiment, the polymer is a copolymer comprising an 
acid-functionalized monomer and a hydrophobic monomer. The polymer to be 
administered can, optionally, further include a monomer comprising a 
neutral hydrophilic group, such as a hydroxyl group or an amide group. 
The present method has several advantages. For example, the polymers 
employed are easily prepared using standard techniques of polymer 
synthesis and inexpensive starting materials. The polymers will not be 
substantially degraded in the gastrointestinal tract and, therefore, can 
be administered orally. Polymer compositions can also be readily varied, 
to optimize properties such as solubility or water swellability and 
antiviral activity. Finally, the polymers to be administered include acid 
functional groups attached to the polymer backbone via aliphatic spacer 
groups. The structural flexibility of such spacer groups minimizes 
backbone constraints on the interaction of the acid groups with viral 
targets. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a method of treating a viral infection in 
an animal, such as a human, by administering to the animal a 
therapeutically effective amount of a polymer comprising a plurality of 
pendant acid functional groups and pendant hydrophobic groups. The acid 
functional group can be directly bonded to the polymer backbone or 
separated from the polymer backbone by an aliphatic spacer group having a 
length of from 1 to about 20 atoms. 
The polymer can be administered in the acid form, in which all acidic 
groups are protonated or in the conjugate base form, wherein the acidic 
functional groups are deprotonated and carry a negative charge. In the 
conjugate base form the negative charge of the polymer will be balanced by 
a suitable number of counter cations, such as alkali metal ions, for 
example, sodium, potassium or cesium ions, alkaline earth metal ions, such 
as magnesium ions, or tetraalkylammonium ions. The polymer can also be 
administered in a partially deprotonated form, in which the extent of 
deprotonation is less than 100%. 
As used herein, a "therapeutically effective amount" is an amount 
sufficient to inhibit or prevent, partially or totally, a viral infection 
or to reverse the development of a viral infection or prevent or reduce 
its further progression. 
The term "monomer", as used herein, refers to both a molecule comprising 
one or more polymerizable functional groups prior to polymerization, and a 
repeating unit of a polymer. A copolymer is said to comprise two or more 
different monomers. 
As used herein, the term "polymer backbone" or "backbone" refers to that 
portion of the polymer which is a continuous chain, comprising the bonds 
which are formed between monomers upon polymerization. The composition of 
the polymer backbone can be described in terms of the identity of the 
monomers from which it is formed, without regard to the composition of 
branches, or side chains, off of the polymer backbone. Thus, poly(acrylic 
acid) is said to have a substituted poly(ethylene) backbone with 
carboxylic acid (--C(O)OH) groups as side chains. 
A "pendant" group is a moiety which forms a side chain or a portion of a 
side chain attached to the polymer backbone. 
The acid-functionalized monomer comprises a pendant acid functional group, 
such as a carboxylic acid group, a sulfonic acid group, a hydrosulfate 
group, a phosphonic acid group, a boronic acid group. The acid functional 
group can also be present in the anionic, or conjugate base, form, in 
combination with a cation. Suitable cations include alkaline earth metal 
ions, such as sodium and potassium ions, alkaline earth ions, such as 
calcium and magnesium ions, and unsubstituted and substituted (primary, 
secondary, tertiary and quaternary) ammonium ions. 
The aliphatic spacer group is a component of the polymer side chain and 
connects the acid functional group to the polymer backbone. The term 
"aliphatic" describes a chemical moiety which is not aromatic and does not 
comprise an aromatic component. The spacer group can be linear, branched 
or cyclic. Suitable aliphatic spacer groups include normal or branched, 
saturated or partially unsaturated hydrocarbyl groups, including alkylene 
groups, for example, polymethylene groups such as --(CH.sub.2).sub.n --, 
wherein n is an integer from 1 to about 20, and cycloalkylene groups, such 
as the 1,4-cyclohexylene group. The alkylene group can be substituted or 
unsubstituted. Suitable alkylene substituents include hydroxyl groups and 
halogen atoms, for example, fluorine, chlorine and bromine atoms. The 
alkylene group can also, optionally, be interrupted at one or more points 
by a heteroatom, such as an oxygen, nitrogen or sulfur atom. Examples 
include the oxaalkylene groups --(CH.sub.2).sub.2 O[(CH.sub.2).sub.2 
O].sub.n (CH.sub.2).sub.2 --, wherein n is an integer ranging from 0 to 
about 3. The aliphatic spacer group can also be a partially unsaturated 
group, such as a substituted or unsubstituted C.sub.2 -C.sub.20 
-alkenylene group or a C.sub.2 -C.sub.20 -alkenylene group interrupted at 
one or more points by a heteroatom. 
