U.S. patent application number 09/493639 was filed with the patent office on 2001-12-20 for ionic polymers as anti-infective agents.
Invention is credited to Holmes-Farley, Stephen Randall, Mandeville III, W. Harry, Neenan, Thomas X..
Application Number | 20010053794 09/493639 |
Document ID | / |
Family ID | 24691770 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053794 |
Kind Code |
A1 |
Mandeville III, W. Harry ;
et al. |
December 20, 2001 |
Ionic polymers as anti-infective agents
Abstract
A method for treating a microbial infection in a mammal, such as
a human, comprising treating the mammal with a therapeutically
effective amount of a polymer comprising an amino group or an
ammonium group attached to the polymer backbone via an aliphatic
spacer group. The polymer can be a homopolymer or a copolymer. In
one embodiment, the polymer is a copolymer comprising a monomer
having a pendant ammonium group and a hydrophobic monomer.
Inventors: |
Mandeville III, W. Harry;
(Lynnfield, MA) ; Neenan, Thomas X.; (Boston,
MA) ; Holmes-Farley, Stephen Randall; (Arlington,
MA) |
Correspondence
Address: |
Carolyn S. Elmore
Hamilton Brook Smith & Reynolds P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Family ID: |
24691770 |
Appl. No.: |
09/493639 |
Filed: |
January 28, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09493639 |
Jan 28, 2000 |
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09286693 |
Apr 6, 1999 |
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09286693 |
Apr 6, 1999 |
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08670764 |
Jun 24, 1996 |
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6034129 |
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Current U.S.
Class: |
514/549 ;
424/78.31; 424/78.37 |
Current CPC
Class: |
A61P 33/04 20180101;
Y02A 50/30 20180101; A61P 33/00 20180101; A61K 31/785 20130101;
A61P 31/04 20180101; A61P 31/10 20180101; A61P 31/00 20180101 |
Class at
Publication: |
514/549 ;
424/78.31; 424/78.37 |
International
Class: |
A61K 031/785; A61K
031/22 |
Goverment Interests
[0002] The invention described herein was supported in whole or in
part by Advanced Technology Program Cooperative Agreement No.
70NANB5H1063 from the National Institute of Standards and
Technology. The United States Government has certain rights in the
invention.
Claims
What is claimed is:
1. A composition for treating a microbial infection in a mammal,
comprising a therapeutically effective amount of a homopolymer
characterized by a polymerized monomer having an amino group or an
ammonium group attached to the polymer backbone by an aliphatic
spacer group.
2. The composition of claim 1 wherein the monomer having an amino
group or an ammonium group attached to the polymer backbone by an
aliphatic spacer group is of Formula I, 2wherein R is a hydrogen
atom or a methyl or ethyl group, X is a covalent bond, a carbonyl
group or a CH.sub.2 group, Y is an oxygen atom or an NH or CH.sub.2
group, Z is an aliphatic spacer group, and R.sub.1, R.sub.2 and
R.sub.3 are each, independently, a hydrogen atom, a normal or
branched, substituted or unsubstituted C.sub.1-C.sub.18-alkyl
group, aryl or arylalkyl group.
3. The composition of claim 2 wherein Z is a normal or branched
C.sub.2-C.sub.12-alkylene group or a C.sub.2-C.sub.12-alkylene
group interrupted at one or more points by a heteroatom.
4. A composition for treating a microbial infection in a mammal,
comprising a therapeutically effective amount of a copolymer of a
polymerized monomer having an amino group or an ammonium group
attached to the polymer backbone by an aliphatic spacer group and a
polymerized neutral hydrophobic monomer.
5. The composition of claim 4 wherein the monomer having an amino
group or an ammonium group attached to the polymer backbone by an
aliphatic spacer group is of Formula I, 3wherein R is a hydrogen
atom or a methyl or ethyl group, X is a covalent bond, a carbonyl
group or a CH.sub.2 group, Y is an oxygen atom or an NH or CH.sub.2
group, Z is an aliphatic spacer group, and R.sub.1, R.sub.2 and
R.sub.3 are each, independently, a hydrogen atom, a normal or
branched, substituted or unsubstituted C.sub.1-C.sub.18-alkyl
group, aryl or arylalkyl group.
6. The composition of claim 5 wherein Z is a normal or branched
C.sub.2-C.sub.12-alkylene group or a C.sub.2-C.sub.12-alkylene
group interrupted at one or more points by a heteroatom.
7. The composition of claim 6 wherein the heteroatom is a nitrogen,
oxygen or sulfur atom.
8. The composition of claim 5 wherein at least one of R.sub.1,
R.sub.2 and R.sub.3 is an aryl group, a benzyl group or a normal or
branched, substituted or unsubstituted C.sub.1-C.sub.18-alkyl
group.
9. The composition of claim 5 wherein the neutral hydrophobic
monomer comprises a straight chain or branched, substituted or
unsubstituted C.sub.3-C.sub.18-alkyl group, an aryl group or an
aralkyl group.
10. The composition of claim 9 wherein the hydrophobic monomer is
selected from the group consisting of styrene,
N-isopropylacrylamide, N-t-butylacrylamide, N-n-butylacrylamide,
heptafluorobutylacrylate, N-n-decylallylamine, N-n-decylacrylamide,
pentafluorostyrene, n-butylacrylate, t-butylacrylate,
n-decylacrylate, N-t-butylmethacrylamide, n-decylmethacrylate,
n-butylmethacrylate, n-decylacrylate, and t-butylacrylate.
11. A composition for treating a microbial infection in a mammal,
comprising a therapeutically effective amount of a copolymer of a
polymerized monomer having an amino group or an ammonium group
attached to the polymer backbone by an aliphatic spacer group, a
polymerized neutral hydrophobic monomer and a polymerized neutral
hydrophilic monomer.
12. The composition of claim 11 wherein the monomer having an amino
group or an ammonium group attached to the polymer backbone by an
aliphatic spacer group is of Formula I, 4wherein R is a hydrogen
atom or a methyl or ethyl group, X is a covalent bond, a carbonyl
group or a CH.sub.2 group, Y is an oxygen atom or an NH or CH.sub.2
group, Z is an aliphatic spacer group, and R.sub.1, R.sub.2 and
R.sub.3 are each, independently, a hydrogen atom, a normal or
branched, substituted or unsubstituted C.sub.1-C.sub.18-alkyl
group, aryl or arylalkyl group.
13. The composition of claim 11 wherein the neutral hydrophilic
monomer is acrylamide, methacrylamide, N-(2-hydroxyethyl)acrylamide
and (2-hydroxyethyl)methacrylate.
14. The composition of claim 12 wherein Z is a normal or branched
C.sub.2-C.sub.12-alkylene group or a C.sub.2-C.sub.12-alkylene
group interrupted at one or more points by a heteroatom.
15. A composition for treating a microbial infection in a mammal,
comprising a therapeutically effective amount of
poly(ethyleneimine) or
poly(decamethylenedimethylammonium-co-ethylenedimethylammonium)
X.sup.-, wherein X.sup.- is an anion.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 09/286,693, filed Apr. 6, 1999, which is a continuation
application of U.S. Ser. No. 08/670,764, filed Jun. 24, 1996. The
teachings of each of these referenced applications are expressly
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] A number of short (ca. 50 amino acid residues or fewer)
linear or cyclic cytotoxic peptides have been isolated recently
from a variety of sources. These include mellitin, from bee venom,
the magainins, from frog skin, and cecropins, from insects (Maloy,
et al., Biopolymers (Peptide Science) 37: 105-122 (1995)). Although
of widely varying peptide sequences and structures, these peptides
all contain multiple lysine and arginine residues, and, at
physiological pH, carry a net positive charge. They also form
amphipathic structures wherein one portion of the structure is
hydrophilic while the other portion is hydrophobic.
