U.S. patent application number 12/687906 was filed with the patent office on 2010-07-08 for anionic polymers as toxin binders and antibacterial agents.
This patent application is currently assigned to GENZYME CORPORATION. Invention is credited to Caroline Isabelle Bacon Kurtz, Richard Fitzpatrick.
Application Number | 20100172861 12/687906 |
Document ID | / |
Family ID | 27384503 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100172861 |
Kind Code |
A1 |
Bacon Kurtz; Caroline Isabelle ;
et al. |
July 8, 2010 |
Anionic Polymers as Toxin Binders and Antibacterial Agents
Abstract
The present invention relates to a method of inhibiting a toxin
in an animal, such as a human, by administering to the animal a
therapeutically effective amount of a polymer having a plurality of
pendant acid functional groups which are directly attached to the
polymer backbone or attached to the polymer backbone by a spacer
group. The spacer group can have a length in the range from 0 to
about 20 atoms. The toxin is, typically, an exotoxin secreted by a
pathogenic microorganism, such as a bacterium.
Inventors: |
Bacon Kurtz; Caroline Isabelle;
(Sudbury, MA) ; Fitzpatrick; Richard; (Marblehead,
MA) |
Correspondence
Address: |
GENZYME CORPORATION;LEGAL DEPARTMENT
15 PLEASANT ST CONNECTOR
FRAMINGHAM
MA
01701-9322
US
|
Assignee: |
GENZYME CORPORATION
Cambridge
MA
|
Family ID: |
27384503 |
Appl. No.: |
12/687906 |
Filed: |
January 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11102576 |
Apr 7, 2005 |
7678369 |
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12687906 |
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10295019 |
Nov 14, 2002 |
6890523 |
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11102576 |
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09901206 |
Jul 9, 2001 |
6517827 |
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10295019 |
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09569276 |
May 11, 2000 |
6290946 |
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09901206 |
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09541268 |
Apr 3, 2000 |
6270755 |
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09569276 |
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60133975 |
May 13, 1999 |
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Current U.S.
Class: |
424/78.08 |
Current CPC
Class: |
Y02A 50/47 20180101;
A61P 31/00 20180101; Y02A 50/471 20180101; Y02A 50/475 20180101;
A61K 38/14 20130101; A61K 31/795 20130101; Y02A 50/478 20180101;
A61K 31/74 20130101; Y02A 50/481 20180101; A61P 1/00 20180101; Y02A
50/469 20180101; Y02A 50/473 20180101; Y02A 50/30 20180101; A61P
31/04 20180101; A61K 31/74 20130101; A61K 2300/00 20130101; A61K
31/795 20130101; A61K 2300/00 20130101; A61K 38/14 20130101; A61K
2300/00 20130101; A61K 38/14 20130101; A61K 31/795 20130101; A61K
38/14 20130101; A61K 31/74 20130101; A61K 31/795 20130101; A61K
31/415 20130101; A61K 31/74 20130101; A61K 31/415 20130101 |
Class at
Publication: |
424/78.08 |
International
Class: |
A61K 31/795 20060101
A61K031/795 |
Claims
1. (canceled)
2. A tablet comprising a. a polystyrene sulfonate polymer and b.
one or more pharmaceutically acceptable carriers, diluents or
excipients.
3. The tablet of claim 2 wherein said polystyrene sulfonate is
uncrosslinked.
4. The tablet of claim 2 wherein said polystyrene sulfonate polymer
is soluble.
5. The tablet of claim 2 wherein said polystyrene sulfonate polymer
has a molecular weight of about 400,000 to 1 million Daltons.
6. The tablet of claim 2 or claim 5 wherein said polystyrene
sulfonate polymer has a molecular weight of about 600,000
Daltons.
7. The tablet of claim 2 wherein said polystyrene sulfonate polymer
is in a protonated form, deprotonated form or a combination
thereof.
8. The tablet of claim 2 wherein said polystyrene sulfonate polymer
is in the deprotonated form in combination with a pharmaceutically
acceptable cation, such as sodium or an alkaline earth ion.
9. The tablet of claim 2 wherein said polystyrene sulfonate polymer
is sodium polystyrene sulfonate.
10. The tablet of claim 2 further comprising an antibiotic.
11. The tablet of claim 10 wherein said antibiotic is vancomycin or
metronidazole.
12. A capsule comprising a. a polystyrene sulfonate polymer and b.
one or more pharmaceutically acceptable carriers, diluents or
excipients.
13. The capsule of claim 12 wherein said polystyrene sulfonate is
uncrosslinked.
14. The capsule of claim 12 wherein said polystyrene sulfonate
polymer is soluble.)
15. The capsule of claim 12 wherein said polystyrene sulfonate
polymer has a molecular weight of about 400,000 to 1 million
Daltons.
16. The capsule of claim 12 or claim 15 wherein said polystyrene
sulfonate polymer has a molecular weight of about 600,000
Daltons
17. The capsule of claim 12 wherein said polystyrene sulfonate
polymer is in a protonated form, deprotonated form or a combination
thereof.
18. The capsule of claim 12 wherein said polystyrene sulfonate
polymer is in the deprotonated form in combination with a
pharmaceutically acceptable cation, such as sodium or an alkaline
earth ion.
19. The capsule of claim 12 wherein said polystyrene sulfonate
polymer is sodium polystyrene sulfonate.
20. The capsule of claim 12 further comprising an antibiotic.
21. The capsule of claim 20 wherein said antibiotic is vancomycin
or metronidazole.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/102,576, filed Apr. 7, 2005, which is a continuation of U.S.
application Ser. No. 10/295,019, filed Nov. 14, 2002, now U.S. Pat.
No. 6,890,523, which is a continuation of U.S. application Ser. No.
09/901,206, filed May 11, 2000, now U.S. Pat. No. 6,517,827, which
is a continuation of U.S. application Ser. No. 09/569,276 filed May
11, 2000, now U.S. Pat. No. 6,290,946, which is a
continuation-in-part of U.S. Application Ser. No. 09/541,268 filed
Apr. 3, 2000, now U.S. Pat. No. 6,270,755, and claims the benefit
of Provisional Application No. 60/133,975, filed May 13, 1999, the
contents of all applications and patents which are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Many pathogens produce toxins which are detrimental, and in
some cases, lethal, to the host organism. Toxins produced by
pathogens can be classified into two general categories, exotoxins
and endotoxins.
[0003] Exotoxins are generally proteins or polypeptides. These
toxins, which are secreted by the pathogen, can travel within the
host and cause damage in regions of the host far removed from the
infection site. Symptoms associated with exotoxins vary greatly and
include hemolysis, systemic shock, destruction of leukocytes,
vomiting, paralysis and diarrhea.
[0004] Enterotoxins are exotoxins which act on the small intestine
and cause massive secretion of fluid into the intestinal lumen,
leading to diarrhea. Enterotoxins are produced by a variety of
bacteria, including the food-poisoning organisms Staphylococcus
aureus, Clostridium perfringens, and Bacillus cereus, and the
intestinal pathogens Vibrio cholerae, Escherichia coli, and
Salmonella enteritidis.
[0005] Endotoxins are lipopolysaccharides/lipoproteins found in the
outer layer of the cell walls of gram-negative bacteria. These
lipopolysaccharides are bound to the cell membrane and are released
upon cytolysis. Symptoms associated with the release of endotoxins
include fever, diarrhea and vomiting. Specifically, endotoxins
stimulate host cells to release proteins, endogenous pyrogens,
which affect the area of the brain which regulates body
temperature. In addition to fever, diarrhea and vomiting, the host
animal may experience a rapid decrease in lymphocyte, leukocyte,
and platelet numbers, and enter into a general inflammatory
state.
[0006] Although endotoxins are less toxic than exotoxins, large
doses of endotoxins can cause death, generally through hemorrhagic
shock and tissue necrosis. Examples of bacteria which produce
endotoxins include bacteria of the genera Escherichia, Shigella,
and especially Salmonella.
[0007] In some cases, the active disease caused by an exotoxin can
be treated by administering an antitoxin to the patient. An
antitoxin comprises antibodies to the toxin derived from the serum
of an animal, typically a horse, which has been immunized by
injection of a toxoid, a nontoxic derivative of the toxin. However,
the effectiveness of antitoxins is limited because toxins are
rapidly taken up by cells and become unavailable to the antibodies.
Furthermore, the patient's immune system can respond to foreign
proteins present in the antitoxin, creating a condition known as
serum sickness.
[0008] Clostridium difficile has become one of the most common
nosocomially-acquired organisms in hospitals and long term care
institutions. The organism typically infects patients whose normal
intestinal flora has been disturbed by the administration of a
broad-spectrum antibiotic. The diarrhea and inflammatory colitis
associated with infection represent a serious medical/surgical
complication leading to increased morbidity and mortality, and
prolonging hospital stays by an average of nearly three weeks. This
is especially true for the elderly and for patients with serious
underlying diseases who are the most likely to develop the
infection. C. difficile associated diarrhea (CDAD) represents a
major economic burden to the healthcare system, conservatively
estimated at $3-6 billion per year in excess hospital costs in the
U.S. alone.
[0009] Currently, treatments for CDAD are inadequate. Such
treatments include discontinuing the antibiotic that caused CDAD to
manifest and allow the normal colonic flora to recover as rapidly
as possible. In most cases however, that is not sufficient and yet
another antibiotic, metronidazole, or vancomycin, are used to kill
the C. difficile organisms. Symptomatic improvement with
metronidazole is slow, typically taking 4 to 8 days. In addition,
because metronidazole also alters the normal flora by eradicating
most anaerobes from the gut, 20% of treated patients have a relapse
or recurrence of CDAD, usually within 1 to 2 weeks of stopping
therapy. In severe or recurrent cases of CDAD, vancomycin may be
used. However, this drug has a similar rate of relapse to
metronidazole and also has the potential for the undesirable side
effect of causing selection for multi-drug resistant enterococci
and staphylococci.
[0010] Diarrhea and colitis are a direct result of intestinal
damage and inflammation caused by C. difficile Toxins A and B. The
Toxins A and B, produced by C. difficile, damage the intestinal
mucosa and are the etiologic agents responsible for the
inflammatory colitis. Currently, no therapies are available to
inhibit the bacterial toxins produced by C. difficile and which are
responsible for the intestinal damage and inflammation leading to
diarrhea and colitis. Pharmaceuticals that can inhibit Toxins A and
B are the most logical approach to CDAD therapy.
[0011] Therefore, a need exists for an improved method of treating
a toxin-mediated condition which significantly reduces or
eliminates the above-mentioned problems.
SUMMARY OF THE INVENTION
[0012] The present invention relates to a method of inhibiting a
toxin in an animal, such as a human, by administering to the animal
a therapeutically effective amount of a polymer having a plurality
of pendant acid functional groups which are directly attached to
the polymer backbone or attached to the polymer backbone by a
spacer group. The spacer group can have a length in the range from
0 to about 20 atoms. The toxin is, typically, an exotoxin secreted
by a pathogenic microorganism, such as a bacterium. In a preferred
embodiment, the polymer is substantially free of acid
anhydrides.
[0013] In another embodiment, the present invention relates to
pharmaceutical compositions comprising anionic polymers and methods
for treatment of CDAD and other antibiotic associated diarrhea
(AAD) in mammals and particularly in humans. The therapeutic
compositions of the invention preferably inactivate both C.
difficile Toxins A and B and are highly effective in preventing the
development of CDAD (prophylactic treatment) as well as to prevent
recurrence and relapse of CDAD when used as a monotherapy or when
used as co-therapy with antibiotics (e.g. metronidazole and
vancomycin).
[0014] As discussed above, the polymers utilized in the described
methods are substituted by acid or anionic groups. Suitable acid
functional groups include carboxylic acid, sulfonic acid,
phosphonic acid, hydrosulfate, hydrophosphate, sulfamic acid and
boronic acid groups. The acid groups can also be present in the
conjugate base form in combination with a suitable cation.
[0015] In one embodiment, the polymer to be administered is a
copolymer characterized by a first monomer or repeat unit having a
pendant acid functional group and a second monomer or repeat unit
having a pendant hydrophobic group. In another embodiment, the
polymer is characterized by a monomer or repeat unit having both a
pendant acid functional group and a pendant hydrophobic group. The
polymer to be administered can, optionally, be further
characterized by a monomer or repeat unit comprising a neutral
hydrophilic group, such as a hydroxyl group or an amide group.
[0016] Preferred therapeutic compositions for use in the methods of
the invention comprise poly(styrenesulfonate) and salts thereof.
Preferred methods of the invention include administering a
therapeutically effective amount of a composition of the invention
to a patient either as a cotherapy with the broad spectrum
antibiotic which may otherwise precipitate the onset of CDAD or
AAD, but for the presence of the polystyrene-containing composition
of the invention. Cotherapy with a broad-spectrum antibiotic will
not interfere with the effectiveness of the antibiotic but at the
same time will prevent the onset of CDAD or AAD.
[0017] In another embodiment, the compositions of the invention may
be used either alone as a monotherapy or as cotherapy with
metronidazole, vancomycin or other antibiotics used to treat CDAD
or AAD, after the onset of disease. In yet another embodiment, the
compositions of the invention may be used either alone or as a
cotherapy with other antibiotics to prevent the recurrence or
relapse of disease.