The pendant hydrophobic group can be a substituted or unsubstituted, 
saturated or partially unsaturated C.sub.2 -C.sub.24 -hydrocarbyl group or 
a substituted or unsubstituted aryl or arylalkyl group. Examples of 
suitable alkyl substituents include halogen atoms, such as fluorine or 
chlorine atoms, and aryl groups, such as a phenyl group. Aryl substituents 
can include halogen atoms, C.sub.1 -C.sub.6 -alkyl groups and C.sub.1 
-C.sub.6 -alkoxy groups. Preferably, the pendant hydrophobic group is a 
normal or branched C.sub.2 -C.sub.24 -alkyl group. 
The polymer to be administered is, preferably, a copolymer comprising an 
acid-functionalized monomer and a hydrophobic monomer. The term 
"hydrophobic monomer", as used herein, is a monomer which comprises a 
pendant hydrophobic group, as described above. Suitable hydrophobic 
monomers include substituted or unsubstituted N--C.sub.3 -C.sub.24 
-alkylacrylamides, such as N-n-decylacrylamide and N-isopropylacrylamide, 
substituted or unsubstituted C.sub.3 -C.sub.24 -alkylacrylates, such as 
n-butylacrylate and n-decylacrylate; and styrene and substituted styrenes, 
such as pentafluorostyrene and 4-fluorostyrene. 
The copolymer can have a wide range of compositions, comprising, for 
example, from about 10 mole % to about 50 mole % of the hydrophobic 
monomer, and from about 90 mole % to about 50 mole % of the 
acid-functionalized monomer. 
In one embodiment, the polymer to be administered is characterized by a 
repeat unit or monomer of the general formula 
##STR1## 
wherein X is an aliphatic spacer group or a direct bond, R is hydrogen or 
an alkyl group, preferably methyl or ethyl, and Y is an acid functional 
group. Examples of suitable monomers of this type include acrylic acid, 
methacrylic acid, 2-ethylacrylic acid, vinylsulfonic acid, vinylphosphonic 
acid, 3-allyloxy-2-hydroxy-1-propanesulfonic acid, vinylacetic acid and 
esters of vinyl and allyl alcohol mineral acids, such as sulfuric, 
phosphoric and boric acids, including vinyl hydrosulfate, vinyl 
dihydrophosphate, allyl hydrosulfate allyl dihydrophosphate and conjugate 
bases thereof. 
In another embodiment, the polymer to be administered is characterized by a 
repeat unit or monomer of the general formula 
##STR2## 
wherein --C(O)--Z--X-- is an aliphatic spacer group wherein Z is oxygen or 
NH and X is an aliphatic group or a direct bond. Y is an acid functional 
group and R is hydrogen or an alkyl group, preferably methyl or ethyl. 
Examples of suitable monomers of this type include 2-acrylamidoglycolic 
acid and 2-acrylamido-2-methyl-1-propanesulfonic acid. 
Suitable copolymers for use in the present method include copolymers of 
acrylic acid and a C.sub.2 -C.sub.20 -alkylacrylate, such as poly(acrylic 
acid-co-n-decylacrylate) and poly(acrylic acid-co-n-butylacrylate). Also 
included are copolymers of acrylic acid and an N--C.sub.2 -C.sub.20 
alkylacrylamide, such as poly(acrylic acid-co-N-isopropylacrylamide) and 
poly(acrylic acid-co-N-n-decylacrylamide), and copolymers of acrylic acid 
with styrene or a substituted styrene, such as pentafluorostyrene or 
4-fluorostyrene. 
In another embodiment, the polymer to be administered is a copolymer 
comprising an acid-functionalized monomer, a hydrophobic monomer and a 
neutral hydrophilic monomer. A neutral hydrophilic monomer is a monomer 
comprising a polar group which is neither appreciably acidic nor 
appreciably basic at physiological pH. Examples of suitable neutral 
hydrophilic monomers include acrylamide, N-(2-hydroxyethyl) acrylamide, 
N-(3-hydroxypropyl)acrylamide, 2-hydroxyethylacrylate, vinyl acetate, 
vinyl alcohol and N-vinylpyrrolidone. A suitable copolymer of this type is 
the terpolymer poly(acrylic acid-co-n-decylacrylate-co-acrylamide). 