[0004] The peptides appear to act solely by direct lysis of the
cell membrane (Maloy et al., supra (1995)). In the current model,
cell lysis is initiated by the electrostatic attraction of the
positive charge on the peptide to the negative phosphate head
groups at the exterior surface of the membrane phospholipid
bilayer. This interaction leads to insertion of the hydrophobic
portion of the protein into the membrane, thereby disrupting the
membrane structure. The lytic peptides are, in general, more active
against prokaryotic cells, such as bacteria and fungi, than
eukaryotic cells. This has led to interest in these peptides as
potential agents for the treatment of infections in humans (Maloy
et al., supra (1995); Arrowood et al., J. Protozool. 38: 161S-163S
(1991); Haynie et al., Antimicrob. Agents Chemotherapy 39: 301-307
(1995).
[0005] The natural cytotoxic peptides, however, suffer from several
disadvantages with respect to their use as human therapeutic
agents. First, it appears that these peptides have evolved to act
at high concentration at specific localized sites. Thus, when
administered as a drug, the dosage necessary to attain an effective
concentration at site of infection can be prohibitively high. A
second disadvantage is the difficulty of isolating useful amounts
of these peptides from the natural sources, along with the high
cost of synthesizing useful amounts of peptides in this size
regime. Finally, these compounds, like other peptides, are degraded
in the gastrointestinal tract, and, thus, cannot be administered
orally.
[0006] There is a need for anti-microbial agents which possess the
broad activity spectrum of the natural cytotoxic peptides, but are
inexpensive to produce, can be administered orally and have lower
concentration requirements for therapeutic activity.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention is a method for treating
a microbial infection in a mammal, comprising administering to the
mammal a therapeutically effective amount of a polymer having an
amine or ammonium group connected to the polymer backbone by an
aliphatic spacer group.
[0008] The polymer to be administered can be a homopolymer or a
copolymer. In one embodiment, the polymer further includes a
monomer comprising a hydrophobic group, such as an aryl group or a
normal or branched C.sub.3-C.sub.18-alkyl group.
[0009] 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.
[0010] Another aspect of the invention is a method for treating a
microbial infection in a mammal, such as a human, comprising
administering to the mammal a therapeutically effective amount of a
polymer comprising a polymethylene backbone which is interrupted at
one or more points by a quaternary ammonium group.
[0011] 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 digestive tract and,
therefore, can be administered orally. Polymer compositions can
also be readily varied, to optimize properties such as solubility
or water swellability and antimicrobial activity. Finally, the
polymers to be administered include amine or ammonium 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 ammonium groups with
anionic targets.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention relates to a method for preventing or
treating a microbial infection in a mammal, such as a human, by
administering to the mammal a therapeutically effective amount of a
polymer comprising a plurality of amino or ammonium groups which
are attached to the polymer backbone via aliphatic spacer
groups.
[0013] As used herein, a "therapeutically effective amount" is an
amount sufficient to inhibit, partially or totally, a microbial
infection or to reverse development of a microbial infection or
prevent or reduce its further progression. The term "polymer"
refers to a macromolecule comprising a plurality of repeat units or
monomers. The term includes homopolymers, which are formed from a
singly type of monomer, and copolymers, which are formed of two or
more different monomers. A "terpolymer" is a copolymer formed from
three different monomers. The term polymer, as used herein, is
intended to exclude proteins, peptides, polypeptides and
proteinaceous materials.
[0014] 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, a poly(acrylamide)
polymer is said to have a poly(acrylamide) backbone, without regard
to the substituents on the acrylamide nitrogen atom, which are
components of the polymer side chains. A
poly(acrylamide-co-styrene) copolymer, for example, is said to have
a mixed acrylamide/styrene backbone.
[0015] The term "polymer side chain" or "side chain" refers to the
portion of a monomer which, following polymerization, forms a
branch off of the polymer backbone. In a homopolymer all of the
polymer side chains are identical. A copolymer can comprise two or
more distinct side chains. When a side chain comprises an ionic
unit, for example, the ionic unit depends from, or is a substituent
of, the polymer backbone, and is referred to as a "pendant ionic
unit". The term "spacer group", as used herein, refers to a
polyvalent molecular fragment which is a component of a polymer
side chain and connects a pendant moiety to the polymer backbone.
The term "aliphatic spacer group" refers to a spacer group which
does not include an aromatic unit, such as a phenylene unit.
[0016] The term "addition polymer", as used herein, is a polymer
formed by the addition of monomers without the consequent release
of a small molecule. A common type of addition polymer is formed by
polymerizing olefinic monomers, wherein monomers are joined by the
formation of a carbon-carbon bonds between monomers, without the
loss of any atoms which compose the unreacted monomers.
[0017] The term "monomer", as used herein, refers to both (a) a
single molecule comprising one or more polymerizable functional
groups prior to or following polymerization, and (b) a repeat unit
of a polymer. An unpolymerized monomer capable of addition
polymerization, can, for example, comprise an olefinic bond which
is lost upon polymerization.
[0018] 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.
[0019] The polymers can be administered, for example, topically,
orally, intranasally, or rectally. The form in which the agent is
administered, for example, powder, tablet, capsule, solution, or
emulsion, depends in part 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.
[0020] Microbial infections which can be treated or prevented by
the method of the present invention include bacterial infections,
such as infection by Streptococcus, including Streptococcus mutans,
Streptococcus salivarius, and Streptococcus sanguis, Salmonella,
Campylobacter, including Campylobacter sputum, Antinomyces,
including Actinomyces naeslundii and Actinomyces viscosus,
Escherichia coli, Clostridium difficile, Staphylococcus, including
S. aureus, Shigella, Pseudomonas, including P. aeruginosa,
Eikenella corrodens, Actinobacillus actinomycetemcomitans,
Bacteroides gingivalis, Capnocytophaga, including Capnocytophaga
gingivalis, Wolinell recta, Bacteriodes intermedius, Mycoplasma,
including Mycoplasma salivarium, Treponema, including Treponema
denticola, Peptostreptococcus micros, Bacteriodes forsythus,
Fusobacteria, including Fusobacterium nucleatum, Selenomonas
sputigena, Bacteriodes fragilis, Enterobacter cloacae and
Pneumocystis. Also included are protozoal infections, such as
infection by Cryptosporidium parvum and Giardia lamblia; ameobic
infections, such as infection by Entameoba histolytica or
Acanthameoba; fungal infections, such as infections by Candida
albicans and Aspergillus fumigatus, and parasitic infections, such
as infections by A. castellani and Trichinella spiralis. The method
is useful for treating infections of various organs of the body,
but is particularly useful for infections of the skin and
gastrointestinal tract.
[0021] Polymers which are particularly suitable for the present
method include polymers which can possess key characteristics of
naturally occurring cytotoxic peptides, in particular, the ability
to form amphipathic structures. The term "amphipathic", as used
herein, describes a three-dimensional structure having discrete
hydrophobic and hydrophilic regions. Thus, one portion of the
structure interacts favorably with aqueous and other polar media,
while another portion of the structure interacts favorably with
non-polar media. An amphipathic polymer results from the presence
of both hydrophilic and hydrophobic structural elements along the
polymer backbone.
[0022] In one embodiment, the polymer to be administered polymer
comprises a monomer of Formula I, 1
[0023] wherein X is a covalent bond, a carbonyl group or a CH.sub.2
group, Y is an oxygen atom, an NH group or a CH.sub.2 group, Z is
an spacer group, R is a hydrogen atom or a methyl or ethyl group,
R.sub.1, R.sub.2 and R.sub.3 are each, independently, a hydrogen
atom, a normal or branched, substituted or unsubstituted
C.sub.1-C.sub.18-alkyl group, an aryl group or an arylalkyl group.
Suitable alkyl substituents include halogen atoms, such as fluorine
or chlorine atoms.