[0018] The present invention has many advantages. For example, the
compositions used in the methods of the invention are easily
prepared using standard techniques of polymer synthesis and
inexpensive starting materials. The methods of the invention
generally do not interfere with the broad spectrum antibiotics
often necessary to treat other infections of the body and thus can
be used in conjunction with broad spectrum antibiotics.
Additionally, the patient can be simultaneously protected from the
adverse side effects of such broad spectrum antibiotics often
leading to CDAD or AAD when the prophylactic treatment regimens of
the invention are used in conjunction with delivery of broad
spectrum antibiotics to the patient. Likewise, the treatment
regimens of the invention generally do not interfere with the
actions of metronidazole or vancomycin and can therefore, also be
used in conjunction with such treatments after the onset of disease
or post treatment to prevent recurrence and relapse of disease.
Additionally, the compositions and methods of the invention may be
used as monotherapy to prevent the onset of disease (prophylactic),
to treat disease after onset, or to prevent relapse. Monotherapy in
accordance with the invention is particularly advantageous when
patients cannot tolerate antibiotic regimens.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
[0020] FIGS. 1 and 2 describe the effects of polystyrene sulfonate
(160-246) on Toxin A in two hamster models, described in more
detail below.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to a method of inhibiting a
pathogenic or microbial toxin in a patient, such as a human, by
administering to the patient a therapeutically effective amount of
a polymer comprising a plurality of pendant acid functional groups.
The acid functional group can be directly bonded to the polymer
backbone or linked to the polymer backbone by an aliphatic spacer
group having a length of from 1 to about 20 atoms.
[0022] As used herein the term "inhibiting a microbial toxin"
refers to inhibiting the activity of a toxin which is associated
with the development of a particular disease state or medical
condition. The microbial toxin is an endotoxin or exotoxin produced
by a microorganism, such as a bacterium, a fungus, a protozoan or a
virus. The toxin can be inhibited by any mechanism, including, but
not limited to, binding of the toxin by the polymer. As used
herein, a "therapeutically effective amount" is an amount
sufficient to inhibit or prevent, partially or totally, tissue
damage or other symptoms associated with the action of the toxin
within or on the body of the patient or to prevent or reduce the
further progression of such symptoms.
[0023] The present invention provides methods and therapeutic
compositions useful for treating AAD, including CDAD, and colitis.
As used herein, the term "treating" diseases of the invention
includes: prophylactic treatment of those mammals susceptible to
AAD, CDAD or inflammatory colitis; treatment at the initial onset
of AAD, CDAD or inflammatory colitis; treatment of ongoing AAD,
CDAD or inflammatory colitis; and treatment of relapsing AAD, CDAD
or inflammatory colitis in susceptible mammals. As used herein a
"susceptible" mammal is a mammal capable of developing disease or
having a relapse of disease for any reason including use of broad
spectrum antibiotics which may disrupt the normal flora leading to
CDAD. Therapeutic compositions of the invention preferably comprise
polystyrene sulfonate, salts and copolymers therefor.
[0024] The term "monomer", as used herein, refers to both a
molecule comprising one or more polymerizable functional groups
prior to polymerization, and a repeat unit of a polymer. A
copolymer is said to characterized by the presence of two or more
different monomers.
[0025] 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 poly(ethylene) backbone which is substituted with
carboxylic acid (--C(O)OH) groups as side chains.
[0026] A "pendant" group is a moiety which forms a side chain or a
portion of a side chain attached to the polymer backbone. A pendant
group can, for example, be bonded directly to one or more atoms
within the polymer backbone or can be connected to the polymer
backbone by way of a spacer group.
[0027] The acid-functionalized monomer comprises a pendant acid
functional group, such as a carboxylic acid group, a sulfonic acid
group, a hydrosulfate group, a phosphonic acid group, a sulfamic
acid group, a hydrophosphate group or a boronic acid group. Acid
functional groups are referred to herein as the acid protonated
form or partially protonated form. However, it is to be understood
that any acid functional group can also exist in the conjugate base
or deprotonated form in combination with a pharmaceutically
acceptable cation. The polymer to be administered can include acid
functional groups in either the protonated form, the deprotonated
form or a combination thereof. Suitable cations include alkali
metal ions, such as sodium, potassium and cesium ions, alkaline
earth ions, such as calcium and magnesium ions, transition metal
ions and unsubstituted and substituted (primary, secondary,
tertiary and quaternary) ammonium ions. In one embodiment, the
cation is a polyvalent metal ion, such as Ca.sup.2+, Mg.sup.2+,
Zn.sup.2+, Bi.sup.3+, Fe.sup.2+ or Fe.sup.3+.
[0028] It is preferred that the polymer is substantially free of
acid anhydride groups. For example, less than 5%, preferably less
than 2%. More preferably none of the acid functional groups within
the polymer are present in the anhydride form.
[0029] The acid functional group can be directly bonded to the
polymer backbone, or can be attached to the polymer backbone via a
spacer group. The spacer group is a component of the polymer side
chain and connects the acid functional group to the polymer
backbone. The spacer group can be linear, branched or cyclic,
aliphatic, aromatic or partially aromatic and partially aliphatic.
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, e.g.
--(CH.sub.2).sub.2O[(CH.sub.2).sub.2O](CH.sub.2).sub.2--, wherein n
is an integer ranging from 0 to about 3. The 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. Suitable aromatic spacer groups include ortho-,
meta- and para-phenylene groups, naphthylene groups and biphenylene
groups.
[0030] In one embodiment, at least a portion of the repeat units
within the polymer further include a pendant hydrophobic group. The
pendant hydrophobic group can be a substituted or unsubstituted,
saturated or partially unsaturated C.sub.2-C.sub.24-hydrocarbyl
group or a substituted or unsubstituted aryl or arylalkyl group.
Examples of suitable alkyl substituents include halogen atoms, such
as fluorine or chlorine atoms, and aryl groups, such as a phenyl
group. Aryl substituents can include halogen atoms,
C.sub.1-C.sub.6-alkyl groups and C.sub.1-C.sub.6-alkoxy groups.
Preferably, the pendant hydrophobic group is a normal or branched
C.sub.2-C.sub.24-alkyl group.
[0031] In one embodiment, the polymer to be administered is a
homopolymer. In another embodiment, the polymer to be administered
is a copolymer which is characterized by 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,
but are not limited to, a substituted or unsubstituted
N--C.sub.3-C.sub.24-alkylacrylamide, 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; styrene and substituted styrenes, such as
pentafluorostyrene and 4-fluorostyrene; vinylnaphthalene and
vinylbiphenyl. 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.
[0032] In a preferred embodiment, the polymer to be administered is
characterized by a repeat unit which comprises one acid functional
group. In this embodiment, no two acid functional groups within the
polymer will be connected to adjacent polymer backbone atoms. In
one embodiment, the polymer to be administered is characterized by
a repeat unit or monomer of the general formula
##STR00001##
wherein X is a spacer group, as described above, or a direct bond,
R.sup.1 and R.sup.2 are each, independently, 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, vinylsulfonic acid, vinylphosphonic acid,
3-allyloxy-2-hydroxy-1-propanesulfonic acid, vinylacetic acid and
esters of vinyl alcohol and allyl alcohol with mineral acids, such
as sulfuric, phosphoric and boric acids, including vinyl
hydrosulfate, vinyl dihydrophosphate, allyl hydrosulfate, allyl
dihydrophosphate and conjugate bases thereof. The monomer can also
be polymerized alkene which is substituted with an acid functional
group, such as undecenoic acid, undecenyl hydrosulfate and
undecenyl sulfonic acid. Other suitable examples include
acid-functionalized styrene, such as styrene sulfonate, styrene
phosphonate and vinylbenzoic acid, acid-functionalized
vinylnaphthalene, such as vinylnaphthalene sulfonate, and
acid-functionalized vinylbiphenyl, such as vinylbiphenyl
sulfonate.
[0033] In another embodiment, the polymer to be administered is
characterized by a repeat unit or monomer of the general
formula
##STR00002##
wherein Z is oxygen or NH and X is a spacer group, as described
above, or a direct bond. Y is an acid functional group and R.sup.1
and R.sup.2 are each, independently, hydrogen or an alkyl group,
preferably methyl or ethyl. Examples of suitable monomers of this
type include 2-acrylamidoglycolic acid and
2-acrylamido-2-methyl-1-propanesulfonic acid.
[0034] Suitable copolymers for use in the present method include
copolymers of acrylic acid and a C.sub.2-C.sub.20-alkylacrylate,
such as poly(acrylic acid-co-n-decylacrylate) and poly(acrylic
acid-co-n-butylacrylate). Also included are copolymers of acrylic
acid and an N--C.sub.2-C.sub.20 alkylacrylamide, such as
poly(acrylic acid-co-N-isopropylacrylamide) and poly(acrylic
acid-co-N-n-decylacrylamide), and copolymers of acrylic acid with
styrene or a substituted styrene, such as pentafluorostyrene or
4-fluorostyrene.
[0035] In another embodiment, the polymer to be administered is a
copolymer comprising an acid-functionalized monomer, a hydrophobic
monomer and a neutral hydrophilic monomer. A neutral hydrophilic
monomer is a monomer comprising a polar group which is neither
appreciably acidic nor appreciably basic at physiological pH.
Examples of suitable neutral hydrophilic monomers include
acrylamide, N-(2-hydroxyethyl)acrylamide,
N-(3-hydroxypropyl)acrylamide, 2-hydroxyethylacrylate, vinyl
acetate, vinyl alcohol and N-vinylpyrrolidone. A suitable copolymer
of this type is the terpolymer poly(acrylic
acid-co-n-decylacrylate-co-acrylamide).
[0036] 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.
[0037] In one embodiment, the polymer to be administered comprises
a first repeat unit which comprises a pendant acid functional group
and a second repeat unit which comprises a pendant acid derivative,
such as an amide group or an ester group. Suitable examples of
polymers of this type include poly(styrenesulfonate) in which a
portion of the sulfonate groups have been converted to sulfonamide
or sulfonate ester groups and polyacrylate in which a portion of
the carboxylate groups have been converted to amide or ester
groups. The properties of the polymer can be varied by varying the
amount and chemical features of the groups introduced into the
polymer via the amidation or esterification process. In one
embodiment, the polymer comprises repeat units having pendant ester
groups, where the ester group is derived from an alcohol, such as
menthol, a bile acid, such as cholic acid or lithocholic acid, or
an alkanol, such as a normal or branched C.sub.4-C.sub.12-alkanol.
In another embodiment, the polymer comprises repeat units having
pendant amide groups, where the amide groups are derived from an
amine, such as an alkylamine, for example, a normal or branched
C.sub.4-C.sub.12-alkylamine or an ammonioalkylamine. Suitable
ammonioalkylamines include compounds of the formula
R.sup.1(R.sup.2)(R.sup.3)N.sup.+(CH.sub.2).sub.nNH.sub.2, where
R.sub.1, R.sub.2 and R.sub.3 are each, independently, hydrogen, a
C.sub.1-C.sub.12-alkyl group or an arylalkyl group, and n is an
integer from 1 to about 12.
[0038] In another embodiment, the polymer to be administered is a
copolymer comprising an acid-functionalized monomer or repeat unit,
a cationic repeat unit and, optionally, a hydrophobic repeat unit
and/or a neutral hydrophilic repeat unit. For example, the
acid-functionalized, hydrophobic and neutral hydrophilic repeat
unit can include any of the repeat units of these types discussed
above. The cationic repeat unit carries a positive charge under
physiological conditions, and, preferably, includes a pendant amino
or ammonium group. Suitable repeat units of this type include those
disclosed in U.S. patent application Ser. No. 08/934,495,
incorporated herein by reference n its entirety. Examples of
suitable cationic repeat units include allylamine, N-substituted
allylamine, quaternized allylamine, diallylamine, N-substituted
diallylamine, quaternized diallylamine, vinylamine, N-substituted
vinylamine, quaternized vinylamine, N-aminoalkylacrylamide and
-methacrylamide, N-ammonioalkylacrylamide and -methacrylamide,
aminoalkyacrylate and -methacrylate, and ammonioalkylacrylate and
-methacrylate. The ratio of anionic and cationic repeat units can
vary widely, for example, from about 95% anionic monomer and 5%
cationic monomer relative to the total charged monomers in the
polymer, to about 5% anionic monomer and 95% cationic monomer
relative to the total charged monomers, preferably 75% or more of
the monomers are anionic.