The polymer to be administered can also be characterized by a repeat unit 
comprising both a pendant hydrophobic group and a pendant acid functional 
group. suitable hydrophobic groups and acid functional groups include 
those discussed above. Polymers of this type include poly(2-alkylacrylic 
acid), wherein the alkyl group comprises from 2 to about 24 carbon atoms. 
One suitable polymer of this type is poly(2-ethylacrylic acid) or a 
conjugate base thereof. The polymer to be administered can also comprise a 
first monomer having a pendant hydrophobic group and a pendant acid 
functional group and a second neutral, hydrophilic monomer, such as the 
neutral hydrophilic monomers previously discussed. 
The polymer to be administered will, preferably, be of a molecular weight 
which is suitable for the intended mode of administration and allows the 
polymer to reach and remain within the targeted region of the body. For 
example, a method for treating an intestinal infection should utilize a 
polymer of sufficiently high molecular weight to resist absorption, 
partially or completely, from the gastrointestinal tract into other parts 
of the body. The polymers can have molecular weights ranging from about 
2,000 Daltons to about 500,000 Daltons, preferably from about 5,000 
Daltons to about 150,000 Daltons. 
The polymers of use in the present method are preferably substantially 
nonbiodegradable and nonabsorbable. That is, the polymers do not 
substantially break down under physiological conditions into fragments 
which are absorbable by body tissues. The polymers preferably have a 
nonhydrolyzable backbone, which is substantially inert under conditions 
encountered in the target region of the body, such as the gastrointestinal 
tract. Polymer backbones which are suitable for the present invention 
include polyacrylamide, polyacrylate, poly(vinyl) and poly(ethyleneimine) 
backbones. A co-polymer of the present invention can comprise a 
combination of two or more of these backbone elements. The polymer to be 
administered can also be an condensation polymer, such as a polyamide or a 
polyester. 
The quantity of a given polymer to be administered will be determined on an 
individual basis and will be determined, at least in part, by 
consideration of the individual's size, the severity of symptoms to be 
treated and the result sought. The polymer can be administered alone or in 
a pharmaceutical composition comprising the polymer, an acceptable carrier 
or diluent and, optionally, one or more additional drugs. 
The polymer can be administered by subcutaneous or other injection, 
intravenously, topically, orally, parenterally, transdermally, or 
rectally. The form in which the polymer will be administered, for example, 
powder, tablet, capsule, solution, or emulsion, will depend on the route 
by which it is administered. The therapeutically effective amount can be 
administered in a series of doses separated by appropriate time intervals, 
such as hours. 
The polymers of the present invention can be prepared via two general 
routes, direct copolymerization of a monomer mixture comprising an 
acid-functionalized monomer and a hydrophobic monomer, and nucleophilic 
side chain substitution on a activated polymer. The monomer mixture can be 
polymerized using, for example, methods of free radical, cationic or 
anionic polymerization which are well known in the art. Due to differences 
in the reactivity ratios of two or more monomers, the mole ratio of the 
monomers in the copolymer product can be different from the mole ratio of 
the monomers in the initial reaction mixture. This reactivity difference 
can also result in a non-random distribution of monomers along the polymer 
chain. 
Another synthetic route to polymers suitable for the present method 
proceeds via an intermediate polymer having labile side chains which are 
readily substituted by a desired side chain. Suitable polymers of this 
type include poly(N-acryloyloxysuccinimide) (pNAS), which reacts with a 
primary amine, for example, to form an N-substituted polyacrylamide. 
Another suitable polymer with labile side chains is 
poly(4-nitrophenylacrylate), which also forms an N-substituted 
polyacrylamide upon reaction with a primary amine. 
For example, a copolymer with a polyacrylamide backbone comprising amide 
nitrogen atoms substituted with an acid functional group and amide 
nitrogen atoms substituted with a hydrophobic group can be prepared by 
treating pNAS with less than one equivalent (relative to 
N-acryloyloxysuccinimide monomer) of a primary amine which terminates in 
an acid functional group, such as an amino acid, for example, glycine. A 
hydrophobic group can then be introduced by reacting at least a portion of 
the remaining N-acryloyloxysuccinimide monomers with a second primary 
amine, such as a C.sub.2 -C.sub.20 -alkylamine. A co-polymer further 
comprising a neutral hydrophilic monomer can be prepared by reacting any 
remaining N-acryloyloxysuccinimide monomers with, for example, 
2-aminoethanol or ammonia. A variety of copolymer compositions can, thus, 
be readily obtained by treating the activated polymer with different 
ratios of selected amines.