[0024] In the case in which at least one of R.sub.1-R.sub.3 is a
hydrogen atom, the monomer can also exist in the free base, or
amino form, that is, as the neutral conjugate base of the ammonium
cation. The polymer comprising such a monomer can be administered
in the protonated, cationic form, such as a salt of a
pharmaceutically acceptable acid, or in the free base form.
Suitable acids include hydrochloric acid, hydrobromic acid, citric
acid, lactic acid, tartaric acid, phosphoric acid, methanesulfonic
acid, acetic acid, formic acid, maleic acid, fumaric acid, malic
acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid,
L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucoronic
acid, oxalic acid, ascorbic acid, and acetylglycine. In either
case, at physiological pH following administration, a plurality of
amino groups will be protonated to become ammonium groups, and the
polymer will carry an overall positive charge.
[0025] The spacer group is a component of the polymer side chain
and connects the amino or ammonium group to the polymer backbone.
The amino or ammonium group is, thus, a pendant group. The spacer
group can be a normal or branched, saturated or unsaturated,
substituted or unsubstituted alkylene group, such as a
polymethylene group --(CH.sub.2).sub.n--, wherein n is an integer
from about 2 to about 15. Suitable examples include the propylene,
hexylene and octylene groups. The alkylene group can also,
optionally, be interrupted at one or more points by a heteroatom,
such as an oxygen, nitrogen (e.g, NH) or sulfur atom. Examples
include the oxaalkylene groups --(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.
[0026] Examples of monomers of Formula I having quaternary ammonium
groups include 2-trimethylammonium-ethylmethacrylate,
2-trimethylammoniumethylac- rylate,
N-(3-trimethylammoniumpropyl)methacrylamide,
N-(6-trimethylammoniumhexyl)acrylamide,
N-(3-trimethylammoniumpropyl)acry- lamide, and
N-(4-trimethylammoniumbutyl)allylamine, each of which also includes
a counter anion. Examples monomers of Formula I having an amino
group include allylamine and
N-(3-dimethylaminopropyl)acrylamide.
[0027] Polymers to be administered which have quaternary ammonium
groups or protonated amino groups will further comprise a
pharmaceutically acceptable counter anion, such as anions which are
conjugate bases of the pharmaceutically acceptable acids discussed
above, for example, chloride, bromide, acetate, formate, citrate,
ascorbate, sulfate or phosphate. The number of counter anions
associated with the polymer prior to administration is the number
necessary to balance the electrical charge on the polymer.
[0028] The polymer can also be a copolymer further comprising a
hydrophobic monomer. The hydrophobic monomer can comprise a side
chain bearing a hydrophobic group, such as a straight chain or
branched, substituted or unsubstituted C.sub.3-C.sub.18-alkyl group
or a substituted or unsubstituted aryl group. Examples of suitable
hydrophobic monomers include styrene, N-isopropylacrylamide,
N-t-butylacrylamide, N-n-butylacrylamide, heptafluorobutylacrylate,
N-n-decylallylamine, N-n-decylacrylamide, pentafluorostyrene,
n-butylacrylate, t-butylacrylate, n-decylacrylate,
N-t-butylmethacrylamide, n-decylmethacrylate, and
n-butylmethacrylate.
[0029] Examples of copolymers comprising a monomer of Formula I and
a hydrophobic monomer include
poly(N-(3-dimethylaminopropyl)acrylamide-co-N-
-(n-butyl)acrylamide) or salts thereof with pharmaceutically
acceptable acids. Other examples of suitable copolymers include
poly(2-trimethylammoniumethylmethacrylate-co-styrene) chloride,
poly(2-trimethylammoniumethylmethacrylate-co-N-isopropylacrylamide)
chloride,
poly(2-trimethyl-ammoniumethylmethacrylate-co-heptafluorobutyla-
cryl) chloride,
poly(3-trimethylammoniumpropylmethacrylate-co-styrene) chloride,
poly(3-trimethylammonium-propylmethacrylate-co-N-t-butylacrylam-
ide) chloride,
poly(3-trimethylammoniumpropylmethacrylate-co-N-n-butylacry-
lamide) chloride, and
poly(N-(3-trimethylammoniumpropyl)allylamine-co-N-n--
decylallylamine). Each of these ionic copolymers can also be
employed with counter ions other than chloride, for example, a
conjugate base of a pharmaceutically acceptable acid.
[0030] In a further embodiment, the polymer to be administered
comprises a monomer of Formula I, a hydrophobic monomer and a
neutral hydrophilic monomer, such as acrylamide, methacrylamide,
N-(2-hydroxyethyl)acrylamide or 2-hydroxyethylmethacrylate.
Examples of polymers of this type include terpolymers of
N-(3-trimethylammonium-propyl)methacrylamide/N-isopropylac-
rylamide/2-hydroxyethyl-methacrylate,
N-(3-trimethylammonium-propyl)methac-
rylamide/N-n-decylacrylamide/2-hydroxyethylmethacrylate,
N-(3-trimethylammoniumpropyl)methacrylamide/N-t-butylmethacrylamide/metha-
crylamide,
N-(3-trimethylammonium-propyl)methacrylamide/n-decylacrylate/me-
thacrylamide,
2-trimethylammoniumethylmethacrylate/n-butyl-acrylate/acryla- mide,
2-trimethylammonium-ethylmethacrylate/t-butylacrylate/acrylamide,
2-trimethylammoniumethylmethacrylate/n-decyl-acrylate/acrylamide,
2-trimethylammonium-ethylmethacrylate/n-decylmethacrylate/methacrylamide,
2-trimethylammoniumethylmethacrylate/N-t-butyl-methacrylamide/methacrylam-
ide and
2-trimethylammoniumethylmethacrylate/N-n-butyl-methacrylamide/meth-
acrylamide.
[0031] The polymer to be administered can be an addition polymer
having a polymer backbone such as a polyacrylate, polyacrylamide
poly(allylalcohol), poly(vinylalcohol), poly(vinylamine),
poly(allylamine), or polyalkyleneimine backbone. The polymer can
have a uniform backbone if it is composed of monomers derived from
a common polymerizable unit, such as acrylamide. If the polymer is
a copolymer, it can also comprise a mixed backbone, for example,
the monomer of Formula I can be an acrylamide derivative, while the
hydrophobic monomer can be a styrene derivative. The polymers
disclosed herein include examples of both uniform and mixed
backbones.
[0032] The polymers of use in the present method also include
condensation polymers, wherein polymerization of monomers is
accompanied by the release of a small molecule, such as a water
molecule. Such polymers include, for example, polyesters and
polyurethanes.
[0033] The polymers of use in the present method are preferably
substantially nonbiodegradable and nonabsorbale. 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
reion of the body, such as the gastrointestinal tract.
[0034] The composition of the copolymers to be administered can
vary substantially. The copolymer can comprise from about 95 mole
percent to about 5 mole percent, preferably from about 20 mole
percent to about 80 mole percent, of a monomer of Formula I. The
copolymer can also comprise from about 95 mole percent to about 5
mole percent, preferably from about 20 mole percent to about 80
mole percent, of a hydrophobic monomer.
[0035] Other examples of polymers which are of use in the present
method are disclosed in U.S. patent application Ser. Nos.
08/482,969, 08/258,477, 08/258,431, 08/469,659 and 08/471,769, the
contents of each of which are incorporated herein by reference.
[0036] 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 a period of time sufficient to
interact with the infecting organism. 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 500 Daltons to about 500,000 Daltons, preferably from about
2,000 Daltons to about 150,000 Daltons.
[0037] The polymers which are useful in the present method can be
prepared by known methods. A first method includes the direct
polymerization of a monomer, such as trimethylammoniumethylacrylate
chloride, or a set of two or more monomers, such as
trimethylammoniumethyl-acrylate chloride, N-n-butylacrylamide and
acrylamide. This can be accomplished via standard methods of free
radical, cationic or anionic polymerization which are well known in
the art. Due to reactivity differences between two monomers, the
composition of a copolymer produced in this way can differ from the
composition of the starting mixture. This reactivity difference can
also result in a non-random distribution of monomers along the
polymer chain.