[0039] Preferred polymers of the invention include but are not
limited to:
Poly(vinylsulfate), poly(propenesulfate), poly(butenesulfate),
poly(pentenesulfate), poly(hexenesulfate), poly(heptenesulfate),
poly(octenesulfate), poly(nonenesulfate), poly(decenesulfate),
poly(undecenesulfate), poly(dodecenesulfate) Poly(vinylsulfonate),
poly(propenesulfonate), poly(butenesulfonate),
poly(pentenesulfonate), poly(hexenesulfonate),
poly(heptenesulfonate), poly(octenesulfonate),
poly(nonenesulfonate), poly(decenesulfonate),
poly(undecenesulfonate), poly(dodecenesulfonate)
Poly(vinylphosphate), poly(propenephosphate),
poly(butenephosphate), poly(pentenephosphate),
poly(hexenephosphate), poly(heptenephosphate),
poly(octenephosphate), poly(nonenephosphate),
poly(decenephosphate), poly(undecenephosphate),
poly(dodecenephosphate) Poly(vinylphosphonate),
poly(propenephosphonate), poly(butenephosphonate),
poly(pentenephosphonate), poly(hexenephosphonate),
poly(heptenephosphonate), poly(octenephosphonate),
poly(nonenephosphonate), poly(decenephosphonate),
poly(undecenephosphonate), poly(dodecenephosphonate) Carrageenan,
heparin, heparan sulfate, dextran sulfate, pentosan sulfate,
laminarin sulfate, chondroitin sulfate, dermatan sulfate
Poly(styrenesulfonate), poly(styrenesulfate),
poly(styrenesulfanilate), poly(sulfophenylalanine),
poly(tyrosinesulfate), poly(sulfophenethylacrylamide),
poly(sulfophenethylmethacrylamide),
poly(vinylnaphthalenesulfonate), poly(vinylnaphthalenesulfate),
poly(vinylbiphenylsulfonate), poly(vinylbiphenylsulfate),
poly(anetholesulfonate), poly(vinylbenzoic acid)
Poly(sulfophenylpropene), poly(sulfophenylbutene),
poly(sulfophenylpentene), poly(sulfophenylhexene),
poly(sulfophenylheptene), poly(sulfophenyloctene),
poly(sulfophenylnonene), poly(sulfophenyldecene),
poly(sulfophenylundecene), poly(sulfophenyldodecene)
Poly(sulfatephenylpropene), poly(sulfatephenylbutene),
poly(sulfatephenylpentene), poly(sulfatephenylhexene),
poly(sulfatephenylheptene), poly(sulfatephenyloctene),
poly(sulfatephenylnonene), poly(sulfatephenyldecene),
poly(sulfatephenylundecene), poly(sulfatephenyldodecene)
Poly(phosphophenylpropene), poly(phosphophenylbutene),
poly(phosphophenylpentene), poly(phosphophenylhexene),
poly(phosphophenylheptene), poly(phosphophenyloctene),
poly(phosphophenylnonene), poly(phosphophenyldecene),
poly(phosphophenylundecene), poly(phosphophenyldodecene)
Poly(phosphatephenylpropene), poly(phosphatephenylbutene),
poly(phosphatephenylpentene), poly(phosphatephenylhexene),
poly(phosphatephenylheptene), poly(phosphatephenyloctene),
poly(phosphatephenylnonene), poly(phosphatephenyldecene),
poly(phosphatephenylundecene), poly(phosphatephenyldodecene)
Sulfonated poly(vinylphenyl ketone), sulfonated
poly(phenylsulfone), sulfonated poly(4-methylstyrene), sulfonated
poly(.alpha.-methylstyrene), sulfonated
poly(styrene-block-ethyleneoxide-block-styrene), sulfonated
poly(ethylene oxide-block-styrene-block-ethyleneoxide), sulfonated
poly(4-methoxystyrene), sulfonated poly(diphenoxyphosphazene),
sulfonated poly(ethyleneoxide-block-styrene), sulfonated
poly(styrene-block-ethylene), sulfonated poly(acenaphthylene),
sulfonated poly(vinylcarbazole), sulfonated
poly(styrene-co-butadiene), sulfonated
poly(styrene-block-(ethylene-co-butylene)-block-styrene)
Poly(styrenesulfonate-co-maleic acid),
poly(styrenesulfonate-co-acrylic acid),
poly(styrenesulfonate-co-methacrylic acid),
poly(styrenesulfonate-co-acrylamidomethylpropanesulfonate),
poly(styrenesulfonate-co-itaconic acid),
poly(styrenesulfonate-co-vinylbenzoic acid)
Poly(styrenesulfonate-co-diallylmethylammonium chloride),
poly(styrenesulfonate-co-diallyldimethylammonium chloride),
poly(styrenesulfonate-co-diallylmethyloctylammonium chloride),
poly(styrenesulfonate-co-allylamine),
poly(styrenesulfonate-co-vinylamine),
poly(styrenesulfonate-co-vinylbenzyltrimethylammonium chloride)
Poly(styrenesulfonate-co-styrene),
poly(styrenesulfonate-co-octylstyrenesulfonamide),
poly(styrenesulfonate-co-menthylstyrenesulfonate),
poly(styrenesulfonate-co-lithocholic acid styrenesulfonate),
[0040] The polymers of use in the present method can be linear or
crosslinked. The polymer can be crosslinked, for example, by the
incorporation within the polymer of a multifunctional comonomer.
Suitable multifunctional co-monomers include diacrylates,
triacrylates and tetraacrylates, dimethacrylates, diacrylamides,
diallylacrylamide, di(methacrylamides), triallylamine and
tetraalkylammonium ion. Specific examples include ethylene glycol
diacrylate, propylene glycol diacrylate, butylene glycol
diacrylate, ethylene glycol dimethacrylate, butylene glycol
dimethacrylate, methylene bis(methacrylamide), ethylene
bis(acrylamide), ethylene bis(methacrylamide), ethylidene
bis(acrylamide), ethylidene bis(methacrylamide), pentaerythritol
tetraacrylate, trimethylolpropane triacrylate, bisphenol A
dimethacrylate, and bisphenol A diacrylate. Other suitable
multifunctional monomers include polyvinylarenes, such as
divinylbenzene. The amount of crosslinking agent is typically
between about 1.0% and about 30% by weight relative to the weight
of the polymer, preferably from about 5% to about 25% by
weight.
[0041] The polymer can also be cross-linked subsequent to
polymerization. For example, a portion of the acid functional
groups can be converted to a reactive derivative, as is known in
the art. For example, carboxylic acid and sulfonic acid groups
react with thionyl chloride to produce, respectively, acyl chloride
and sulfonyl chloride groups. These reactive groups can then be
reacted with a diamine, a dialcohol or an amino alcohol, preferably
diamine, a dialcohol or an amino alcohol in which the amino and/or
hydroxyl groups are separated by an alkylene chain, such as a
C.sub.3-C.sub.18-alkylene chain. This reaction results in the
formation of ester and/or amide groups on a given polymer chain
which are linked to similar groups on adjacent polymer chains. The
extent of cross-linking can be controlled, for example, by
controlling the fraction of acid functional groups which are
converted to reactive groups.
[0042] The molecular weight of the polymer is not critical, but is,
preferably, 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 or degree of cross-linking to resist absorption, partially
or completely, from the gastrointestinal tract into other parts of
the body. Preferably, if linear, the polymer to be administered has
a molecular weight ranging from about greater than 1 to about 1
million Daltons or more, such as 2,000 Daltons to about 500,000
Daltons, 5,000 Daltons to about 150,000 Daltons, or about 25,000
Daltons to about 1 million Daltons. Alternatively, the molecular
weight can be from about 100,000 to about 1 million or between
about, 400,000 to about 1 million Daltons.
[0043] The polymers of use in the present method are preferably
substantially nonbio-degradable 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. A
particularly preferred polymer is polystyrene sulfonate.
Preferably, the polymer is a soluble, uncrosslinked polystyrene
sulfonate polymer having a molecular weight between about 400,000
and 1 million Daltons, such as 600,000 Daltons. Alternatively,
polymer backbones which are suitable for the present invention
include polyacrylamide, polyacrylate, poly(vinyl) and
poly(ethyleneimine), polystyrene 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.
[0044] 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 identity of
the known or suspected pathogenic organism, 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 and one or more pharmaceutically acceptable carriers,
diluents or excipients. The pharmaceutical composition can also,
optionally, include one or more additional drugs, such as
antibiotics, anti-inflammatory agents or analgesics.
[0045] The polymer can be administered by subcutaneous or other
injection, intravenously, topically, orally, parenterally,
transdermally, or rectally through feeding tube. Preferably, the
polymer or the pharmaceutical composition comprising the polymer is
administered orally. The form in which the polymer is 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 single
dose or in a series of doses separated by appropriate time
intervals, such as hours.
[0046] For oral delivery, polymers may be administered at a doseage
of about 0.1 to 10 g/Kg/day and more preferably from 1.0-7.0
g/Kg/day and even more preferably from 2.0 to 6.6 g/Kg/day.
[0047] The polymer can also administered in combination with one or
more antimicrobial agents, for example, selected from among
antibiotics which are known in the art. The antibiotic to be
administered is, generally, selected based on the identity or
suspected identity of the pathogenic microorganism, as is known in
the art. For example, if the pathogenic microorganism is C. parvum,
one suitable antibiotic which can be administered in combination
with the polymer is paromomycin. The polymer and the antimicrobial
agent can be administered simultaneously, for example, in separate
dosage forms or in a single dosage form, or in sequence separated
by appropriate time intervals.
[0048] The term "antimicrobial agent" is intended to include
antibacterial agents, antifungal agents, antiseptics and the like.
Suitable antimicrobial agents are known in the art and include
isoniazid, rifampin, pyrazinamide, ethambutol, erythromycin,
vancomycin, tetracycline, chloramphenicol, sulfonamides,
gentamicin, amoxicillin, penicillin, streptomycin,
p-aminosalicyclic acid, clarithromycin, clofazimine, minocycline,
sulfonamides, ethionamide, cycloserine, kanamycin, amikacin,
capreomycin, viomycin, thiacetazone, rifabutin and the quinolones,
such as ciprofloxacin, ofloxacin and sparfloxicin. The term
"antibacterial agent" includes but is not limited to: naturally
occurring antibiotics produced by microorganisms to suppress the
growth of other microorgansims, and agents synthesized or modified
in the laboratory which have either bactericidal or baceriostatic
activity, e.g., .beta.-lactam antibacterial agents including, e.g.
carbencillim; ampicillin, cloxacillin, oxacillin and pieracillin,
cephalosporins and other cephems including, e.g. cefaclor,
cefamandole, cefazolin, cefoperazone, ceftaxime, cefoxitin,
ceftazidime, ceftriazone and carbapenems including, e.g., imipenem
and meropenem; and glycopeptides, macrolides, quinolones (e.g.
nalidixic acid), tetracyclines, aminoglycosides (e.g. Gentamicin
and Paromomycin) and further includes antifungal agents. In general
if an antibacterial agent is bacteriostatic, it means that the
agent essentially stops bacterial cell growth (but does not kill
the bacteria); if the agent is bacteriocidal, it means that the
agent kills bacterial cells (and may stop growth before killing the
bacteria).
[0049] Thus, the polymers and compositions described herein can be
used in medicine, for example, in the manufacture of a medicament
for the therapies and treatments described herein.
[0050] In one embodiment, the polymer which comprises a plurality
of pendant acid functional groups is administered in combination
with a cationic polymer, preferably a polymer comprising amino
and/or ammonium groups. Examples of suitable polymers of this type
are disclosed in copending application Ser. No. 08/934,495,
incorporated herein by reference in its entirety. Suitable cationic
polymers can be linear or cross-linked. Included are polymers
comprising repeat units or monomers such as allylamine,
diallylamine, diallylmethylamine, vinylamine,
N-aminoallcylacrylamide, N-aminoalkylmethacrylamide,
aminoalkylacrylate, aminoalkylmethacrylate and acid addition salts
and monoalkylated, dialkylated and trialkylated (quaternized)
derivatives thereof. Suitable cationic polymers include
homopolymers of these repeat units and copolymers including at
least one of these repeat units and, optionally, one or more
hydrophobic monomers and/or neutral hydrophilic monomers, as
discussed above. The acid-functionalized polymer and the cationic
polymer can be administered in varying ratios by weight and can be
administered simultaneously, for example, in a single dosage form
or in separate dosage forms, or in a sequence separated by minutes
or hours. Suitable dosages and administration methods can be
readily determined by one of skill in the art. In one embodiment,
the anionic polymer is poly(styrensulfonate) and the cationic
polymer is poly(diallylmethylamine) or poly(diallylmethylamine) in
which a portion of the repeat units have been alkylated, for
example with a C.sub.4-C.sub.12-alkyl group, such as an octyl group
or a decyl group.
[0051] The polymers of the present invention can be prepared via
methods known in the art, for example, by direct polymerization of
an acid-functionalized monomer or copolymerization of a monomer
mixture comprising an acid-functionalized monomer and at least one
additional co-monomer, such as a second acid-functionalized
monomer, a hydrophobic monomer, a neutral hydrophilic monomer, a
multifunctional cross-linking monomer or a combination thereof. 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.
[0052] The polymers can also be synthesized by nucleophilic side
chain substitution on a activated polymer. This 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.
[0053] For example, a copolymer with a polyacrylamide backbone
comprising amide nitrogen atoms substituted with an acid functional
group and amide nitrogen atoms substituted with a hydrophobic group
can be prepared by treating pNAS with less than one equivalent
(relative to N-acryloyloxysuccinimide monomer) of a primary amine
which terminates in an acid functional group, such as an amino
acid, for example, glycine. A hydrophobic group can then be
introduced by reacting at least a portion of the remaining
N-acryloyloxysuccinimide monomers with a second primary amine, such
as a C.sub.2-C.sub.20-alkylamine. A co-polymer further comprising a
neutral hydrophilic monomer can be prepared by reacting any
remaining N-acryloyloxysuccinimide monomers with, for example,
2-aminoethanol or ammonia. A variety of copolymer compositions can,
thus, be readily obtained by treating the activated polymer with
different ratios of selected amines.
