Note: Descriptions are shown in the official language in which they were submitted.
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FACTORS THAT BIND BACTERIAL TOXINS
PRIORITY CLAIM
This claims the benefit of U.S. Provisional Application No. 60/409,742, filed
September 10, 2002, which is incorporated by reference in its entirety.
FIELD
The invention relates to diagnosis and treatment of bacterial infections and
their
symptoms. More specifically, the invention concerns the use of compositions
derived
from hop bracts to neutralize bacterial toxins, such as Shiga-toxins.
BACKGROUND
In a large number of enteric diseases, caused by bacterial infection, toxins
elaborated by the organism appear to be responsible for the clinical
presentation. Thus,
vaccination against the toxic products of the organism may be sufficient for
prevention
of disease. For example, for tetanus, diphtheria and pertussis, immunization
prevents
the overt signs of infection. However, for enteric diseases, such as cholera
and certain
E. coli infections, immunization is not as effective because symptoms largely
result
from the effects of toxins on intestinal cells.
Strong epidemiological evidence supports an association of Shiga toxin-1
(Stxl)-producing Escherichia coli strains (STEC) with outbreaks of hemorrhagic
colitis,
hemolytic uremic syndrome (HUS), and encephalopathy. Stxl is the dominant
virulence factor in diseases caused by STEC. In general, antibiotics are used
for STEC
infections. However, following antibiotic administration, STEC, such as E.
coli
0157:H7, often produce massive amounts of Stxl, leading to a worsening of the
clinical
condition. Furthermore, although antibiotics have saved the lives of many
patients,
their administration has resulted in new drug-resistant bacteria such as
methicillin-
resistant Staphylococcus aureus (MRSA) and vancornycin-resistant
enterobacteria
(VRE), leaving some conditions untreatable.
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The biological activities of Stxl are well characterized. It is cytotoxic for
Vero
cells and a certain line of HeLa cells, lethal for mice and other small
rodents, and
enterotoxic, causing fluid accumulation in rabbit ileal loop assays. Stxl
consists of two
subunits, an A-subunit and five B-subunits. The A-subunit (StxA) is a 33-kDa
enzyme
that blocks protein synthesis in eukaryotic cells through its RNA N
glycosidase activity.
StxA cleaves an N glycosidic bond of adenosine at position 4,324 from the 5'-
terminus
of the 28S ribosomal RNA [60S ribosomal subunit in rabbit reticulocytes]. The
Stxl B-
subunits (StxB) bind to Gb3 globotriaosylceramide on the cell surface,
facilitating
STxA translocation into the cytosol. Recent reports describe substances that
inhibit
StxB binding to Gb3, but an effective inhibitor of StxA enzymatic activity has
not been
previously identified.
SUMMARY
Methods are described for treating a subject suffering from a condition caused
by exposure to a toxin, such as an enterotoxin, for example, a Shiga toxin or
a cholera
toxin. The disclosed methods include enterically administering, such as
administering
intraluminally, a polyphenolic component of, or a fraction of, an extract of
the bracts of
Humulus luplus (Hops) to neutralize pathogenic bacterial toxins.
Administration of the
hop component in combination with antibiotics reduces the effect of increased
toxin
production associated with antibiotic treatement of enterohemorrhagic
diseases. Also
disclosed are methods and devices for isolating polyphenolic compounds that
bind
bacterial toxins, and methods and devices for detecting the presence of
bacterial toxins
in biological samples. Fractions, subfractions and components of hop bract
tannin that
may be used in any of the disclosed methods and devices also are disclosed.
The foregoing and other features and advantages will become more apparent
from the following detailed description of several embodiments, which proceeds
with
reference to the accompanying figures.
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BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1 a-d are bar graphs illustrating the effect of hop bract extract (HBE,
Fig.
la), hop bract tannin (HBT, Fig. lb), and hop-bract extract low molecular
weight
fraction (HBE-LMW, Fig. lc) on RNA N glycosidase activity of Stxl, the effect
of
HBT on StxA N glycosidase activity (Fig. 1 d), and the effect of added EDTA on
N
glycosidase activity in the presence of HBT.
FIGS. 2a-d are a set of graphs illustrating the effects of HBT on protein
synthesis (Fig. 2a) and cell viability (Figs 2b, 2c and 2d) for Vero cells in
the presence
of Stxl.