[0038] A second method proceeds via the intermediacy of an
activated polymer comprising labile side chains which are readily
substituted by a desired side chain. An example of a suitable
activated polymer is the succinimide ester of polyacrylic acid,
poly(N-acryloyloxysuccinimide) (also referred to hereinafter as
"pNAS"), which reacts with nucleophiles such as a primary amine to
form a N-substituted polyacrylamide. Another suitable activated
polymer is poly(para-nitrophenylacrylate), which react with amine
nucleophiles in a similar fashion.
[0039] Polymers suitable for use in the present method can also be
prepared by addition of a side chain to a preformed polymer. For
example, poly(allylamine) can be alkylated at the amino nitrogen by
one or more alkylating agents. For example, one fraction of amino
groups can be alkylated using a normal or branched
C.sub.3-C.sub.18-alkyl halide, such as n-decyl bromide, while
another fraction can be alkylate by a quatemary ammonium-containing
alkyl halide, such as 1-trimethylammonium-4-bromombut- ane.
[0040] A copolymer having a polyacrylamide backbone comprising
amide nitrogens bearing two different substituents can be prepared
by treating p(NAS) with less than one equivalent (relative to
N-acryloyloxysuccinimid- e monomer) of a first primary amine,
producing a poly(N-substituted
acrylamide-co-N-acryoyloxysuccinimide) copolymer. Remaining
N-acryoyloxysuccinimide monomer can then be reacted with, for
example, an excess of a second primary amine to produce a
polyacrylamide copolymer having two different N-substituents. A
variety of copolymer compositions can, thus, be obtained by
treating the activated polymer with different proportions of two or
more amines.
[0041] An additional aspect of the present invention is a method
for treating a microbial infection in a mammal, such as a human,
comprising administering to the mammal a therapeutically effective
amount of a polymer having an amino group or an ammonium group
within the polymer backbone. The polymer can have, for example, a
polymethylene backbone which is interrupted by one or more amino or
ammonium groups. An example of a polymer of this type is
poly(decamethylenedimethylammonium-co-ethyle- nedimethylammonium)
bromide, which is synthesized via the reaction of
N,N,N',N'-tetramethylethylenediamine and 1,10-dibromodecane. The
polymer can also be administered in association with anions other
than bromide, such as chloride or acetate anions. Other examples
include poly(alkyleneimines), for example, poly(ethyleneimine).
Such polymers can comprise secondary or tertiary amino groups,
salts of such groups with pharmaceutically acceptable acids, and/or
quaternary ammonium groups.
[0042] As discussed below in Example 35, several polymers described
herein were tested for in vitro activity against Cryptosporidium
parvum infectivity in mammalian cell culture. Of these,
poly(TMAEMC-co-styrene), described in Example 7, was most active,
exhibiting greater than 90% inhibition of C. parvum infectivity
relative to the control when applied as a 0.1 mg/mL solution in
dimethylsulfoxide. The remaining polymers tested also showed
significant anti-Cryptosporidium activity.
[0043] The invention will now be further and specifically described
by the following examples.
EXAMPLES
[0044] The following abbreviations are used throughout the examples
to denote the following monomers: MAPTAC,
N-(3-trimethylammoniumpropyl)metha- crylamide chloride; TMAEMC,
2-trimethylammoniumethylmethacrylate chloride; HEMA,
2-hydroxyethylmethacrylate; TMAEAC,
2-trimethylammoniumethylacrylat- e chloride.
[0045] The copolymers and terpolymers of the following examples are
given nominal compositions which correspond to the molar ratios of
starting monomers in the copolymerization mixture.
Example 1
Synthesis of Poly(N-acryloyloxysuccinimide) (pNAS)
[0046] A solution of N-acryloyloxysuccinimide (25.0 g, 148 mmole)
in 100 mL dry DMF was degassed by nitrogen purging and
simultaneously heated to 60.degree. C. To the reaction mixture was
added azobisisobutyronitrile (AIBN) (120 mg, 0.005 equivalents with
respect to monomer). The reaction was allowed to proceed for 24
hours at 60.degree. C. The polymer solution was cooled to room
temperature and poured into rapidly stirred THF. The resulting
white precipitate was filtered, washed with THF and dried in
vacuo.
Example 2
Synthesis of
Poly(N-(3-dimethylamino-propyl)acrylamide-co-N-n-butylacrylam-
ide)
[0047] To a solution of 3.0 g (17.75 mmole) pNAS in 20 mL dry DMF
was added 0.6 g (3.55 mmole) n-butylamine. The resulting solution
was stirred at room temperature for 14 hours, and then heated at
60.degree. C. for 4 hours. After the solution was cooled to room
temperature, 9.05 g (89 mmole) 3-dimethylaminopropylamine was
added, and the resulting solution was stirred at room temperature
for 2 hours, then heated to 60.degree. C. for 20 hours. After
cooling to room temperature, the solution was diluted with 25 mL
water, and dialyzed against water for 24 hours. The solution was
then lyophilized to afford
poly(N-(3-dimethylaminopropyl-acrylamide)-- co-N-n-butylacrylamide)
as a tacky white solid.
Example 3
Synthesis of
Poly(N-(3-trimethylammoniumpropyl)acrylamide-co-N-n-butylacry-
lamide) Iodide
[0048] To a suspension of
poly(3-dimethylaminopropyl-acrylamide-co-N-n-but- ylacrylamide in
methanol was added 0.5 g methyl iodide. The resulting mixture was
stirred for 3 hours, and gradually became homogeneous. After
stirring for another 12 hours, the solvent was removed under
reduced pressure and the polymer was washed with dry hexane.
Example 4
Synthesis of
Poly(N-(2-hydroxyethyl)acrylamide-co-N-(6-trimethylammoniumhe-
xyl)acrylamide) Bromide
[0049] To a solution of 2.48 g (15 mmole) pNAS in 5 mL DMF was
added 1.00 g (3 mmole) 1-trimethylammonium-6-hexanamine bromide.
The solution was stirred at room temperature for 4 hours and then
heated at 60.degree. C. for 20 hours. The solution was cooled to
room temperature, and then 8.95 g (150 mmole) 2-ethanolamine was
added. The resulting mixture was heated to 80.degree. C. for 20
hours, cooled to room temperature and diluted with 10 mL water. The
solution was dialyzed against water for 24 hours, then lyophilized,
yielding the polymer as a brittle white solid.
Example 5
Synthesis of Poly(TMAEAC)
[0050] A solution of 48.25 g (0.25 mol)
2-trimethylammoniumethylacrylate chloride in 400 mL isopropanol was
degassed by nitrogen purging and heated to 35.degree. C. To this
stirred solution was added a solution of 0.8 g potassium persulfate
in 10 mL distilled water. A slight exotherm was observed. The
solution was stirred at 35.degree. C. for 6 hours, then cooled to
room temperature. The solution was added to hexanes and the
resulting precipitate was isolated by filtration.
Example 6
Synthesis of
Poly(decamethylenedimethylammonium-co-ethylenedimethylammoniu- m)
Bromide
[0051] N,N,N'N'-tetramethylethylenediamine (10.0 g, Aldrich),
1,10-dibromodecane (25.8 g, Aldrich) and methanol (100 mL) were
placed into a three-neck 250 mL round bottom flask. The mixture was
heated with gentle stirring to 65.degree. C. for 6 days, at which
point methanol (40 mL) was added, and the mixture was refluxed for
an additional 2 days. The mixture was then dripped into acetone,
forming a solid that was collected by filtration, rinsed with
acetone, and dried in a vacuum oven to yield 30.9 g of product.