[0054] The polymers of use in the present method can also be
synthesized by functionalization of a precursor polymer with an
acid functional group. For example, a polymer having side chains
which include aryl groups can be sulfonated using known methods to
produce a polymer having pendant sulfonic acid groups. Precursor
polymers which include hydroxyl groups, such as poly(vinyl alcohol)
and poly(allyl alcohol) can be sulfated using known methods to form
polymers comprising sulfate ester groups.
[0055] Polymers having both acid functional groups and hydrophobic
groups can also be synthesized using this general approach. For
example, a poly(vinylarene) polymer, such as polystyrene can be
sulfonated by reaction with, for example, fuming sulfuric acid, to
form poly(styrene sulfonate).
[0056] An acid-functionalized polymer can be modified by converting
at least a portion of the acid groups to an acid derivative, such
as an amide or an ester. For example, poly(styrenesulfonate) can be
reacted with a substoichiometric amount, based on sulfonate groups,
of thionyl chloride, thereby converting a portion of the sulfonate
groups to sulfonyl chloride groups. The resulting polymer can, for
example, be reacted with an excess of a primary amine to convert
the sulfonyl chloride groups to N-substituted-sulfonamide groups or
with an alcohol to convert the sulfonyl chloride groups to
sulfonate ester groups. The hydrophobicity of the resulting polymer
can be varied by varying either or both of the N-substituent or
ester functionality and the extent of conversion of sulfonate
groups to sulfonamide or sulfonate ester groups. Pathogenic toxins
which can be inhibited by the method of the present invention
include, but are not limited to, toxins, such as exotoxins and/or
endotoxins produced by Streptococcus spp., including Streptococcus
pneumoniae, Streptococcus pyogenes and Streptococcus Sanguis;
Salmonella spp., including Salmonella enteritidis; Campylobacter
spp., including Campylobacter jejuni; Escherichia spp., including
E. coli; Clostridia spp., including Clostridium difficile and
Clostridium botulinum; Staphylococcus spp., including
Staphylococcus aureus; Shigella spp., including Shigella
dysenteriae; Pseudomonas spp., including Pseudomonas aeruginosa;
Bordatella spp., including Bordatella pertussis; Listeria spp.,
including Listeria monocytogenes; Vibrio cholerae; Yersinia spp.,
including Yersinia enterocolitica; Legionella spp., including
Legionella pneumophilia; Bacillus spp., including Bacillus
anthracis; Helicobacter spp.; Corynebacteria spp.; Actinobacillus
spp.; Aeromonas spp.; Bacteroides spp. including Bacteroides
fragilis; Neisseria spp, including N. meningitidis; Moraxella spp.,
such as Moravella catarrhalis and Pasteurella spp. Also included
are protozoal toxins, such as toxins produced by Entameoba
histolytica and Acanthameoba; and parasitic toxins.
[0057] The method of the invention can also be used to inhibit a
viral toxin, such as a toxin produced by rotavirus, human
immunodeficiency virus, influenza virus, polio virus, vesicular
stomatitis virus, vaccinia virus, adenovirus, piavirus, togaviruses
(such as sindbis and semlikifores viruses), paramyxoviruses,
papillomaviruses. Toxins which can be inhibited using the method of
the invention include viroporin molecules produced by any of these
viruses. A preferred toxin which can be inhibited using the method
of the invention is the rotavirus NSP4 protein. Other toxins which
can be inhibited include influenza M2 protein, HIV Vpu and gp41
proteins, picornavirus 3A protein, togavirus 6K protein,
respiratory syncitial virus SH protein, coronavirus D3 protein and
adenovirus E5 protein.
[0058] The infection can be a systemic infection or a localized
infection. Preferably, the infection is localized to one or more of
the oral cavity, the eye, the gastrointestinal tract, including the
throat, the skin and the ear, such as the ear canal or the middle
ear.
[0059] 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.
[0060] The polymer can be administered systemically or
non-systemically, for example, by subcutaneous or other injection,
intravenously, topically, orally, parenterally, transdermally, or
rectally. The route of administration selected will generally
depend upon whether the infection is systemic or localized 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. Preferably, the polymer is administered
non-systemically, for example, orally or topically, for example, by
application to the skin, the eye, oral tissue, such as the oral
mucosa, or gastrointestinal mucosa.
[0061] In a preferred embodiment, the toxin is an exotoxin produced
by a pathogenic bacterial strain. Of particular pathogenic
importance are Escherichia coli, for example, E. coli strains
06:H-, 0157:H7, 0143 and other clinical isolates, and Clostridium
difficile. Enterohemorrhagic E. coli (EHEC), such as 0157:H7, can
cause a characteristic nonfebrile bloody diarrhea known as
hemorrhagic colitis. EHEC produce high levels of one or both of two
related cytotoxins which resemble a Shiga toxin in structure and
function and are referred to as Shiga-like toxins (SLT I or SLT
II). These Shiga-like toxins are believed to damage the intestinal
mucosa, resulting in the manifestation of hemorrhagic colitis.
[0062] In a preferred embodiment, the microbial toxin or toxins are
produced by Clostridium difficile. C. difficile produces two
toxins, Toxin A and Toxin B. Toxin A is an enterotoxin which
stimulates infiltration of neutrophils and release of mediators of
inflammation, resulting in fluid secretion, altered membrane
permeability and hemorrhagic necrosis. Toxin B is a cytotoxin. C.
difficile is associated with many cases of antibiotic-associated
diarrhea and most cases of pseudomembranous colitis, a severe,
potentially fatal inflammation of the colon. Treatment of C.
difficile infection typically involves administration of vancomycin
or metronidazole. In one embodiment, the condition to be treated is
C. difficile induced gastroenteritis, such as antibiotic-associated
diarrhea or pseudomembranous colitis. In this embodiment, the
polymer can, optionally be administered in combination with one or
more antibiotic agents which are effective, at least partially,
against C. difficile, such as vancomycin and metronidazole.
[0063] As used herein "treatment" of C. difficile associated
diarrhea (CDAD) includes: prophylactic treatment of those patients
susceptible to CDAD; treatment at initial onset of CDAD; treatment
of ongoing CDAD and treatment of relapsing CDAD in susceptible
patients. As used herein a "therapeutically effective amount" is an
amount sufficient to prevent, diminish or eradicate symptoms of
disease.
[0064] In a preferred embodiment, a therapeutic/prophylactic CDAD
treatment regimen (which results in prevention of disease,
diminution or eradication of disease after onset, or prevention of
relapse of disease) comprises administration of a therapeutically
effective amount of a therapeutic composition comprising
polystyrene sulfonate, preferably soluble, uncrosslinked
polystyrene sulfonate and even more preferably, soluble,
uncrosslinked polystyrene sulfonate having a molecular weight of
between 400,000 and 1 million, and most preferably a molecular
weight of 600,000. While not intending to be limited to any
mechanism, compositions in accordance with the invention bind the
toxins, e.g. toxins produced by C. difficile. Toxin A is an
enterotoxin which stimulates infiltration of neutrophils and
release of mediators of inflammation, resulting in fluid secretion,
altered membrane permeability and hemorrhagic necrosis. Toxin B is
a cytotoxin. These toxins are believed to be responsible for the
symptoms of CDAD and other AAD.
[0065] The present invention also contemplates a prophylactic
treatment regimen comprising administering a therapeutic
composition comprising polystyrene sulfonate as a cotherapy with
broad spectrum antibiotic therapy. As used herein "cotherapy" means
a treatment regiment wherein two drugs are administered
simultaneously or sequentially, separated by minutes, hours or
days, but in some way act together to provide the desired
therapeutic response.
[0066] The invention will now be further and specifically described
by the following examples.
EXAMPLES
Example 1
Synthesis of Acrylic Acid/styrene copolymer (2:1)
[0067] 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,
azobisisobutyronitrile (AIBN) (1.47 g, 3 mol % with respect to
total monomer) was added. The solution was degassed for a further
thirty minutes and the reaction was then heated to 70.degree. C.
for 14 h. The solution was cooled and precipitated into n-hexane
(800 mL). The hexane was decanted from the fibrous white product,
the product was washed with ethyl acetate (300 mL) followed by
washing with a further aliquot of hexane (200 mL). The polymer was
dried in vacuo to yield 21.6 g, 84.6% of a brittle white solid.
Example 2
Synthesis of Acrylic Acid/decylacrylate (96:4) Copolymer
[0068] 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
hr. The solution was cooled and precipitated into ethyl acetate
(600 mL). The ethyl acetate was decanted from the fibrous white
product, the product was washed with ethyl acetate (300 mL) and
then with hexane (200 mL). The polymer was dried in vacuo to yield
9.0 g, 81% of a brittle white solid.
Example 3
Synthesis of Acrylic Acid/n-butylacrylate (9:1) Copolymer
[0069] 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.
[0070] The corresponding copolymer of acrylic acid and
n-butylacrylate (10:3) was made by the same procedure.
Example 4
Synthesis of Acrylic Acid/n-decylacrylate/acrylamide (70:7.5:22.5)
Terpolymer
[0071] A solution was prepared of acrylic acid (10.0 g, 133 mmol),
n-decylacrylate (3.0 g, 14.2 mmol) and acrylamide (3.0 g, 42.2
mmol) in dioxane (200 mL). After the solution was degassed with a
rapid stream of nitrogen, AIBN (1.3 g) was added. The solution was
degassed for a further thirty minutes and the reaction was then
heated to 70.degree. C. for 17 h. The polymer precipitated as a
fibrous white solid as the reaction proceeded. The solution was
cooled and the dioxane decanted. The residue was washed with ethyl
acetate (600 mL) and the ethyl acetate was discarded. The polymer
was finally washed with hexanes (300 mL) and dried in vacuo.
Example 5
Synthesis of Acrylic Acid/n-butylacrylate/acrylamide (60:15:25)
Terpolymer
[0072] 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 white fibrous solid. The solution was
cooled and the dioxane was decanted. The polymer was washed with
ethyl acetate (600 mL), then with hexanes (300 mL) and dried in
vacuo.
Example 6
Synthesis of co-polymer of acrylic acid and decylacrylate
(10:2)
[0073] 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 hr. 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
Preparation of 2% Cross-Linked poly(ethyleneglycolmethacrylate
phosphate) Gel
[0074] Poly(ethyleneglycolmethacrylate phosphate) gel was prepared
by polymerizing ethyleneglycolmethacrylate phosphate (29.4 mmoles,
6.178 g) with divinylbenzene ("DVB") (0.926 mmoles, 0.1319 mL) in
ethanol/water (50/50) using about 1 mole % AIBN as initiator. The
resulting resilient gel was split in 2 portions in two 50 mL
centrifuge tubes and washed 4 times with ethanol for a total of
about 120 mL of ethanol. The gel was dried overnight in a
forced-air oven at 70.degree. C. The dried gel was ground and
sieved and washed 3 times in water in a 50 mL centrifuge tube. The
gel was dried overnight in a forced-air oven at 70.degree. C.
Example 8
Preparation of Sulfonated Polystyrene Gels
[0075] Polystyrene gels were prepared by polymerizing styrene with
divinyl benzene in toluene using about 1 mole % AIBN as initiator
as follows:
[0076] Polystyrene gel (6% DVB). Styrene (282 mmole, 3.23 mL) was
added to a 40 mL vial fitted with a septum cap. Toluene (5 mL) was
added and the solution was degassed for 15 min. A solution of AIBN
(0.9852 g in 10 mL of toluene) was prepared and 0.5 mL was added to
the solution. The solution was further degassed for 5 min and then
maintained at 60.degree. C. for 21 hr. The resulting clear
colorless gel was washed 5 times with ethanol in a 50 mL centrifuge
tube and dried overnight in a 70.degree. C. forced air oven.
[0077] Polystyrene gels were also prepared using this procedure
with the following cross-linking levels: 4% DVB; 2% DVB; 1.5% DVB;
1% DVB; and 0.5% DVB.
Sulfonation of Polystyrene Gel
[0078] Dried polystyrene gel was transferred to a 40 mL glass vial.
Concentrated sulfuric acid (10 mL) was added and the mixture was
heated at 100.degree. C. for 1 hr. The resulting brown, swollen gel
was allowed to cool to room temperature and was washed exhaustively
with methanol until the pH was 4-5. The gel was dried overnight in
a 70.degree. C. forced air oven. The dried gel was then ground in a
coffee grinder, transferred to a 50 mL centrifuge tube, and washed
several times with water.
Example 9
Preparation of sulfonated poly(2-vinylnaphthalene) Gels
[0079] Poly(2-vinylnaphthalene) gels were prepared by polymerizing
2-vinyl-naphthalene with divinyl benzene in toluene using .about.1
mole % AIBN as initiator as follows.