The invention will now be further and specifically described by the 
following examples. 
EXAMPLES 
Example 1 
Synthesis of Acrylic Acid/Styrene Copolymer (2:1) 
A solution was prepared of acrylic acid (15.0 g, 0.2 mol) and styrene (10.4 
g, 0.1 mol) in tetrahydrofuran (200 mL). After the solution was degassed 
with a rapid stream of nitrogen, azobis(isobutyrylnitrile) (AIBN, 1.47 g, 
3 mol % with respect to total monomer) was added. The solution was 
degassed for a further thirty minutes and the reaction was then heated to 
70.degree. C. for 14 h. The solution was cooled and precipitated into 
n-hexane (800 mL). The hexane was decanted from the fibrous white product, 
the product was washed with ethyl acetate (300 mL) followed by washing 
with a further aliquot of hexane (200 mL). The polymer was dried in vacuo 
to yield 21.6 g, 84.6% of a brittle white solid. 
Example 2 
Synthesis of Acrylic Acid/Decylacrylate (96:4) Copolymer 
A solution was prepared of acrylic acid (10.0 g, 133 mmol) and 
n-decylacrylate (1.0 g, 4.71 mmol) in dioxane (200 mL). The solution was 
degassed by passing a rapid stream of nitrogen through it, and to the 
solution was added AIBN (0.6 g, 5 mol % with respect to total monomer). 
The solution was degassed for a further thirty minutes and the reaction 
was then heated to 70.degree. C. for 16 h. The solution was cooled and 
precipitated into ethyl acetate (600 mL). The ethyl acetate was decanted 
from the fibrous white product, the product was washed with ethyl acetate 
(300 mL) and then with hexane (200 mL). The polymer was dried in vacuo to 
yield 9.0 g, 81% of a brittle white solid. 
Example 3 
Synthesis of Acrylic Acid/n-butylacrylate (9:1) Copolymer 
A solution was prepared of acrylic acid (10.0 g, 133 mmol) and 
n-butylacrylate (2.0 g, 14.41 mmol) in dioxane (200 mL). The solution was 
degassed by passing a rapid stream of nitrogen through it, and to the 
solution was added AIBN (0.6 g, 5 mol % with respect to total monomer). 
The solution was degassed for a further thirty minutes and the reaction 
was then heated to 70.degree. C. for 17 h. The solution was cooled and 
precipitated into ethyl acetate (600 Ml). The ethyl acetate was decanted 
from the fibrous white product, the product was washed with ethyl acetate 
(300 Ml) followed by washing with hexane (200 Ml). The polymer was dried 
in vacuo to yield 9.0 g (81%) of a brittle white solid. 
The corresponding polymer of acrylic acid and n-butylacrylate (10:3) was 
made by the same procedure. 
Example 4 
Synthesis of Acrylic Acid/n-decylacrylate/Acrylamide (70:7.5:22.5) 
Terpolymer 
A solution was prepared of acrylic acid (10.0 g, 133 mmol), n-decylacrylate 
(3.0 g, 14.2 mmol) and acrylamide (3.0 g, 42.2 mmol) in dioxane (200 mL). 
After the solution was degassed with a rapid stream of nitrogen, AIBN (1.3 
g) was added. The solution was degassed for a further thirty minutes and 
the reaction was then heated to 70.degree. C. for 17 h. The polymer 
precipitated as a fibrous white solid as the reaction proceeded. The 
solution was cooled and the dioxane decanted. The residue was washed with 
ethyl acetate (600 mL) and the ethyl acetate was discarded. The polymer 
was finally washed with hexanes (300 mL) and dried in vacuo. 
Example 5 
Synthesis of Acrylic Acid/n-butylacrylate/Acrylamide (60:15:25) Terpolymer 
A solution was prepared of acrylic acid (10.0 g, 133 mmol), n-butylacrylate 
(4.0 g, 31.4 mmol) and acrylamide (4.0 g, 56.3 mmol) in dioxane (200 mL). 
After the solution was degassed with a rapid stream of nitrogen, AIBN (1.3 
g) was added. The resulting solution was degassed for a further thirty 
minutes and was then heated to 70.degree. C. for 17 h. As the reaction 
proceeded, the polymer precipitated as a fibrous white solid. The solution 
was cooled and the dioxane was decanted. The polymer was washed with ethyl 
acetate (600 mL), then with hexanes (300 mL) and dried in vacuo. 