Example 7
Synthesis of Poly(TMAEMC-co-styrene) 75/25
[0052] A 500 mL round bottomed flask was charged with
trimethylammoniumethylmethacrylate chloride (26.0 g of a 70 wt %
aqueous solution, 18.2 g), styrene (6.0 g) and isopropanol (150
mL). The solution was degassed by the addition of a rapid stream of
nitrogen for 10 minutes, followed by the addition of AIBN (0.5 g).
The solution was degassed for a further thirty minutes and, while
continuing the addition of nitrogen, the solution was heated to
70.degree. C., and the temperature maintained for 17 h. The polymer
began to precipitate within 2 h, and by the completion of the
reaction a sticky white precipitate had formed. The reaction
mixture was cooled, the isopropanol was decanted from the polymer,
and the polymer was dissolved in methanol. Dropwise addition of the
methanol solution to ethyl acetate (1200 mL) caused the polymer to
precipitate as a fine white powder which was recovered by
filtration.
Example 8
Synthesis of Poly(TMAEMC-co-N-isopropylacrylamide) (67/33)
[0053] A 500 mL round bottomed flask was charged with
trimethylammoniumethylmethacrylate chloride (14.5 g of a 70 wt %
aqueous solution, 10.0 g), N-isopropylacrylamide (5.0 g) and
isopropanol (150 mL). The solution was degassed by the addition of
a rapid stream of nitrogen for 10 minutes, followed by the addition
of AIBN (0.5 g). The solution was degassed for a further 60
minutes. The reaction mixture was heated to 70.degree. C., and the
temperature maintained for 16 h. The polymer partially precipitated
over the course of the reaction. Upon cooling, the propanol was
decanted from the polymer, and the polymer was dissolved in
methanol. Precipitation of the methanol solution dropwise into
ethyl acetate (1200 mL) caused the polymer to be deposited as white
curds which were recovered by filtration, washed with ethyl
acetate, and dried in vacuo.
[0054] Additional TMAEMC/N-isopropylacrylamide copolymers were
prepared by a similar method with the starting monomers in the
following ratios: TMAEMC/N-isopropylacrylamide =40/60, 25/75 and
15/85.
Example 9
Synthesis of Poly(MAPTAC-co-styrene) 75/25
[0055] To isopropanol (150 mL) was added a solution of
N-(3-trimethylammoniumpropyl)methacrylamide chloride in water (50
wt % solution, 24.0 g, 12.0 g of monomer). To this solution was
added styrene (6.0 g), followed by the addition of AIBN (0.5 g).
The homogeneous solution was degassed by bubbling a stream of
nitrogen through it for 30 minutes. The solution was heated to
70.degree. C. for 15 h. The polymer partially precipitated as the
reaction proceeded. The solution was cooled, the isopropanol was
decanted off, the white solid was washed with propanol (50 mL). The
propanol was decanted a second time, and the solid was dissolved in
methanol (150 mL). The clear solution was added dropwise to ethyl
acetate, causing the polymer to be precipitated as a white powder.
The polymer was recovered by filtration, washed with 50 mL of
ethylacetate and air dried.
[0056] An additional MAPTAC/styrene copolymer was prepared by a
similar method employing a 50/50 mixture of starting monomers.
Example 10
Synthesis of Poly(TMAEMC-co-heptafluorobutylacrylate) 75/25
[0057] A 500 mL round bottomed flask was charged with
2-trimethylammoniumethylmethacrylate chloride (26.0 g of a 70 wt %
aqueous solution, 18.2 g), heptafluorobutylacrylate (6.0 g) and
isopropanol (150 mL). The solution was degassed by the addition of
a rapid stream of nitrogen for 10 minutes, followed by the addition
of AIBN (0.5 g). The solution was degassed for a further thirty
minutes and, continuing the addition of nitrogen, the solution was
heated to 70.degree. C. The temperature was maintained for 17 h.
The polymer began to precipitate within 1 h, and by the completion
of the reaction a sticky white precipitate had formed. The reaction
mixture was cooled, the propanol was decanted from the polymer, and
the polymer was dissolved in methanol (100 mL). Precipitation of
the methanol solution dropwise into ethyl acetate (1200 mL) caused
the polymer to be deposited as a white solid which was recovered by
filtration.
Example 11
Synthesis of Poly(MAPTAC-co-N-t-butylacrylamide) 75/25
[0058] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 36.4 g of a
50% aqueous solution of
N-(3-trimethylammonium-propyl)methacrylamide chloride and 6 g of
N-t-butyl-acrylamide followed by 150 mL of isopropanol. The
solution was purged with nitrogen for 1 hour and 0.5 g AIBN was
added. The mixture was purged for .about.15 minutes until all of
the AIBN dissolved. The solution was heated to 75.degree. C. under
nitrogen for 16 hours.
[0059] The resulting reaction mixture consisted of two phases. The
turbid liquid phase was decanted from the bulk of the reaction
which was a white sticky solid phase. The liquid was precipitated
into 1200 mL of ethyl acetate and filtered by vacuum filtration
through a Buchner funnel. The white hygroscopic precipitate was
dried in vacuo. The solid phase was dissolved in methanol and
precipitated into 1200 mL of ethyl acetate and filtered by vacuum
filtration to yield a white powder which was stored under
vacuum.
[0060] Additional MAPTAC/N-t-butylacrylamide copolymers were
prepared by a similar method beginning with the starting monomers
in the following ratios:
N-(3-trimethylammoniumpropyl)methacrylamide/N-t-butyl-acrylamide=-
60/40, 50/50, 40/60, and 25/75.
Example 12
Synthesis of
Poly(N-decylallylamine-co-N-(4-trimethylammoniumbutyl)allylam-
ine)
[0061] To a solution of poly(allylamine).HCl (20.15 g of a 50 wt %
aqueous solution) was added sodium hydroxide (5.64 g) as a solid.
The solution was stirred for 40 minutes, filtered and the filter
cake was washed with methanol (15 mL). The solution was further
diluted with methanol (25 mL) and to the solution was added
1-bromodecane (7.73 g, 35 mmol) and
(1-trimethylamino-4-bromobutane) chloride (9.13 g, 35 mmol). A
solution was prepared of sodium hydroxide (2.8 g, 70 mmol) in water
(5 mL). This solution was added to the reaction mixture in four
portions at thirty minute intervals. The solution was then stirred
at room temperature for 24 h, followed by dialysis against
deionized water and freeze-dried. A total of 23.2 g of a glassy,
hygroscopic solid was recovered.
Example 13
Synthesis of Poly(TMAEMC-co-N-t-butylacrylamide) 57/43
[0062] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 18.20 g of a
70% aqueous solution of 2-trimethylammonium-ethylnethacrylic
chloride and 9.7 g of N-t-butylacrylamide followed by 150 mL of
isopropanol. The solution was purged with nitrogen for 1 hour and
0.5 g AIBN was added. The mixture was purged for .about.15 minutes
until all of the AIBN dissolved. The solution was heated to
75.degree. C. under nitrogen for 16 hours.
[0063] The resulting reaction mixture consisted of two easily
separable phases. The liquid phase was decanted from the bulk of
the reaction which was a white solid. The liquid was precipitated
into 1200 mL of ethyl acetate and filtered by vacuum filtration
through a Buchner funnel. The white precipitate was dried in vacuo
and weighed: fraction A, 10.1 g (45.1% yield based on 22.4 g
monomers added). The solid phase was dissolved in methanol and
precipitated into 600 mL of ethyl acetate and filtered by vacuum
filtration to yield fraction B, 5.81 g of a white powder (25.9%
yield) which was dried under vacuum.
[0064] TMAEMC/N-t-Butylacrylamide copolymers were also prepared by
a similar method with the starting monomers in the following
ratios: TMAEMC/N-t-Butylacrylamide=63/37, 50/50, 40/60, 25/75,
15/85 and 5/95.