Poly(2-vinyl naphthalene) Gel (2% DVB)
[0080] 2-Vinylnaphthalene (29.4 mmoles, 4.534 g) and divinylbenzene
(0.6 mmoles, 85.46 microL) was added to a 40 mL vial fitted with a
septum-cap. Toluene (10 mL) was added and the solution was heated
to dissolve the monomer. The solution was degassed for 15 min. A
solution of AIBN (0.9852 g in 10 mL in toluene) was prepared and
0.5 mL was added to the polymerization solution. The solution was
further degassed for 5 min and then maintained at 60.degree. C. for
21 h. The resulting clear brown gel was washed with ethanol (2 L
total) by gravity filtration and dried for 2 days in a 70.degree.
C. forced air oven.
Sulfonation of poly(2-vinylnaphthalene) Gel
[0081] Dried poly(2-vinyl naphthalene) gel was transferred to a 40
mL glass vial. Concentrated sulfuric acid (10 mL) was added and the
mixture was heated at 100.degree. C. for 1 h. The resulting brown,
swollen gel was allowed to cool to room temperature and was washed
exhaustively with methanol by gravity filtration until the pH was
4-5. The gel was washed several times with water. The gel was dried
for 2 days in a 70.degree. C. forced air oven.
Example 10
Preparation of sulfonated poly(4-vinylbiphenyl) Gels
[0082] Poly(4-vinylbiphenyl) gels were prepared by polymerizing
4-vinylbiphenyl with divinyl benzene in toluene using .about.1 mole
% AIBN as initiator as follows:
Poly(4-vinylbiphenyl) Gel (2% DVB)
[0083] 4-Vinylbiphenyl (29.4 mmoles, 5.299 g) and divinylbenzene
(0.6 mmoles, 85.46 microL) were added to a 40 mL vial fitted with a
septum cap. Toluene (10 mL) was added and the solution was heated
to dissolve the monomer. The solution was degassed for 15 min. A
solution of AIBN (0.9852 g in 10 mL of toluene) was prepared and
0.5 mL was added to the polymerization solution. The solution was
further degassed for 5 minutes and then maintained at 60.degree. C.
for 21 h. The resulting clear brown gel was washed with ethanol (2
L total) by gravity filtration and dried for 2 days in a 70.degree.
C. forced air oven.
Sulfonation of 2% Cross-Linked poly(4-vinylbiphenyl) Gel
[0084] Dried poly(4-vinylbiphenyl) gel was transferred to a 40 mL
glass vial. Concentrated sulfuric acid (10 mL) was added and the
mixture was heated at 100.degree. C. for 1 h. The resulting brown,
swollen gel was allowed to cool to room temperature and was washed
exhaustively with methanol by gravity filtration until the pH was
4-5. The gel was washed several times with water and then dried for
2 days in a 70.degree. C. forced air oven.
Example 11
Preparation of
poly(styrenesulfonate-co-styrene-n-N-octylsulfonamide)
[0085] Sodium poly(styrenesulfonate) (114.9 mmoles, 20 g) was
dispersed in N,N-dimethylformamide ("DMF",100 mL, anhydrous).
Thionyl chloride (114.9 mmoles, 9.95 mL) was added and the mixture
was heated at 60.degree. C. for 16 h. The mixture was poured over
ice and neutralized with 50% NaOH (aq) until the pH was about 6.5.
The solution was dialyzed through SpectraPor 6-8K MWCO dialysis
tubing in 4.times.3 gallons of deionized water until the
conductivity of the dialysate was 0.00 mS/cm. The sample was
lyophilized to yield a white powder.
Poly(styrenesulfonate) w/10 mole % n-octylsulfanamide
[0086] Sodium poly(styrenesulfonate) (114.9 mmoles, 20 g) was
dispersed in DMF (100 mL, anhydrous). Thionyl chloride (114.9
mmoles, 9.95 mL) was added and the mixture was heated at 60.degree.
C. for 16 h. n-Octylamine (11.486 mmoles, 1.8980 mL) was added and
the mixture was stirred at for 5.5 h. The mixture was poured over
ice and neutralized with 50% NaOH (aq) until the pH was 6.1. The
solution was dialyzed through SpectraPor 6-8K MWCO dialysis tubing
in 4.times.3 gallons of DI water until the conductivity of the
dialysate was 0.00 mS/cm. The sample was lyophilized to yield a
white powder.
Poly(styrenesulfonate) w/20 mole % n-octylsulfanamide
[0087] Poly(styrenesulfonate, Na) (114.9 mmoles, 20 g) was
dispersed in DMF (100 mL, anhydrous). Thionyl chloride (114.9
mmoles, 9.95 mL) was added and the mixture was heated at 60.degree.
C. for 16 h. n-Octylamine (22.97 mmoles, 3.7967 mL) was added and
the mixture was stirred at rt for 5.5 h. The mixture was poured
over ice and neutralized with 50% NaOH (aq) until the pH was 6.7.
The solution was dialyzed through SpectraPor 6-8K MWCO dialysis
tubing in 4.times.3 gallons of DI water until the conductivity of
the dialysate was 0.00 mS/cm. The sample was lyophilized to yield a
white powder.
Poly(styrenesulfonate) w/30 mole % n-octylsulfanamide
[0088] Poly(styrenesulfonate, Na) (114.9 mmoles, 20 g) was
dispersed in DMF (100 mL, anhydrous). Thionyl chloride (114.9
mmoles, 9.95 mL) was added and the mixture was heated at 60.degree.
C. for 16 h. n-Octylamine (34.457 mmoles, 5.6950 mL) was added and
the mixture was stirred at rt for 5.5 h. The mixture was poured
over ice and neutralized with 50% NaOH (aq) until the pH was 6.5.
The solution was dialyzed through SpectraPor 6-8K MWCO dialysis
tubing in 4.times.3 gallons of deionized water until the
conductivity of the dialysate was 0.00 mS/cm. The sample was
lyophilized to yield a white powder.
Poly(styrenesulfonate) w/40 mole % n-octylsulfonamide
[0089] Sodium poly(styrenesulfonate) (114.9 mmoles, 20 g) was
dispersed in DMF (100 mL, anhydrous). Thionyl chloride (114.9
mmoles, 9.95 mL) was added and the mixture was heated at 60.degree.
C. for 16 h. n-Octylamine (55.131 mmoles, 7.5934 mL) was added and
the mixture was stirred at room temperature for 5.5 h. The mixture
was poured over ice and neutralized with 50% NaOH (aq) until the pH
was about 6.7. The solution was dialyzed through SpectraPor 6-8K
MWCO dialysis tubing in 4.times.3 gallons of deionized water until
the conductivity of the dialysate was 0.00 mS/cm. The sample was
lyophilized to yield a white powder.
Example 12
Synthesis of poly(styrenesulfonate) Calcium Salt
[0090] To a 500 mL 3-necked round bottomed flask were added 2 g of
poly(sodium 4-styrene sulfonate) and 100 mL of deionized water. The
mixture was stirred for several minutes until a homogeneous
solution was obtained. To this polymer solution was added 6.46 mL
of a 0.225 M solution of CaCl.sub.2. The reaction mixture was
allowed to stir at room temperature for 15 hr.
[0091] The reaction mixture was purified by membrane centrifugation
using molecular weight 3K cut-off filters. The solution was dried
at 70.degree. C. in a forced air oven for 24 hours, yielding 1.4 g
of the polymer as an off white solid.
Example 13
Preparation of cross-linked styrenesulfonate copolymers with
hydrophobic co-monomers
[0092] Polystyrenesulfonate/hydrophobe gels were prepared by
copolymerizing styrene sulfonate with acrylamide,
n-butylacrylamide, n-decylacrylamide, or styrene with either
divinylbenzene (2%) or N,N'-methylenebisacrylamide (8%) as the
crosslinker as follows:
Polystyrenesulfonate Gel (2% Cross-Linked)
[0093] Polystyrenesulfonate (29.4 mmoles, 5.119 g) and
divinylbenzene (0.6 mmoles, 85.5 microL) were dissolved in 10 mL
ethanol and 10 mL water in a 40 mL vial fitted with a septum cap.
The solution was degassed by bubbling nitrogen through and 1 mole %
AIBN was added as a solution. The polymerization solution was
further degassed and the placed in a heated reaction block at
60.degree. C. for 18 h. A clear, colorless gel formed.
Polystyrenesulfonate-co-acrylamide Gel (75 mole %:23 mole %:2%
Cross-Linked)
[0094] Polystyrenesulfonate (22.5 mmoles, 3.918 g), acrylamide
(6.90 mmoles, 0.490 g), and divinylbenzene (0.6 mmoles, 85.5
microL) were dissolved in 10 mL ethanol and 10 mL water in a 40 mL
vial fitted with a septa cap. The solution was degassed by bubbling
nitrogen through and 1 mole % AIBN was added as a solution. The
polymerization solution was further degassed and the placed in a
heated reaction block at 60.degree. C. for 18 h. A clear, colorless
gel formed.
Polystyrenesulfonate-co-n-butylacrylamide Gel (75 mole %:23 mole
%:2% Cross-Linked)
[0095] Polystyrenesulfonate (22.5 mmoles, 3.918 g),
n-butylacrylamide (6.90 mmoles, 0.878 g), and divinylbenzene (0.6
mmoles, 85.5 microL) were dissolved in 15 mL ethanol and 5 mL water
in a 40 mL vial fitted with a septa cap. The solution was degassed
by bubbling nitrogen through and 1 mole % AIBN was added as a
solution. The polymerization solution was further degassed and the
placed in a heated reaction block at 60.degree. C. for 18 h. A
clear, colorless gel formed.
Polystyrenesulfonate/acrylamide/n-butylacrylamide Gel (75 mole
%:11.5 mole %:11.5 mole %:2% Cross-Linked)
[0096] Polystyrenesulfonate (22.5 mmoles, 3.918 g), acrylamide
(3.45 mmoles, 0.245 g), n-butylacrylamide (3.45 mmoles, 0.439 g)
and divinylbenzene (0.6 mmoles, 85.5 microL) were dissolved in 15
mL ethanol and 5 mL water in a 40 mL vial fitted with a septa cap.
The solution was degassed by bubbling nitrogen through and 1 mole %
AIBN was added as a solution. The polymerization solution was
further degassed and the placed in a heated reaction block at
60.degree. C. for 18 h. A clear, light yellow gel formed.
Polystyrenesulfonate-co-n-decylacrylamide Gel (75 mole %:23 mole
%:2% Cross-Linked)
[0097] Polystyrenesulfonate (22.5 mmoles, 3.918 g),
n-decylacrylamide (6.90 mmoles, 1.458 g), and divinylbenzene (0.6
mmoles, 85.5 microL) were dissolved in 15 mL ethanol and 5 mL water
in a 40 mL vial fitted with a septa cap. The solution was degassed
by bubbling nitrogen through and 1 mole % AIBN was added as a
solution. The polymerization solution was further degassed and the
placed in a heated reaction block at 60.degree. C. for 18 h. A
creamy yellow gel formed.
Polystyrenesulfonate/acrylamide/n-decylacrylamide Gel (75 mole
%:11.5 mole %:11.5 mole %:2% Cross-Linked)
[0098] Polystyrenesulfonate (22.5 mmoles, 3.918 g), acrylamide
(3.45 mmoles, 0.245 g), n-decylacrylamide (3.45 mmoles, 0.729 g)
and divinylbenzene (0.6 mmoles, 85.5 microL) were dissolved in 15
mL ethanol and 5 mL water in a 40 mL vial fitted with a septa cap.
The solution was degassed by bubbling nitrogen through and 1 mole %
AIBN was added as a solution. The polymerization solution was
further degassed and the placed in a heated reaction block at
60.degree. C. for 18 h. A creamy yellow gel formed.
Polystyrenesulfonate-co-styrene Gel (75 mole %:23 mole %:2%
Cross-Linked)
[0099] Polystyrenesulfonate (22.5 mmoles, 3.918 g), styrene (6.90
mmoles, 0.7906 mL), and divinylbenzene (0.6 mmoles, 85.5 microL)
were dissolved in 10 mL ethanol and 10 mL water in a 40 mL vial
fitted with a septum cap. The solution was degassed by bubbling
nitrogen through and 1 mole % AIBN was added as a solution. The
polymerization solution was further degassed and the placed in a
heated reaction block at 60.degree. C. for 18 h. A clear, colorless
gel formed.
Polystyrenesulfonate/acrylamide/styrene Gel (75 mole %:11.5 mole
%:11.5%; 2% Cross-Linked)
[0100] Polystyrenesulfonate (22.5 mmoles, 3.918 g), acrylamide
(3.45 mmoles, 0.245 g), styrene (3.45 mmoles, 0.3953 mL) and
divinylbenzene (0.6 mmoles, 85.5 microL) were dissolved in 10 mL
ethanol and 10 mL water in a 40 ml, vial fitted with a septum cap.
The solution was degassed by bubbling nitrogen through and 1 mole %
AIBN was added as a solution. The polymerization solution was
further degassed and the placed in a heated reaction block at
60.degree. C. for 18 h. A clear, colorless gel formed.