FIGS. 3a-b are a digital image (Fig. 3a) and a bar graph (Fig. 3b)
demonstrating
the counteracting effect of HBT on Stxl-induced fluid accumulation in a rabbit
ileal
loop model.
FIGS. 4a-b are graphs illustrating the kinetics of HBT neutralization of
Stxl's
effects on protein synthesis in rabbit reticulocyte lysate (raw data, Fig. 4a;
Lineweaver-
Burke plot, Fig. 4b).
FIGS. Sa-c are a graph, a digital image and a pair of diagrams demonstrating
and
illustrating the formation of specific HBT-Stxl complexes. In Fig. Sa, the
signal
generated using a Biacore sensor having HBT as the bioreceptor demonstrates
the
specificity of HBT complex formation with Stxl relative to other proteins. HBT-
Stxl
complex formation and precipitation is shown in Fig. Sb. Figs. Sc and Sd show,
respectively, a polyphenolic component of HBT and a model that may explain the
behavior observed in Figs. Sa and Sb.
FIG. 6 is a series of light micrographs (top panels) and fluorescent
micrographs
(bottom panels) showing binding of fluorescent-labeled Stxl to a Vero cell
surface in
the absence of HBT and showing no binding of the labeled Stxl to Vero cell
surfaces in
the presence of HBT.
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DETAILED DESCRIPTION
I. Abbreviations
HBT (HBE-HMV - hop bract tannin [hop bract extract, high molecular
weight fraction (Mw > 5 kDa) or a polyphenolic component or subfraction
thereofJ.
Stx - Shiga toxin, also known as verotoxin or Shiga-like toxin.
Stxl - Shiga toxin 1.
Stx2 - Shiga toxin 2.
StxA - the catalytic A-subunit of a Shiga-toxin.
StxB - membrane binding B-subunit of a Shiga-toxin.
HBE - an extract of hop bracts comprising polyphenolic compounds.
HBE-LMW - hop bract extract, low molecular weight fraction.
STEC - Shiga toxin producing Eschericia coli
II. Terms
Unless otherwise explained, all technical and scientific terms used herein
have
the same meaning as commonly understood by one of ordinary skill in the art to
which
this invention belongs. Definitions of common terms in molecular biology may
be
found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994
(ISBN
0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology,
published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference,
published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
In order to facilitate review of the various embodiments of the invention, the
following explanations of specific terms are provided:
The terms "enteric" and "enterically" refer to the gastrointestinal tract,
whereas
the terms "intraluminal" and "intraluminally" refer specifically to the
intestines (small
andlor large). The term "enteric administration" refers to delivery of an
agent to at
least a part of the gastrointestinal tract. For example, enteric
administration includes,
without limitation, administration through an enteric tube (for example,
through an
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endoscope or plastic tube introduced through the gastrointestinal tract), or
in an oral
formulation, such as a tablet or liquid. The teen "theranostic" refers to a
treatment
having both a diagnostic and therapeutic component. Theranostic, for example,
may
refer to a treatment with an agent, where the agent is selected based on the
results of a
diagnostic test designed to reveal which particular agent is expected to
provide the most
efficacious treatment. The term "exotoxin" refers to a toxin produced by a
microorganism and the term "enterotoxin" refers to a toxin that shows toxicity
toward
intestinal cells.
The singular terms "a," "an," and "the" include plural referents unless
context
clearly indicates otherwise. Similarly, the word "or" is intended to include
"and"
unless the context clearly indicates otherwise. It is further to be understood
that all
base sizes or amino acid sizes, and all molecular weight or molecular mass
values,
given for nucleic acids or polypeptides are approximate, and are provided for
description. Although methods and materials similar or equivalent to those
described
herein can be used in the practice or testing of the present invention,
suitable methods
and materials are described below. The term "comprises" means "includes."
All publications, patent applications, patents, and other references mentioned
herein are incorporated by reference in their entirety. In case of conflict,
the present
specification, including explanations of terms, will control. In addition, the
materials,
methods, and examples are illustrative only and not intended to be limiting.
Various
embodiments are illustrated by the following non-limiting Examples.