Example 14
Synthesis of Poly(MAPTAC-co-N-n-decylacrylamide) 75/25
[0065] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 36.4 g of a
50% aqueous solution of N-(3-trimethylammoniumpropyl)methacrylamide
chloride and 6 g of N-n-decylacrylamide followed by 150 mL of
isopropanol. The solution was purged with nitrogen for 1 hour and
0.5 g AIBN was added. The mixture was purged for .about.15 minutes
until all of the AIBN dissolved. The solution was heated to
75.degree. C. under nitrogen for 16 hours.
[0066] The reaction mixture consisted of two easily separable
phases. The clear, yellow liquid phase was precipitated into 1200
mL of ethyl acetate. The precipitate was isolated by filtration and
dried under vacuum to yield 2.14 g of a yellow powder, fraction A
(8.84% yield). Methanol was added to the creamy yellow reaction
precipitate and the resulting turbid yellow solution was
precipitated into 1200 mL of ethyl acetate. The white precipitate
was isolated by filtration and dried under vacuum to yield fraction
B, 17.22 g, as a slightly yellow powder (71.2% yield).
[0067] Additional MAPTAC/N-n-decylacrylamide copolymers were
prepared by a similar method with the starting monomers in the
following ratios: MAPTAC/N-n-decylacrylamide=60/40, 50/50, and
40/60.
Example 15
Synthesis of Poly(TMAEMC-co-pentafluorostyrene) 75/25
[0068] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 26.0 g of a
70% aqueous solution of 2-trimethylammonium-ethylmethacrylate
chloride and 6 g of pentafluorostyrene followed by 150 mL of
isopropanol. The solution was purged with nitrogen for 1 hour and
0.5 g AIBN was added. The mixture was purged for .about.15 minutes
until all of the AIBN dissolved. The solution was heated to
75.degree. C. under nitrogen for 16 hours.
[0069] The reaction mixture consisted of two phases. The turbid
solution was discarded. The bulk of the reaction, consisting of a
white solid mass at the bottom of the flask, was dissolved in
methanol. The resulting clear solution was precipitated into 1200
mL of ethyl acetate. The white precipitate was isolated by vacuum
filtration to yield 20.39 g of a fine white powder (84.3%
yield).
[0070] Additional TMAEMC/pentafluorostyrene copolymers were
prepared by a similar method with the starting monomers in the
following ratios: TMAEMC/pentafluorostyrene=60/40 and 50/50.
Example 16
Synthesis of Poly(MAPTAC-co-pentafluorostyrene) 75/25
[0071] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 36.3 g of a
50% aqueous solution of N-(3-trimethylammoniumpropyl)methacrylamide
chloride and 6 g of pentafluorostyrene followed by 150 mL of
isopropanol. The solution was purged with nitrogen for 1 hour and
0.5 g AIBN was added. The mixture was purged for .about.15 minutes
until all of the AIBN dissolved. The solution was heated to
75.degree. C. under nitrogen for 16 hours.
[0072] The reaction mixture consisted of a turbid solution with a
white precipitate. The supematent was disgarded. The white reaction
precipitate was dissolved in methanol and the resulting clear
solution was precipitated into 1200 mL of ethyl acetate. The white
precipitate was isolated by filtration and dried under vacuum to
yield 12.81 g of a fine white powder (52.9% yield).
[0073] Additional MAPTAC/pentafluorostyrene copolymers were
prepared by a similar method with the starting monomers in the
following ratios: MAPTAC/pentafluorostyrene=60/40 and 50/50.
Example 17
Synthesis of MAPTAC/N-t-Butylacrylamide/HEMA Terpolymer
33/33/33
[0074] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.1 g of a 50% aqueous solution of
N-(3-trimethylammoniumpropyl)methacrylamide chloride, 8 g of
N-t-butylacrylamide, and 8 g of 2-hydroxyethylmethacrylate. The
solution was purged with nitrogen for 1 hour and 0.5 g of AIBN was
added. The mixture was purged for .about.15 min until all of the
AIBN dissolved. The solution was heated to 75.degree. C. under
nitrogen for 16 hours.
[0075] The reaction mixture consisted of a turbid solution with a
white latex in the bottom of the flask. The solution was
precipitated into 1200 mL of ethyl acetate. The white precipitate
was isolated by filtration to yield a sticky white powder which was
dried under vacuum to yield 10.43 g of a lumpy white solid
(fraction A) (43.1% yield). The white reaction precipitate was
dissolved in methanol and precipitated into 1200 mL of ethyl
acetate. The precipitate was isolated by filtration and dried under
vacuum to yield 8.89 g of a fine white powder (fraction B) (36.7%
yield).
[0076] Additional MAPTAC/N-t-butylacrylamide/HEMA terpolymers were
prepared by a similar method beginning with the following ratios of
the starting monomers: MAPTAC/N-t-Butylacrylamide/HEMA=28/43/28,
23/53/23, and 18/63/18.
Example 18
Synthesis of MAPTAC/N-Isopropylacrylamide/HEMA Terpolymer
18/63/18
[0077] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 8.9 g of a 50% aqueous solution of
N-(3-trimethylammoniumpropyl)methacrylamide chloride, 15.3 g of
N-iso-propylacrylamide, and 4.4 g of 2-hydroxyethylmethacrylate.
The solution was purged with nitrogen for 1 hour and 0.5 g of AIBN
was added. The mixture was purged for .about.15 min until all of
the AIBN dissolved. The solution was heated to 75.degree. C. under
nitrogen for 16 hours.
[0078] The clear slightly pink reaction solution was precipitated
into 1200 mL of ethyl acetate. The precipitate was isolated by
filtration to yield a sticky white solid which was dried under
vacuum to yield 14.42 g of a hard clear/white granular solid (59.6%
yield).
Example 19
Synthesis of MAPTAC/N-Decylacrylamide/HEMA Terpolymer 33/33/33
[0079] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.1 g of a 50% aqueous solution of
N-(3-trimethylammoniumpropyl)methacrylamide chloride, 8 g of
N-decylacrylamide, and 8 g of 2-hydroxyethylmethacrylate. The
solution was purged with nitrogen for 1 hour and 0.5 g of AIBN was
added. The mixture was purged for .about.15 min until all of the
AIBN dissolved. The solution was heated to 75.degree. C. under
nitrogen for 16 hours.
[0080] The reaction mixture consisted of two phases. The clear
yellow solution was precipitated into 1200 mL of ethyl acetate. The
precipitate was isolated by filtration. The sticky yellow
precipitate was dried under vacuum and the resulting brittle clear
yellow foam was crushed to yield 4.98 g of a fine yellow granular
powder (fraction A) (20.6% yield). The white reaction latex was
dissolved in methanol and precipitated into 1200 mL of ethyl
acetate. The precipitate was isolated by filtration and dried under
vacuum to yield 10.24 g of a slightly yellow granular solid
(fraction B) (42.3% yield).
[0081] Additional MAPTAC/N-Decylacrylamide/HEMA terpolymers were
prepared by a similar method beginning with the following ratios of
starting monomers: MAPTAC/N-Decylacrylamide/HEMA=28/43/28,
23/53/23, and 18/63/18.
Example 20
Synthesis of TMAEAC/n-Butylacrylate/Acrylamide Terpolymer
10/30/60
[0082] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 4.84 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 7.26 g of
n-butylacrylate, and 14.52 g of acrylamide. The solution was purged
with nitrogen for 1 hour and 0.5 g AIBN was added. The mixture was
purged for .about.15 minutes until all of the AIBN dissolved. The
solution was heated to 75.degree. C. under nitrogen for 16
hours.
[0083] The resulting white reaction mixture was filtered by vacuum
filtration through a Buchner funnel to yield a white powder. The
powder was washed with isopropanol and dried under vacuum to yield
21.57 g of a fine white powder (89.1% yield based on 24.2 g of
monomers).
[0084] Additional TMAEAC/n-butylacrylate/acrylamide terpolymers
were prepared by a similar method beginning with the following
ratios of starting monomers:
TMAEMC/n-butylacrylate/acrylamide=20/20/60 and 30/10/60.