Polystyrenesulfonate-co-acrylamide Gel (50 mole %:48 mole %:2%
Cross-Linked)
[0101] Polystyrenesulfonate (15.0 mmoles, 2.612 g), acrylamide
(14.4 mmoles, 1.024 g), and divinylbenzene (0.6 mmoles, 85.5
microL) were dissolved in 5 mL ethanol and 15 mL water in a 40 mL
vial fitted with a septa cap. The solution was degassed by bubbling
nitrogen through and 1 mole % AIBN was added as a solution. The
polymerization solution was further degassed and the placed in a
heated reaction block at 60.degree. C. for 18 h. A clear, colorless
gel formed.
Polystyrenesulfonate/acrylamide/n-butylacrylamide Gel (50 mole %:24
mole %:24 mole %:2% Cross-Linked)
[0102] Polystyrenesulfonate (15.0 mmoles, 2.612 g), acrylamide (7.2
mmoles, 0.512 g), n-butylacrylamide (7.2 mmoles, 0.916 g) and
divinylbenzene (0.6 mmoles, 85.5 microL) were dissolved in 5 mL
ethanol and 15 mL water in a 40 mL vial fitted with a septum cap.
The solution was degassed by bubbling nitrogen through and 1 mole %
AIBN was added as a solution. The polymerization solution was
further degassed and the placed in a heated reaction block at
60.degree. C. for 18 h. A clear, colorless gel formed.
Polystyrenesulfonate/acrylamide/styrene Gel (50 mole %:24 mole %:24
mole %:2% Cross-Linked)
[0103] Polystyrenesulfonate (15.0 mmoles, 2.612 g), acrylamide (7.2
mmoles, 0.512 g), styrene (7.2 mmoles, 0.8250 mL) and
divinylbenzene (0.6 mmoles, 85.5 microL) were dissolved in 10 mL
ethanol and 10 mL water in a 40 mL vial fitted with a septa cap.
The solution was degassed by bubbling nitrogen through and 1 mole %
AIBN was added as a solution. The polymerization solution was
further degassed and the placed in a heated reaction block at
60.degree. C. for 18 h. A clear, colorless gel formed.
Polystyrenesulfonate-co-acrylamide Gel (25 mole %:73 mole %:2%
Cross-Linked)
[0104] Polystyrenesulfonate (7.5 mmoles, 1.306 g), acrylamide (21.9
mmoles, 1.557 g), and divinylbenzene (0.6 mmoles, 85.5 microL) were
dissolved in 5 mL ethanol and 15 mL water in a 40 mL vial fitted
with a septa cap. The solution was degassed by bubbling nitrogen
through and 1 mole % AIBN was added as a solution. The
polymerization solution was further degassed and the placed in a
heated reaction block at 60.degree. C. for 18 h. A clear, colorless
gel formed.
[0105] All samples were purified by splitting the gel into 2
portions in two 50 mL centrifuge tubes. The gels were washed a
minimum of three times with ethanol or until the supernatant was
clear and colorless. The total volume of ethanol used was roughly
between 75 mL and 100 mL depending on the swelling index of the
gel. The gels were dried in a forced-air oven at 60.degree. C. for
2 days.
[0106] Gels were ground in a coffee grinder and sieved through 140,
230 mesh sieves. A 0.5-1 g sample of the 140-230 size gel particles
was washed in 50 mL centrifuge tubes 3 times with water. Some
samples were highly absorbent and had to be split up over multiple
tubes. Generally, material in each tube was washed with a total of
20-80 mL of water. Samples were then washed 1.times. with MeOH,
centrifuged, decanted, and dried for two days at 70.degree. C. Gel
was washed with a total of 20-80 mL of water.
Example 14
Preparation of poly(4-vinylbiphenylsulfonate)
[0107] Polymerization of 4-vinylbiphenyl 4-Vinylbiphenyl (166.4
mmoles, 30 g) was added to a 500 mL, 3-neck, round-bottom flask
equipped with a reflux condenser, a J-Kem thermocouple, and a
septum. Toluene (60 mL) was added and the solution was degassed for
1 h. AIBN (1 mole %, 0.294 mmoles, 0.2733 g) was added and the
solution was degassed for a further 15 min. The polymerization
mixture was heated at 60.degree. C. for 21 h. The resulting clear
brown solution was poured into 2 L of methanol and stirred for
several hours. The fine brown powder was filtered and washed
3.times.500 mL methanol and dried overnight in a forced-air oven at
70.degree. C. A fine brown powder was obtained (28.02 g, 93.40%
yield).
Sulfonation of Poly(4-vinylbiphenyl)
[0108] Poly(4-vinylbiphenyl) was mixed with concentrated sulfuric
acid (100 mL) and heated at 100.degree. C. for 8 h. The mixture
eventually turned to a clear brown viscous solution. The polymer
solution was poured into ice and neutralized to pH 6.2 with 50%
aqueous NaOH. The solution was dialyzed through dialysis membrane
have a molecular weight cut-off of 3.5 K in 4 times 5 L deionized
water. The conductivity of the dialysate was <0.1 mS/cm. The
water was removed by distillation on a rotary evaporator to yield a
clear brown flaky solid.
Example 15
Preparation of Poly(2-vinylnaphthalenesulfonate)
[0109] Polymerization of 2-vinylnaphthalene 2-Vinylnaphthalene
(194.5 mmoles, 30 g) was added to a 500 mL, 3-neck, round-bottom
flask equipped with a reflux condenser, a J-Kem thermocouple, and a
septum. Toluene (60 mL) was added and the solution was degassed for
1 h. AIBN (1 mole %, 0.294 mmoles, 0.3195 g) was added and the
solution was degassed for a further 15 min. The polymerization
mixture was heated at 60.degree. C. for 21 h. The resulting clear
brown solution was poured into 2 L of methanol and stirred for
several hours. The fine brown powder was filtered and washed
3.times.500 mL methanol and dried overnight in a forced-air oven at
70.degree. C. A fine brown powder was obtained (28.45 g, 94.83%
yield) Sulfonation of Poly(2-vinylnaphthalene)
[0110] Poly(2-vinylnaphthalene) was mixed with concentrated
sulfuric acid (100 mL) and heated at 100.degree. C. for 8 h. The
mixture eventually turned to a clear brown viscous solution. The
polymer solution was poured into ice and neutralized to pH 6.4 with
50% aqueous NaOH. The solution was dialyzed through dialysis
membrane have a molecular weight cut-off of 3.5 K in 4 times 5 L
deionized water. The conductivity of the dialysate was <0.1
mS/cm. The water was removed by distillation on a rotary evaporator
to yield a clear brown flaky solid.
Example 16
Poly(sodium 4-styrene sulfonate-co-(-)-menthyl-4-styrene
sulfonate), 5% (-)-menthol
[0111] To a mixture of poly(sodium 4-styrene sulfonate) (30.0 g;
0.145 mol of sulfonate) in 300 mL of anhydrous DMF stirred at room
temperature thionyl chloride (17.3 g; 0.145 mol) was added. The
addition was done slowly insuring that the temperature did not go
above 50.degree. C. Stirring was continued overnight and then one
third of the reaction mixture was treated with pyridine (0.764 g;
00966 mol). After stirring at room temperature for 2.5 h,
(-)-menthol (0.375 g; 0.00240 mol) was added and the resulting
reaction mixture was stirred at room temperature overnight and then
at 50.degree. C. for 3 h. The mixture was then poured slowly into
one liter of water containing sodium bicarbonate (5 g). After the
addition was complete more sodium bicarbonate was added until the
bubbling stopped and the pH was neutral. Exhaustive dialysis
followed by drying with a flow of air gave a white solid.
Example 17
Synthesis of Poly(sodium 4-styrene sulfonate-co-Lithocholyl
acid-4-styrene sulfonate), 5% Lithocholic Acid
[0112] To a mixture of poly(sodium 4-styrene sulfonate) (30.0 g;
0.145 mol of sulfonate) in 300 mL of anhydrous DMF stirred at room
temperature thionyl chloride (17.3 g; 0.145 mol) was added. The
addition was done slowly insuring that the temperature did not go
above 50.degree. C. Stirring was continued overnight and then one
third of the reaction mixture was treated with pyridine (0.764 g;
00966 mol). After stirring at room temperature for 2.5 h,
lithocholic acid (0.904 g; 0.00240 mol) was added and the resulting
reaction mixture was stirred at room temperature overnight and then
at 50.degree. C. for 3 h. The mixture was then poured slowly into
one liter of water containing sodium bicarbonate (5 g). After the
addition was complete more sodium bicarbonate was added until the
bubbling stopped and the pH was neutral. Exhaustive dialysis
followed by drying with a flow of air gave a white solid.
[0113] The in vivo hamster assay was also used as described above
to assess the efficacy of the following polymers, as shown in Table
1.
Example 18
Poly(sodium 3-styrene sulfonate) with 40% Metronidazole
[0114] Poly(sodium 4-styrene sulfonate (160-246-001, 25 g) was
dissolved in deionized water (500 ml) in a 3 liter plastic bucket
using a magnetic stir plate. Into a separate, appropriately sized
beaker, metronidazole (8.32 g) was dissolved in deionized water
(600 ml) and 1 M HCl (0.75 eq). This metronidazole solution was
slowly poured into the poly(sodium 4-styrene sulfonate) solution.
The resulting solution was stirred at room temperature for
approximately 17 hours. It was placed in dialysis bags (6K-8K MWCO)
until a conductivity of <0.05 was obtained. The polymer solution
was dried in a forced air oven at 70.degree. C. Approximately 22 g
of the polymer was obtained. Analysis by HPLC showed a 13.8%
loading of metronidazole.
Example 19
Sulfonation of Poly(4-methoxystyrene)
[0115] Poly(4-methoxystyrene) (1 g) was placed in a 30 ml vial.
Concentrated sulfuric acid (5 ml) was added to the vial. While
stirring, this was heated at 100.degree. C. for 8 hours. After the
8 hours, the polymer was dissolved (clear, dark solution).
Deionized water was added (50 ml). the sample was neutralized by
adding NaOH (50% soln.) drop-wise. It was placed in dialysis bags
(6K-8K MWCO) until a conductivity of <0.1 was reached. The
polymer solution was filtered and then dried on the speed vac.
Example 20
Sulfonation of Poly(diphenoxyphosphazene)
[0116] Poly(diphenoxyphosphazene) (1 g) was placed in a 30 ml vial.
Concentrated sulfuric acid (5 ml) was added to the vial. While
stirring, this was heated at 100.degree. C. for 8 hours. After the
8 hours, the polymer was dissolved (clear, yellow solution).
Deionized water was added (50 ml). the sample was neutralized by
adding NaOH (50% soln.) drop-wise. It was placed in dialysis bags
(6K-8K MWCO) until a conductivity of <0.1 was reached. The
polymer solution was filtered and then dried on the speed vac.
Example 21
Sulfonation of Ethylene Oxide-Styrene-Ethylene Oxide Block
Copolymer
[0117] Ethylene oxide-styrene-ethylene oxide block copolymer (900
mg) was placed in a 30 ml vial. Concentrated sulfuric acid (5 ml)
was added to the vial. While stirring, this was heated at
100.degree. C. for 8 hours. After the 8 hours, the polymer was not
dissolved. More concentrated sulfuric acid (5 ml) was added to the
vial. It was then heated at 110.degree. C., with stirring, for
another 8 hours. This was repeated two more times. A total of 20 ml
of concentrated sulfuric acid was added (5 ml, 100.degree. C., 8
hours; 15 ml, 110.degree. C., 24 hours). Deionized water was added
(50 ml). The sample was neutralized by adding NaOH (50% soln.)
drop-wise. It was placed in dialysis bags (6K-8K MWCO) until a
conductivity of <0.1 was reached. The polymer was filtered (a
lot of insoluble material remained) and then dried on the speed
vac.
Example 22
Co-Administration of Poly(styrenesulfonate) and
Poly(diallylmethylamine)
[0118] Soluble polystyrene sulfonate and cross-linked
C.sub.8-alkylated polydiallylmethylamine (PDMA) are both able to
reduce mortality from C. difficile infection in the hamster model
of C. difficile colitis. These polymers exhibit different toxin
binding properties in vitro. Polystyrene sulfonate binds C.
difficile Toxin A and protects cells in culture from Toxin A
mediated cell rounding. Cross-linked C.sub.8 alkylated PDMA binds
Toxins A and B in vitro and can protect cells in culture from toxin
mediated cell rounding. This polymer is about five times more
potent for binding Toxin B than binding Toxin A in vitro. To gain
the benefit of optimal Toxin A and B binding, these polymers were
tested in combination in the hamster model of C. difficile colitis.
Treatment with polymer began 24 hours prior to infection with C.
difficile. Hamsters were give 3 daily doses of polymer at 8 am, 12
pm, and 4 pm each day. Hamsters were treated for a total of 7 days
with saline (control), polystyrene sulfonate alone, cross-linked,
C.sub.8 alkylated PDMA alone, or a combination of the two polymers
administered in separate doses (2 doses of polystyrene sulfonate, 1
dose of C.sub.8 PDMA). Animals were observed for a total of 7 days.
