U.S. patent application number 09/878724 was filed with the patent office on 2002-02-07 for antiviral polymers comprising acid functional groups and hydrophobic groups.
This patent application is currently assigned to GelTex Pharmaceuticals, Inc.. Invention is credited to Mandeville,, W. Harry III, Neenan, Thomas X..
Application Number | 20020015946 09/878724 |
Document ID | / |
Family ID | 25465340 |
Filed Date | 2002-02-07 |
United States Patent
Application |
20020015946 |
Kind Code |
A1 |
Neenan, Thomas X. ; et
al. |
February 7, 2002 |
Antiviral polymers comprising acid functional groups and
hydrophobic groups
Abstract
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.
Inventors: |
Neenan, Thomas X.; (Boston,
MA) ; Mandeville,, W. Harry III; (Lynnfield,
MA) |
Correspondence
Address: |
Carolyn S. Elmore
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Assignee: |
GelTex Pharmaceuticals,
Inc.
Waltham
MA
|
Family ID: |
25465340 |
Appl. No.: |
09/878724 |
Filed: |
June 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09878724 |
Jun 11, 2001 |
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09491008 |
Jan 25, 2000 |
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6268126 |
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09491008 |
Jan 25, 2000 |
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08934313 |
Sep 19, 1997 |
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6060235 |
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Current U.S.
Class: |
435/5 ;
424/78.27; 424/78.35; 526/315 |
Current CPC
Class: |
A61K 31/79 20130101;
A61K 31/785 20130101 |
Class at
Publication: |
435/5 ; 526/315;
424/78.27; 424/78.35 |
International
Class: |
C12Q 001/70; A61K
031/74; A61K 031/795; C08F 016/34; C08F 216/34 |
Claims
We claim:
1. A method of treating a viral infection in a mammal, comprising
administering to the mammal a therapeutically effective amount of a
polymer comprising a plurality of pendant hydrophobic groups and a
plurality of pendant acid functional groups, said acid functional
groups being directly connected to the polymer backbone or
connected to the polymer backbone by an aliphatic spacer group
having a length from 1 to about 20 atoms.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 09/491,008, filed Jan. 25, 2000, which is a continuation of
U.S. application Ser. No. 08/934,313, filed Sep. 19, 1997, the
entire teachings of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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
[0009] 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.
[0010] 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%.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] A "pendant" group is a moiety which forms a side chain or a
portion of a side chain attached to the polymer backbone.
[0015] The acid-finctionalized 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.
[0016] 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
[0017]
-(CH.sub.2).sub.2O[(CH.sub.2).sub.2O].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.
[0018] The pendant hydrophobic group can be a substituted or
unsubstituted, saturated or partially unsaturated
C.sub.2-C.sub.24-hydroc- arbyl 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.
[0019] 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-alkylac- rylamides, 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.
[0020] 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.
[0021] In one embodiment, the polymer to be administered is
characterized by a repeat unit or monomer of the general formula
1
[0022] 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 sulfric,
phosphoric and boric acids, including vinyl hydrosulfate, vinyl
dihydrophosphate, allyl hydrosulfate allyl dihydrophosphate and
conjugate bases thereof.
[0023] In another embodiment, the polymer to be administered is
characterized by a repeat unit or monomer of the general formula
2
[0024] 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-acrylarnidoglycolic acid and
2-acrylamido-2-methyl-1-propanesulfonic acid.
[0025] 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-decylacrylami- de), and copolymers of acrylic acid with
styrene or a substituted styrene, such as pentafluorostyrene or
4-fluorostyrene.
[0026] In another embodiment, the polymer to be administered is a
copolymer comprising an acid-finctionalized 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).
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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-acryloyloxysuccininide 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.
[0035] The invention will now be further and specifically described
by the following examples.
EXAMPLES
Example 1
[0036] Synthesis of acrylic acid/styrene copolymer (2:1)
[0037] A solution was prepared of acrylic acid (15.0 g, 0.2 mol)
and styrene (10.4 g, 0.1 mol) in THF (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
[0038] Synthesis of acrylic acid/decylacrylate (96:4) copolymer
[0039] 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
[0040] Synthesis of acrylic acid/n-butylacrylate (9:1)
copolymer
[0041] 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.
[0042] The corresponding polymer of acrylic acid and
n-butylacrylate (10:3) was made by the same procedure.
Example 4
[0043] Synthesis of acrylic acid/n-decylacrylate/acrylamide
(70:7.5:22.5) terpolymer
[0044] 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
[0045] Synthesis of acrylic acid/n-butylacrylate/acrylamide
(60:15:25) terpolymer
[0046] 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
[0047] Synthesis of co-polymer of acrylic acid and decylacrylate
(10:2).
[0048] 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
[0049] In vitro assessment of rotavirus inhibition activity
[0050] 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:
[0051] 1. MA104 cells (3.times.10.sup.4) were plated in 96 well
microtiter plates (Coming) 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 6. The cells were permeabilized by adding 1% Triton X100 for
3 minutes, followed by washing twice with Hanks Balanced Salt
Solution.
[0057] 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.
[0058] 8. The wells were washed twice with HBSS 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.
[0059] 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.
[0060] 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
[0061] 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.
* * * * *