Example 21
Synthesis of TMAEAC/t-Butylacrylate/Acrylamide Terpolymer
10/30/60
[0085] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 4.84 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 7.26 g of
t-butylacrylate, and 14.52 g of acrylamide. The solution was purged
with nitrogen for 1 hour and 0.5 g AIBN was added. The mixture was
purged for .about.15 minutes until all of the AIBN dissolved. The
solution was heated to 75.degree. C. under nitrogen for 16
hours.
[0086] The resulting white reaction mixture was filtered by vacuum
filtration through a Buchner funnel to yield a white powder. The
powder was washed with isopropanol and dried under vacuum to yield
21.13 g of a white powder (87.3% yield).
[0087] Additional TMAEAC/t-butylacrylate/acrylamide terpolymers
were prepared by a similar method beginning with the following
ratios of starting monomers:
TMAEAC/t-butylacrylate/acrylamide=20/20/60 and 30/10/60.
Example 22
Synthesis of TMAEAC/n-Decylacrylate/Acrylamide Terpolymer
10/30/60
[0088] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 4.84 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 7.26 g of
n-decylacrylate, and 14.52 g of acrylamide. The solution was purged
with nitrogen for 1 hour and 0.5 g AIBN was added. The mixture was
purged for .about.15 minutes until all of the AIBN dissolved. The
solution was heated to 75.degree. C. under nitrogen for 16
hours.
[0089] The resulting white reaction mixture was filtered by vacuum
filtration through a Buchner funnel to yield a white powder. The
powder was washed with isopropanol and dried under vacuum to yield
21.52 g of a fine white powder (89% yield).
[0090] Additional TMAEAC/n-decylacrylate/acrylamide terpolymers
were prepared by a similar method beginning with the following
ratios of starting monomers:
TMAEAC/n-decylacrylate/acrylamide=20/20/60, and 30/10/60.
Example 23
Synthesis of MAPTAC/N-t-Butylmethacrylamide/Methacrylamide
Terpolymer 10/30/60
[0091] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 4.84 g of a 50% aqueous solution of
N-(3-trimethylammoniumpropyl)methacrylamide chloride, 7.26 g of
N-t-butylmethacrylamide, and 14.52 g of methacrylamide. The
solution was purged with nitrogen for 1 hour and 0.5 g of AIBN was
added. The mixture was purged for .about.15 min until all of the
AIBN dissolved. The solution was heated to 75.degree. C. under
nitrogen for 16 hours.
[0092] The white reaction mixture was too difficult to filter by
vacuum filtration so centrifugation techniques were employed
instead. The reaction mixture was poured into 50 mL centrifuge
tubes and centrifuged. The supernatant was discarded. Isopropanol
was added to the polymer and the mixture was stirred and
centrifuged. The supernatant was discarded and the white solids
were combined and dried under vacuum to yield 14.99 g of a slightly
buff powder (61.9% yield).
[0093] Additional MAPTAC/N-t-butylmethacrylamide/methacrylamide
terpolymers were prepared by a similar method beginning with the
following ratios of starting monomers:
MAPTAC/N-t-butylmethacrylamide/met- hacrylamide=20/20/60, 33/33/33
and 30/10/60.
Example 24
Synthesis of MAPTAC/n-Decylmethacrylate/Methacrylamide Terpolymer
10/30/60
[0094] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 4.84 g of a 50% aqueous solution of
N-(3-trimethylammoniumpropyl)methacrylamide chloride, 7.26 g of
n-decylmethacrylate, and 14.52 g of methacrylamide. The solution
was purged with nitrogen for 1 hour and 0.5 g of AIBN was added.
The mixture was purged for .about.15 min until all of the AIBN
dissolved. The solution was heated to 75.degree. C. under nitrogen
for 16 hours.
[0095] The isopropanol was decanted leaving a white chunky powder.
Isopropanol was added and the mixture was poured into 50 mL
centrifuge tubes and centrifuged. The supernatant was discarded.
Isopropanol was added to the polymer and the mixture was stirred
and centrifuged. The supernatant was discarded and the white solids
were combined and dried under vacuum to yield 18.50 g of a granular
white solid (76.4% yield).
[0096] Additional MAPTAC/N-decylmethacrylamide/methacrylamide
terpolymers were prepared by a similar method beginning with the
following ratios of starting monomers:
MAPTAC/N-decylmethacrylamide/methacrylamide=20/20/60, 33/33/33 and
30/10/60.
Example 25
Synthesis of TMAEMC/n-Decylmethacrylate/Methacrylamide Terpolymer
10/30/60
[0097] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 3.46 g of a 70% aqueous solution of
2-trimethylammoniumethylmethacrylate chloride, 7.26 g of
n-decylmethacrylate, and 14.52 g of methacrylamide. The solution
was purged with nitrogen for 1 hour and 0.5 g AIBN was added. The
mixture was purged for .about.15 minutes until all of the AIBN
dissolved. The solution was heated to 75.degree. C. under nitrogen
for 16 hours.
[0098] The white reaction mixture was poured into 50 mL centrifuge
tubes and centrifuged. The supernatant was discarded. Isopropanol
was added to the polymer and the mixture was stirred and
centrifuged. The supernatant was discarded and the white solids
were combined and dried under vacuum to yield 10.29 g of a hard
white solid (42.5% yield).
[0099] Additional TMAEMC/N-n-decylmethacrylamide/methacrylamide
terpolymers were prepared by a similar method beginning with the
following ratios of starting monomers:
TMAEMC/N-n-decylmethacrylamide/met- hacrylamide=20/20/60, 33/33/33
and 30/10/60.
Example 26
Synthesis of TMAEMC/N-t-Butylmethacrylamide/Methacrylamide
Terpolymer 10/30/60
[0100] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 3.46 g of a 70% aqueous solution of
2-trimethylammoniumethylmethacrylate chloride, 7.26 g of
N-t-butylmethacrylamide, and 14.52 g of methacrylamide. The
solution was purged with nitrogen for 1 hour and 0.5 g AIBN was
added. The mixture was purged for 15 minutes until all of the AIBN
dissolved. The solution was heated to 75.degree. C. under nitrogen
for 16 hours.
[0101] The white reaction mixture was poured into 50 mL centrifuge
tubes and centrifuged. The supernatant was discarded. Isopropanol
was added to the polymer and the mixture was stirred and
centrifuged. The supernatant was discarded and the white solids
were combined and dried under vacuum to yield 18.35 g of a fine
white powder (75.8% yield).
[0102] Additional TMAEMC/N-t-butylmethacrylamide/methacrylamide
terpolymers were prepared by a similar method beginning with the
following ratios of starting monomers:
TMAEMC/N-t-butylmethacrylamide/met- hacrylamide=20/20/60, 33/33/33
and 30/10/60.
Example 27
Synthesis of TMAEMC/n-Butylmethacrylate/Methacrylamide Terpolymer
10/30/60
[0103] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 3.46 g of a 70% aqueous solution of
2-trimethylammoniumethylmethacrylate chloride, 7.26 g of
n-butylmethacrylate, and 14.52 g of methacrylamide. The solution
was purged with nitrogen for 1 hour and 0.5 g AIBN was added. The
mixture was purged for .about.15 minutes until all of the AIBN
dissolved. The solution was heated to 75.degree. C. under nitrogen
for 16 hours.
[0104] The white reaction mixture was poured into 50 mL centrifuge
tubes and centrifuged. The supernatant was discarded. Isopropanol
was added to the polymer and the mixture was stirred and
centrifuged. The supernatant was discarded and the white solids
were combined and dried under vacuum to yield 20.99 g of a clumpy
white powder (86.7% yield).
[0105] Additional TMAEMC/N-n-butylmethacrylamide/methacrylamide
terpolymers were prepared by a similar method beginning with the
following ratios of starting monomers:
TMAEMC/N-n-butylmethacrylamide/met- hacrylamide=20/20/60 and
30/10/60.