None of the saline treated animals survived treatment. The
polystyrene sulfonate treated animals had a 80% survival on day 7,
with the survivors showing mild or moderate disease. The C.sub.8
alkylated PDMA animals had a 50% survival on day 7, with the
survivors showing moderate or no disease. The combination of the
polystyrene sulfonate and the C.sub.8 alkylated PDMA resulted in a
90% survival on day 7, with 80% of the animals showing no disease.
Therefore, combination of soluble polystyrene sulfonate and C.sub.8
alkylated PDMA appears to be an effective therapy for C. difficile
colitis in vivo.
Example 23
Toxin Binding Assays
ELISA
[0119] An enzyme linked immunosorbant assay (ELISA) is available
commercially for diagnosis of C. difficile toxin levels in stool
samples. This assay uses microtiter plates coated with purified
monoclonal antibodies to C. difficile toxins A and B to bind toxin
in solution. The bound toxins are then detected with an affinity
purified polyclonal antisera that has been linked with the enzyme
horseradish peroxidase ("HRP"). Unbound antibody is washed away and
the antibody-bound toxin is then detected with a colored substrate
for the HRP. The assay is sensitive to nanogram quantities of
Toxins A and B. This ELISA assay was used to determine free toxin
levels after incubation of purified Toxin A or B with a variety of
polymers in the presence of hamster cecal contents, which were used
to provide physiologically relevant conditions predictive of in
vivo toxin binding.
[0120] To perform the ELISA assay, polymer (10 mg) was weighed out
into each of four 1.5 ml Eppendorf tubes. 500 .mu.L cecal contents
were then added to each tube, and the tubes were then mixed on a
Vortex and placed on the nutator for 1 hour. 500 .mu.L of a 200
ng/mL or 2000 ng/mL solution of Toxin A or Toxin B in phosphate
buffer was then added to each tube to produce a final toxin
concentration of 1000 ng/mL or 100 ng/mL. The tubes were then
vortexed again and then placed on the nutator for another hour. The
tubes were then centrifuged.
[0121] 100 .mu.l of diluted supernatant from each tube was added to
each well of a plate, avoiding solid material. 100 .mu.l of
conjugate 1 was added to each well. The wells were covered with a
plate sealer and incubated at 37.degree. C. for 1 hour. The wells
were aspirated into a biohazard receptacle (reservoir containing
wescodyne or 10% bleach in water). The plate was washed using a
plate washer, filling the wells with 300 .mu.l, of wash buffer.
After the final wash the plate was inverted and banged to remove
any residual wash buffer on paper towels. 100 .mu.l of diluted step
2 conjugate was added to each well. The plate was covered and
incubated 20 min. at room temperature. The wells were washed five
times using the plate washer, as above, and then banged to remove
residual buffer. 100 .mu.l of substrate mix was added to each well,
then the plate was sealed and incubated 15 min. at room
temperature. 150 .mu.L of stop solution was then added to each
well, and the plate was mixed gently by swirling plate on bench.
The absorbance at 450 nm was read immediately.
Cell Culture Assay
[0122] C. difficile toxins cause rounding of cells in culture. This
property can be used to screen for inhibition of toxin activity by
polymeric compounds. Sensitivities to toxins A and B differ among
different cell lines. In the present case, Vero cells (ATCC cell
line) were used. These cells are sensitive to 600 pg of Toxin A and
less than 2 pg of Toxin B. The assay was run by plating Vero cells
in 12 well transwell plates or 96 well microtiter plates. The cells
were seeded 24 hours prior to testing, and were confluent
monolayers at the time of toxin addition. Polymers (5 mg/ml) were
incubated in tissue culture media (Minimal Essential Media with 10%
fetal bovine serum) with Toxin A or Toxin B for 1 hour at room
temperature with rocking. Following incubation, samples were
handled differently, depending on whether they were insoluble gels
or soluble polymers. Insoluble gels were added to transwells (0.5
ml/well), since direct addition of the gels to the monolayer would
obscure the cells, preventing detection of cell rounding. Soluble
polymers were added directly to cell monolayers in 96 well
microtiter plates (0.1 ml/well). Cells were incubated at 37.degree.
C. for 18 hours and observed for cell rounding. The endpoint of the
assay was scored as the lowest concentration of polymer that can
protect 50% and 100% of the monolayer from cell rounding at 18
hours incubation. Controls included an active polymer incubated
with toxins A and B, toxins A and B alone, and each polymer alone
without toxin.
Rat Ileal Loop Model Assay
[0123] The objective of this assay was to measure the ability of
polymeric compounds to prevent Toxin A mediated fluid accumulation
and permeability in a ligated section of rat ileum. Rats were
anesthetized and a 5 cm section of rat ileum was ligated with silk
suture. Polymer (1-5 mg) and 5 .mu.g of purified Toxin A were
injected into this section. The rat also received an intravenous
injection 10 .mu.Ci of .sup.3H mannitol as a marker for intestinal
permeability. Toxin A increases vascular permeability in the
intestine, allowing the mannitol to enter the loop. Four hours
after injection of Toxin A and polymer, the ileal loop sections
were removed, weighed and total fluid accumulation was measured.
Accumulation of .sup.3H mannitol was measured by liquid
scintillation counting. Polymeric compounds that bind Toxin A will
block the intestinal fluid accumulation and permeability to .sup.3H
mannitol. The endpoint of the assay is the concentration of polymer
that completely inhibits Toxin A mediated fluid accumulation and
permeability. A modification of this assay involved administration
of the polymer by oral gavage. Rats received 250 mg/kg in solution
by oral gavage 90 minutes prior to preparation of ileal loops.
Toxin was injected into ileal loops as described above.
Results
[0124] Results of the ELISA, cell culture, rat ileal loop and
hamster assays are presented in Table 1 for a variety of polymers.
Also included are corresponding data for cholestyramine, a cationic
polymer which has been used clinically to neutralize C. difficile
toxins.
TABLE-US-00001 TABLE 1 Results of biological assays Toxin Binding
(in vitro) concentration Rat Loop of toxin neutralized Dose
Inhibiting Hamster by 5 mg/ml polymer 5.mu. Toxin A % survival
Polymer solution (direct) (gavage) on day 5 Sodium A = 10 ng/ml 2-5
mg 250 mg/kg 90% polystyrene B = 0.004-0.008 ng/ml sulfonate
Polystyrene A = 10 ng/ml <0.5 mg ND 80% sulfonate, B = ND 15%
Ca++ Polystyrene A = 10 ng/ml 2.5 mg ND 90% sulfonate 5% B = 0.004
ng/ml menthol Cholestyr- A = <0.015 ng/ml >20 mg ND 10% amine
B = <0.015 ng/ml
The data presented for the hamster model indicate percent survival
at day 5 following inoculation with C. difficile.
[0125] The results presented in Table 1 indicate that each of the
polystyrenesulfonate polymers tested is more effective in each of
the assays than cholestyramine.
In Vivo Tests
Basic Protocol
[0126] Hamsters are highly sensitive to infection with C. difficile
and develop a fatal colitis when disease is initiated by treatment
with antibiotics. Compounds were evaluated for their ability to
inhibit the activity of C. difficile toxins the hamster model of C.
difficile colitis. Male hamsters (80-100 g) were purchased
Biobreeders Inc. (Falmouth Mass.). All animals were maintained in
groups of five in microisolator cages on autoclaved bedding (Beta
Chips) and given free access to autoclaved chow and autoclaved,
filtered water. Animals were rested approximately 1 week following
arrival and prior to initiation of a study. A C. difficile strain
initially isolated from a human C. difficile colitis was used to
infect the animals. This strain (HUC2-4) produces moderate to high
levels of toxins in vitro and in vivo. A suspension of
10.sup.6-10.sup.7 bacterial cells/ml was prepared from an overnight
broth culture and 0.1 ml was administered by oral gavage on day -1.
Twenty-four hours later, each animal was injected subcutaneously
with Cleocin Phosphate IV.RTM. at a dose of 10 mg/kg. Polymers were
administered to groups of ten animals by gavage three times daily
(t.i.d). The dose of polymers was 25-100 mg/day, administered in
three divided doses of 0.75 ml each. Initiation and duration of
dosing varied according to the treatment regimens as described
below. Animals were observed daily for morbidity or mortality.
Additional parameters assessed were general appearance, time to
onset of clinical signs, and presence or absence of diarrhea ("wet
tail"). Animals judged to be in extremis and surviving animals at
the end of the study were painlessly euthanized by asphyxiation
with 100% CO.sub.2. Cecal contents were obtained in some studies
and frozen for later toxin analysis.
[0127] Administration of clindamycin (on day 0) predictably induced
disease in hamsters infected with C. difficile (on day -1) by
eliminating the normal colonic flora and allowing C. difficile to
proliferate. C. difficile leads to fatal colitis in 90-100% of
infected hamsters within 2-4 days. The primary endpoint of the
assay is the protection of the hamsters from mortality. This is a
very severe and challenging model and CDAD in hamsters is much more
aggressive disease, in both severity and time course, compared with
the human form of the disease. Pharmaceutical compositions of the
invention have been evaluated using the hamster model of CDAD using
two different treatment paradigms. In the prophylaxis model, the
studies have been designed to determine the potential for
pretreatment with the toxin binders to prevent CDAD, despite the
development of a toxingenic C. difficile infection. In the
therapeutic model, the compositions are administered after the
development of CDAD.
Prophylaxis Model
[0128] In this model four different doses (25, 50, 75 or 100 mg/day
administered orally in three doses of 0.750 ml) of a pharmaceutical
composition of polystyrene sulfonate (or saline control) was
administered to male Syrian golden hamsters (ten animals per test
group and ten animals per test group) orally for 7 days, from day
-2 to day 5. hamsters were inoculated with C. difficile (Onderdonk
strain (G69)) on day -1 and treated with clindamycin on day 0. In
animals that received placebo control, 100% developed disease and
were dead at day 3. In contrast, prophylaxis with the composition
led to survival of 70% and 90% of the animals in the 50 mg/day dose
and 100 mg/day dose groups, respectively. Although all of the
animals experienced a toxin-producing C. difficile infection, the
composition comprising polystyrene sulfonate effectively inhibits
the toxins and minimizes the colitis during the period of altered
colon flora. As the normal flora recover, C. difficile appears
unable to compete and is reduced to a non-pathogenic level. Thus
having a toxin binder on board during the initial stages of toxin
exposure and preventing colitis before it developes.
Therapeutic Model
[0129] Polymer therapy was initiated on day 1 at a dose of 25, 50,
75 or 100 mg/day. Polymer was administered three times daily in
0.7-1.5 ml of sterile saline t.i.d. from day 1 through day 7 for a
total of 7 days of treatment. Animals were observed for morbidity,
mortality and clinical signs of disease for 7-14 days following the
end of treatment.
Combination Therapy Model
[0130] Animals were administered a combination of 50-100 mg/day of
polymer and antibiotic in a total volume of 0.75 ml saline from day
1 (48 hours after administration of pathogen) through day 5. The
antibiotic was either metronidazole or vancomycin. The dose of
metronidazole was 21 mg/day. The dose of vancomycin was 3 mg/day.
Following combination therapy, animals were dosed for an additional
4 days with 50-100 mg/day of polymer alone. Animals were observed
for morbidity, mortality and clinical signs of disease for 7-14
days following the end of treatment.
[0131] Metronidazole is effective in preventing death from CDAD in
hamsters as long as the drug is being actively administered.
However, at the conclusion of therapy, 80-90% of animals have a
relapse/recurrence of CDAD and die within 3-6 days of discontinuing
metronidazole. Experiments show the effectiveness of a polystyrene
sulfonate (PSS) composition admininstered as treatment after CDAD
onset and in the prevention of relapse. Mortality from C. difficile
relapse after withdrawal of metronidazole was prevented in 90% of
the animals treated with a high dose of a composition comprising
polystyrenesulfonate. Relapse was prevented in 70% of animals in
the low dose polystyrene sulfonate group. None of the saline
treated controls survived. Only 20% of the animals treated with
metronidazole alone survived 16 days after the last dose of
antibiotic (surviving animals contained to have diarrhea at day
21). Thus, inhibition of the toxins by the PSS composition
prevented colitis and other associated pathology during the time
necessary for the normal flora to recover after metronidazole
therapy appears to prevent the development of relapse.
Monotherapy with PSS Compositions
[0132] Treatment with PSS composition alone was also studied and
compared with the current standard of care, metronidazole alone.
The PSS composition in accordance with the invention was superior
to metronidazole, preventing 40% of animals compared with 20% in
the metronidazole alone group. This result in a rigorous model
suggests that the PSS composition is superior to metronidazole as
monotherapy. PSS compositions did not appear to interfere with the
activity of antibiotics.
Procedure for Vero Cell Culture Assay:
[0133] For all screens Vero cells were used in the 96 well format
with 4.times.10.sup.4 cells per well, with 100 .mu.l per well. The
plates were incubated overnight at 37.degree. C. to be utilized the
following day. All media, plates, pipettes and equipment used for
the culture was kept sterile. Plates were covered and sprayed with
EtOH before being placed in the incubator.