Example 6 
Synthesis of Co-polymer of Acrylic Acid and Decylacrylate (10:2) 
A solution was prepared of acrylic acid (10.0 g, 133 mmol) and 
decylacrylate (5.64 g, 26.6 mmol) in dioxane (300 mL). After the solution 
was degassed with a rapid stream of nitrogen, AIBN (0.8 g) was added. The 
resulting solution was degassed for a further thirty minutes and the 
reaction mixture was heated to 70.degree. C. for 16 h. The solution was 
cooled and added to ethyl acetate (600 mL). The ethyl acetate was decanted 
from the resulting fibrous white product. The product was then redissolved 
in dioxane (150 mL), precipitated with ethyl acetate (500 mL), filtered, 
washed with cold hexanes (300 mL) and dried in vacuo. 
Example 7 
In Vitro Assessment of Rotavirus Inhibition Activity 
The ability of several compounds to inhibit the infection of cells by 
rotavirus was assessed via a Focus Forming Unit Assay. The Focus Forming 
Unit Assay measures the ability of a compound to inhibit primary infection 
of cells with rotavirus, using the Rhesus Rotavirus strain (RRV). The 
protocol for the Focus Forming Unit Assay is as follows: 
1. MA104 cells (3.times.10.sup.4) were plated in 96 well microtiter plates 
(Corning) at 3 days before infection. Serial dilutions of polymers to be 
tested were prepared in Medium 199 in a concentration range between 10 and 
0.01 mg/ml and adjusted to pH 7 with 2M NaOH solution. 
2. 100 .mu.l of thawed RRV virus was added to 900 .mu.l of Medium 199 
(Gibco/BRL), followed by the addition of 2.5 .mu.l of 2 mg/ml trypsin and 
the solution was incubated at 37.degree. C. for 20 minutes. This process 
activated the virus for infection. 
3. The virus was diluted 1:125 (1:1250 final) in Medium 199 without Fetal 
Bovine Serum. 250 .mu.l of diluted polymer and incubated at 37.degree. C. 
for 1 hour. Controls included mixing virus with media alone and with 
neutralizing monoclonal antibody. 
4. The medium was aspirated from the wells and washed once with 100 .mu.l 
of the virus/polymer dilution mixture was added to each well, plating each 
dilution of polymer in quadruplicate. The plates were incubated on a 
rocking platform for 20 hours at 37.degree. C. 
5. The medium was aspirated from the wells and to the wells was added 100 
.mu.l of 10% Formalin. The plates were incubated 1 hour at room 
temperature. 
6. The cells were permeabilized by adding 1% Triton X100 for 3 minutes, 
followed by washing twice with Hanks Balanced Salt Solution. 
7. 80-100 .mu.l of primary antibody (DAKO rabbit anti-rotavirus serum 
diluted 1:250) were added to the wells and incubated at 37.degree. C. for 
1 hour on a rocking platform. 
8. The wells were washed twice with Hanks Balanced Salt Solution followed 
by the addition of 100 .mu.l per well of 1:10,000 dilution of peroxidase 
conjugated anti-rabbit serum (Sigma). The wells were incubated at 
37.degree. C. for 1 hour on a rocking platform. 
9. The wells were washed twice with HBSS. 100 .mu.l of AEC substrate (3 
amino-9-ethylcarbazole dissolved to 4 mg/ml in dimethylformamide and 
diluted to 20% in pH 5.2 0.1 M acetate buffer) was then added. After 
incubating for 20 minutes at room temperature, the reaction was stopped by 
washing once with HBSS. Infected cells appeared red and were quantified by 
counting the number of foci relative to control. 
The polymers of Examples 2, 3, 5 and 6 were examined via the Focus Forming 
Unit Assay. Each of these polymers had an ED.sub.50 (the concentration at 
which the extent of infection was 50% that of the control) of less than 
0.08 mg/mL. 
EQUIVALENTS 
While this invention has been particularly shown and described with 
references to preferred embodiments thereof, it will be understood by 
those skilled in the art that various changes in form and details may be 
made therein without departing from the spirit and scope of the invention 
as defined by the appended claims. Those skilled in the art will recognize 
or be able to ascertain using no more than routine experimentation, many 
equivalents to the specific embodiments of the invention described 
specifically herein. Such equivalents are intended to be encompassed in 
the scope of the claims.