Example 28
Synthesis of MAPTAC/n-Butylmethacrylate/Methacrylamide Terpolymer
10/30/60
[0106] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 4.84 g of a 50% aqueous solution of
N-(3-trimethylammoniumpropyl)methacrylamide chloride, 7.26 g of
n-butylmethacrylate, and 14.52 g of methacrylamide. The solution
was purged with nitrogen for 1 hour and 0.5 g of AIBN was added.
The mixture was purged for .about.15 min until all of the AIBN
dissolved. The solution was heated to 75.degree. C. under nitrogen
for 16 hours.
[0107] The white reaction mixture was filtered by vacuum filtration
to yield a white powder. The powder was washed with isopropanol and
dried under vacuum to yield 22.20 g of a white powder (91.7%
yield).
[0108] Additional MAPTAC/n-butylmethacrylate/methacrylamide
terpolymers were prepared by a similar method beginning with the
following ratios of starting monomers:
MAPTAC/n-butylmethacrylate/methacrylamide=20/20/60 and
30/10/60.
Example 29
Synthesis of TMAEAC/n-Decylacrylamide/Acrylamid Terpolymer
33/33/33
[0109] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.13 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 8.06 g of
n-decylacrylamide, and 8.06 g of acrylamide. The solution was
purged with nitrogen for 1 hour and 0.5 g AIBN was added. The
mixture was purged for .about.15 minutes until all of the AIBN
dissolved. The solution was heated to 75.degree. C. under nitrogen
for 16 hours.
[0110] The reaction mixture was precipitated into 1200 mL of ethyl
acetate. The fine precipitate was filtered by vacuum filtration to
yield a sticky yellow material. The light yellow solid was
dissolved in methanol and precipitated into 1200 mL of ethyl
acetate. The precipitate was filtered by vacuum filtration to yield
10.85 g of a sticky, slightly yellow powder (44.8% yield).
Example 30
Synthesis of TMAEAC/N-t-Butylacrylamide/Acrylamide Terpolymer
33/33/33
[0111] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.13 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 8.06 g of
N-t-butylacrylamide, and 8.06 g of acrylamide. The solution was
purged with nitrogen for 1 hour and 0.5 g AIBN was added. The
mixture was purged for .about.15 minutes until all of the AIBN
dissolved. The solution was heated to 75.degree. C. under nitrogen
for 16 hours.
[0112] The reaction mixture consisted of a clear colorless solution
with a small amount of white sticky solid. The clear solution was
precipitated into 1200 mL of ethyl acetate. The white precipitate
was filtered, dissolved in water, and lyophilized to yield 6.65 of
a white powder (27.5% yield).
Example 31
Synthesis of TMAEAC/Styrene/Acrylamide Terpolymer 33/33/33
[0113] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.13 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 8.06 g of styrene, and
8.06 g of acrylamide. The solution was purged with nitrogen for 1
hour and 0.5 g AIBN was added. The mixture was purged for .about.15
minutes until all of the AIBN dissolved. The solution was heated to
75.degree. C. under nitrogen for 16 hours.
[0114] The reaction mixture consisted of a clear colorless solution
and a white solid. The clear solution was disgarded. The solid was
dissolved in methanol, and precipitated into ethyl acetate (1200
mL). A white precipitate formed which settled out of the solution
as a sticky white solid. The ethyl acetate was decanted and the
solid dried by passing nitrogen through the flask. The solid was
dissolved in water and lyophilized to yield 18.14 g of a fine white
powder (75% yield).
Example 32
Synthesis of TMAEAC/n-Butylacrylate/Acrylamide Terpolymer
33/33/33
[0115] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.13 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 8.06 g of
n-butylacrylate, and 8.06 g of acrylamide. The solution was purged
with nitrogen for 1 hour and 0.5 g AIBN was added. The mixture was
purged for .about.15 minutes until all of the AIBN dissolved. The
solution was heated to 75.degree. C. under nitrogen for 16
hours.
[0116] The reaction mixture consisted of a clear colorless solution
and a white chunky solid. The solution phase was disgarded and the
white solid dissolved in water, filtered and lyophilized to yield
12.84 of a fine white powder (53.1% yield).
Example 33
Synthesis of TMAEAC/n-Decylacrylate/Acrylamide Terpolymer
33/33/33
[0117] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.13 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 8.06 g of
n-decylacrylate, and 8.06 g of acrylamide. The solution was purged
with nitrogen for 1 hour and 0.5 g AIBN was added. The mixture was
purged for .about.15 minutes until all of the AIBN dissolved. The
solution was heated to 75.degree. C. under nitrogen for 16
hours.
[0118] The white reaction mixture was precipitated into 1200 mL of
ethyl acetate. The turbid solution was decanted and the polymer was
dried with nitrogen, dissolved in water, and lyophilized to yield
8.79 g of fine white powder (36.3% yield).
Example 34
Synthesis of TMAEAC/t-Butylacrylate/Acrylamide Terpolymer
33/33/33
[0119] To a 500 mL round-bottom, three-neck flask fitted with a
thermocouple, reflux condenser, and septum was added 150 mL of
isopropanol followed by 16.13 g of a 50% aqueous solution of
2-trimethylammoniumethylacrylate chloride, 8.06 g of
t-butylacrylate, and 8.06 g of acrylamide. The solution was purged
with nitrogen for 1 hour and 0.5 g AIBN was added. The mixture was
purged for .about.15 minutes until all of the AIBN dissolved. The
solution was heated to 75.degree. C. under nitrogen for 16
hours.
[0120] The white reaction mixture was precipitated into 1200 mL of
ethyl acetate. The turbid solution was decanted and the polymer was
dried with nitrogen, dissolved in water, and lyophilized to yield
6.51 g of fine white powder (26.9% yield).
Example 35
In vitro activity of selected polymers against C. Parvum
infectivity
[0121] Confluent MDBK cell monolayers were grown on 16 well slides,
and infected with 5.times.10.sup.5 of C. parvum oocysts per well.
Various dilutions of the test reagents in dimethylsulfoxide (DMSO)
were added to the monolayers and cultures were incubated at
37.degree. C.(8% CO.sub.2) for 48 hours. The level of C. parvum
infections was determined and analysed by an indirect
immunofluorescence (IF) assay at 48 hours. Anti-C. parvum
sporozoite rabbit serum (1:1000) was used as the primary antibody,
and fluorscein-conjugated anti rabbit goat serum (1:100) was used
as the secondary antibody. Each dilution was tested in quadruple,
and each assay was performed at least two times. The monolayers
were methanol fixed and, after IF labelling, the number of
parasites observed in 10 high power fields (HPF) per well in each
of the four wells per dilution was counted, statistically analysed
and compared with infected wells which contained DMSO only.
Paromomycin was used as the positive control drug. The results are
presented in the following Table.
1TABLE Concentration Polymer (mg/mL) % Inhibition
poly(TMAEMC-co-styrene) 25/75, 0.1 91.7 Example 7 0.033 83.2 0.011
38.9 0.0037 3.95 poly(TMAEMC-co-N- 10 100 t-butylacrylamide), 15/85
Example 13 1.0 100 0.1 59.1 0.01 38.0 poly(MAPTAC-co-N- 10 100
n-decylacrylamide), 40/60 Example 14 1.0 100 0.1 64.3 0.01 35.5
poly(MAPTAC-co-N- 10 70.2 t-butylacrylamide-co-HEMA) 33/33/33
Example 17 1.0 57.4 0.1 52.1 0.01 18.4 Poly(TMAEMC-co- 0.1 91.35
heptafluorobutylacrylate 60/40, Example 10 0.033 53.0 0.011 23.5
0.0037 4.2 paromomycin 2 79.4
[0122] Equivalents
[0123] 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
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
* * * * *