Regular Toxin A and B Screen
[0134] Polymers were weighed out in 15 ml. conical tubes with 10
mg/ml to start, with the final concentration in the wells at 5
mg/ml. Usually the tubes had about 40 mg to 4 ml of media, as
diluent. The excess was kept in the 4.degree. C. refrigerator in
case a re-test needs was desired. The media was pipetted into the
tubes sterilely and the tubes were then vortexed thoroughly. If the
polymer was not in solution, the tubes were placed in a 37.degree.
C. water bath for 15-20 minutes. For this experiment the Toxin A
concentrations were 10 ng/ml and the Toxin B was 1 ng/ml final. The
Toxins and polymer were made up 2.times. and mixed 1:1 to give a
final concentration of 1.times. for both. These solutions were made
in conical tubes and placed on ice.
[0135] Once the polymers were thoroughly dissolved, sterile 96-well
plates were gathered and media placed at 100 .mu.l per well in rows
G-H as well as columns 2-6 and 8-12 in rows A-F. Four polymers were
screened per plate and each polymer had three rows, one for polymer
and media alone, one for Toxin A and polymer and one for Toxin B
and polymer. The set-up is as follows: [0136] Polymer 1: Polymer
alone--Row A, col. 1-6 [0137] Polymer+A--Row B, col. 1-6 [0138]
Polymer+B--Row C, col. 1-6 [0139] Polymer 2: Polymer alone--Row D,
col. 1-6 [0140] Polymer+A--Row E, col. 1-6 [0141] Polymer+B--Row F,
col. 1-6 [0142] Polymer 3: Polymer alone--Row A, col. 7-12 [0143]
Polymer+B--Row C, col. 7-12 [0144] Polymer+A--Row B, col. 7-12
[0145] Polymer 4: Polymer alone--Row D, col. 7-12 [0146]
Polymer+A--Row E, col. 7-12 [0147] Polymer+B--Row F, col. 7-12
[0148] After the media was added, 200 .mu.l of the polymer solution
was added to the first columns in each segment (col. 1 or 7) and
diluted two-fold across the plate to the end of the section (col. 6
or 12). Media was added to rows A, D, and H and Toxin A was added
to rows B, E, and G (col. 1-6). Toxin B was added to rows C, F and
G (col. 7-12). Toxin and media were both added at 100 .mu.l per
well, bringing the total volume in the well up to 200 ti. The last
row (H) was not used in the assay. The plates were then placed on a
shaker and incubated at room temperature for one hour.
[0149] Plates with cells that had been kept overnight in the
incubator were then taken out and the media from the wells was
pipetted off using a vacuum suction apparatus. Media is removed
from only one plate at a time, as the cells tended to dry out
during the process. After removal of media, the toxin/polymer
mixture was pipetted onto the cells. Solution was added at 100
.mu.l per well from the least amount of toxin to the most
concentrated wells, using the same pipette tips throughout the
procedure. The pipette tips were changed for each new polymer run.
Row H was left untouched and the plates were then examined under
the microscope to insure that the monolayer of cells was not
damaged during suction. The plates were then put back into the
37.degree. C. incubator and checked at 18 hours post polymer/toxin
treatment and scored for cell rounding, no cell rounding or partial
(50%) cell rounding.
Toxin B Dose-Down
[0150] Polymers were weighed at 10 mg/ml (5 mg/ml final) in conical
tubes. Usually about 70 mg to 7 ml media (the diluent used) was
adequate to complete a run. They were vortexed into solution.
Polymers that were not in solution were placed in the 37.degree. C.
water bath for 15-20 minutes. Solutions that were still unable to
dissolve completely were noted and gels were run with the gel
procedure. Toxin B was made starting at 2 ng/ml (1 ng/ml final) and
kept on ice. Sterile 96-well plates were used and media was added
at 100 .mu.l per well to all wells except the first column. Each
polymer was tested on three different rows (two polymers per
plate), the first row for polymer alone with no toxin and the next
two with polymer and Toxin B. Row G was a dose-down of Toxin B
alone and row H was left untreated. The set-up was as follows:
[0151] Polymer 1: Row A (1-12)--Polymer alone [0152] Row B
(1-12)--Polymer with Toxin B [0153] Row C (1-12)--Polymer with
Toxin B [0154] Polymer 2: Row D (1-12)--Polymer alone [0155] Row E
(1-12)--Polymer with Toxin B Row F (1-12)--Polymer with Toxin B
[0156] Extra rows: Row (1-12)--Dose down of Toxin B alone [0157]
Row H--Untreated
[0158] 200 .mu.l of either media or Toxin B were placed in the
first column (media for rows with polymer alone) and this was
diluted two-fold across the plate, carrying over 100 .mu.l to each
new column. Polymer mixture was added at 100 .mu.l to all well
except for row G and H. Row substituted 100 .mu.l of media and row
H was left blank. These plates were put on a shaker for one hour
and the procedure from above was followed from this step on.
[0159] When toxin and polymer had been added to the cells, the
plates were then sprayed with EtOH, placed in the 37.degree. C.
incubator overnight, and scored 18 hours later. Scoring was
positive for cell rounding, negative for normal cells and +/- for
wells with the morphology at least 50% normal.
Procedure for Gels
[0160] Gels were run at two different concentrations of polymers
with concentrations of either 5 or 10 mg/ml. Concentrations of
toxins were at 100, 10, 1 for Toxin A and B. These concentrations
varied, depending on polymer type. Twelve Eppendorf tubes were
weighed for each polymer. Toxins were made at the 1.times. dilution
and 1 ml. of toxin added to the appropriate Eppendorf tube. Control
tubes were used with polymer alone (at all concentrations) and with
toxins alone. The tubes were then nutated at room temperature for
one hour and centrifuged for five minutes at 14,000 rpm. 200 .mu.l
of each sample was placed in a well on sterile 96-well plates.
Plates with Vero cells were removed from the incubator and the
media removed via suction (one plate at a time). 100 .mu.l of the
toxin/polymer mixture was added directly to the plates with cells
and the plates were covered and placed into the incubator overnight
for scoring the next day. Scoring employed the procedures
above.
Rat Beal Loop Experiments
[0161] This protocol describes the preparation of ileal loops in
anesthetized rats. This experimental model can be used to test the
effects of enterotoxic agents, such as bacterial toxins on
intestinal structure and function; the effects of putative
protective agents can also be determined.
[0162] Two intestinal loops about 5 cm in length were prepared in
each animal by ligating the ileum with silk suture. The renal
pedicles were ligated to prevent the excretion of mannitol, and
radiolabelled mannitol (.sup.3H-mannitol) injected i.v. Test agents
(.+-.toxin.+-.polymer) were then injected into these loops. Four
hours later the animals were sacrificed and the loops harvested.
Intestinal fluid secretion (an index of secretory diarrhea) was
estimated by weighing the loops and measuring their length. The
permeability of the intestinal mucosa was estimated by measuring
the accumulation of radiolabelled mannitol (3H-mannitol) into the
loop. Tissue samples were also placed in fixative for subsequent
morphological evaluation of mucosal injury and inflammation.
Animal Preparation:
[0163] Male Wistar rats 200-250 g were fasted overnight (18-22
hours) in wire bottom cages, to minimize copraphagy. Water was
available ad libidum. The ileal loops were substantially free from
luminal contents (food and bile) that could bind to or denature the
toxin or the test agents used.
Solution Preparation:
[0164] Each ileal loop received a 0.5 ml volume of a test agent or
agents in PBS. Four sets of labelled tubes and labelled syringes
were prepared (.+-.toxin.+-.polymer). Toxin A adheres to
plasticware, thus reducing its effective concentration. For this
reason tubes and syringes should be reused between loops.
Toxin A
[0165] Toxin A was commercially prepared by TechLab Inc. and was
provided in 0.5 ml vials containing 2 mg/ml. Each loop receiving
Toxin A was administered 5 .mu.g in total or 2.5 .mu.l of the above
solution.
Polymer
[0166] Loops receiving polymer received a total of 10 mg in a
volume of 0.5 ml PBS (20 mg/ml). New polymer solutions/suspensions
were prepared before each experiment.
.sup.3H-Mannitol
[0167] A stock solution 1 mCi/ml .sup.3H-mannitol (Mannitol,
D-[1-.sup.3H(N)]--, NEN Catalog No. NET101) was refrigerated. Each
animal received 10 .mu.Ci of .sup.3H-mannitol injected i.v: in a
volume of 200 .mu.l (ie. 10 .mu.l stock .sup.3H-mannitol+190 .mu.l
PBS per animal).
Anesthesia
[0168] Animals were anesthetized with 35-60 mg/kg pentobarbital
i.p. (50 mg/ml solution.) Thus, an i.p. injection of 0.2-0.3 ml
anesthetizes a 200-250 g rat. As an alternative, a
ketamine/xylazine cocktail can be used. This cocktail can be
prepared by mixing 5.4 ml of ketamine (100 mg/ml) and 0.45 ml of
xylazine (100 mg/ml). Animals were anesthetized with 0.25-0.3 mls
of this cocktail. Ten-fifteen minutes for anesthesia to be induced
was allowed. Surgical stage anesthesia was confirmed by the lack of
response from a painful stimulus (toe pinch).
Surgical Preparation
[0169] A midline abdominal incision was made and the cecum and
small bowel exteriorized. A length of 3.0 silk was placed around
the left renal pedicle and the vasculature ligated. The viscera was
then positioned so as to expose the right kidney and the renal
pedicle ligated, being careful not to damage the vena cava. The
cecum was then positioned so as to visualize the small bowel and
the vasculature supplying it. Two 5 cm lengths of ileum were
identified that were free of luminal contents (foodstuff or bile)
and ligatures tied so as to form two blind loops. The loops were
then injected with the desired test agents. Agents were injected
into the loop below the ligature distal to the cecum. The viscera
was then positioned so as to visualize the vena cava and 200 .mu.l
of the .sup.3H-mannitol solution injected i.v. Light pressure was
applied to the puncture wound after withdrawing the needle to
promote clotting. Following injection of mannitol viscera were
placed back into the abdomen and the muscle and skin layers of the
wound closed with surgical staples.
Injecting Beal Loops:
[0170] 5 .mu.g of Toxin A was dissolved in 0.5 ml of PBS
(.+-.polymer) and the mixture loaded into a syringe and injected
into a loop. The contents of the syringe are then quickly injected
into the intestinal loop.
Maintaining the Animals
[0171] Following surgery, animals were placed in a shoebox cage
with wood chips and allowed to recover for a 4-hour period. Animals
regained consciousness during this period. Any animals showing
overt signs of pain or distress were euthanized immediately.
Harvesting and Processing the Loops
[0172] Four hours after test substances were administered, the
ileal loops was harvested. Animals were sacrificed using 100%
CO.sub.2, the abdomen opened and the loops identified visually and
by using the suture markers. Loops were trimmed of excess tissue
and surgically excised, making sure not to lose any of the loop
contents. The loops were laid on a piece of weighing paper, their
length measured and the loop weight measured. The distal end of the
loop was then placed in a pre-weighed vial and cut open so as to
collect its contents. 500 .mu.l of PBS was then injected into the
proximal end of the loop to help wash the contents through. The
loop was then cut in half and a 0.5 cm section isolated, opened and
placed in fixative.
Sample Preparation for Scintillation Counting
[0173] Tubes were reweighed to determine the volume of the loop
contents. The tubes were vortexed vigorously and 200 .mu.l sample
aliquoted to a 7 ml plastic scintillation vial. These samples were
mixed with 5 mls of Ultima Gold scintillation cocktail and
vortexed. Samples were allowed to equilibrate overnight (to
minimize chemiluminescence), vortexed and counted the next day.
MIC Assay
[0174] The minimum inhibitory concentration (MIC) assay determines
the minimum concentration of an antimicrobial agent required to
inhibit growth of the test organisms. MIC assays were performed
against a standard panel of organisms as a screening tool to
identify compounds that have antimicrobial activity. The MIC assay
was subsequently repeated against other specialized microbial
panels; compounds were tested for biocidal activity, tested for
time course of killing, tested for toxicity against tissue culture
cells grown in vitro, and in some cases tested for antimicrobial
activity in vivo.
[0175] The MIC assay was performed according to the Performance
Standards for Antimicrobial Susceptibility Testing, 1998, vol.
M100-S8, Eighth Informational Supplement, NCCLS, 940 West Valley
Road, Suite 1400, Wayne, Pa. 19087.
[0176] Briefly, polymers to be tested were dissolved in 0.85%
saline to a final concentration of up to 5000 .mu.g/ml, the pH was
adjusted to 7.0 and the solution was filter-sterilized through a
0.22 .mu.m filter. Two-fold serial dilutions of polymer were
prepared in Mueller-Hinton broth with cations aliquotted into
96-well microtiter plates. The plates were then inoculated with
5.times.10.sup.5 cells/well of target organism, and incubated 18-24
hr at 35.degree. C. The optical density (OD) was then read at 590
nm, and microorganism growth was scored (OD>0.1 is considered to
be growth; OD<0.1 is considered growth inhibition). The MIC
value was defined as the lowest concentration of compound which
inhibits growth.
[0177] Organisms tested include a broad panel of aerobic gram
negative and gram positive bacterial strains of clinical
significance, a single anaerobe species (Clostridium difficile) as
well as several strains of Candida spp.
[0178] 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.
* * * * *