III. Examples
Hop bract tannin (HBT) specifically binds toxin molecules, such as cholera
toxin, E. coli heat-labile enterotoxin and Stx, and enables methods for
treating a subject
suffering from an infection caused by a toxin-producing bacteria. These
methods
include administering to the subject a therapeutically effective amount of hop
bract
tannin. Administration of HBT may be accompanied by administration of a
therapeutically effective amount of an antibiotic that is capable of killing
at least a
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portion of the toxin-producing organisms. The HBT may be administered
enterically,
such as intraluminally, to block the action of the toxin. Enteric
administration includes,
without limitation, administration through an enteric tube (for example,
through an
endoscope or plastic tube introduced through the gastrointestinal tract), or
in an oral
formulation, such as a tablet or liquid. The oral formulation can be designed
to dissolve
enterically. In a particular example, the oral formulation is enterically
coated to
dissolve in a target region of the gastrointestinal tract, for example, the
intestines, for
example the small intestine or the large intestine. The infection to be
treated may
present itself clinically as severe diarrhea, hemorrhagic colitis, hemolytic
uremic
syndrome, thrombotic thrombocytopenic purpura and combinations thereof.
The HBT may be the high molecular weight fraction (>_ 5 kDa) of a hop bract
extract, a particular component thereof, or a subfraction thereof (e.g., a
fraction
obtained from the high molecular weight fraction of a hop bract extract, and
described
by an weight-average molecular mass between 5 kDa and 30 kDa). In one
embodiment,
the HBT comprises a catechin polymer, or a mixture of one or more such
polymers,
such as a polycatechin selected from the group of 10-mers to 30-mers, and
mixtures
thereof. In particular embodiments, the polycatechin may have the formula
where n=8 to 28. In other particular embodiments, the polycatechin may have
the formula
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where n = 8 to 28.
In yet other particular embodiments, the polycatechin may be a catechin
polymer where the linkages between individual catechin molecules are any
combination
of the linkages shown in the two structures above. For example, if the
polycatechin is a
30-mer, there may be anywhere from 1 to 28 linkages of one type, and anywhere
from
28 to 1 linkages of the other type.
In other embodiments, the HBT comprises a high molecular weight fraction
isolated from hop bract extract (HBE), such as a fraction having a weight-
average
molecular mass between 5 kDa and 30 kDa.
The Stx-producing organism causing an infection may be an Stxl- or Stx2-
producing organism. In particular embodiments the Stx-producing organism is a
Shiga
toxin-producing Esehericia eoli (STEC).
Also disclosed are theranostic methods of treating a subject having an
infection
caused by an Stx-producing organism. In these methods, a HBT having an
affinity for
the particular Stx produced by the Stx-producing organism is selected and then
administered to the subject enterically, such as intraluminally, in an amount
effective to
alleviate a clinical presentation of the infection. Desirably, selection is
used to identify
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the HBT fraction or HBT polyphenol that is most effective against the
particular Stx
produced by the infecting organism.
Selection of the appropriate HBT may be accomplished by affinity
chromatography using a chromatographic matrix derivatized with the particular
Stx (or
combination of Stxs) produced by the infecting organism. Alternatively,
selecting the
HBT is accomplished simply by obtaining a high molecular weight fraction of a
hop
bract extract, for example, by selecting a fraction having a weight-average
molecular
weight of 5 kDa or greater. HBT fractions that most effectively bind the Stx
may be
more precisely determined, for example, by determining the fractions that
precipitate
the most Stx (detected visually or electrophoretically).
Particular hop bract polyphenolic compounds, or fractions that have an
affinity
for the Stx, may also be selected by measuring their affinity for the Stx
using a
biosensor, where the HBT polyphenol or HBT fraction serves as the bioreceptor
portion
of the biosensor. Polyphenols that may serve as the bioreceptor include
polycatechins,
such as between 10-mer and 30-mer polycatechins. HBT fractions may be
fractions
having a weight-average molecular weight between 5 kDa and 30 kDa. Desirably,
the
fraction or compounds that provide the greatest signal when used as the
biosensor's
bioreceptor are selected for administration to the subject.
The specificity of the HBT/Stx interaction also enables methods for detecting
the presence of an Stx in a biological sample by contacting the biological
sample with
hop bract tannin, and detecting a macromolecular complex between the Stx and
the hop
bract tannin. The macromolecular complex rnay be detected by observing
formation of
a precipitate when the biological sample is contacted with the HBT. Complex
formation may also be detected by electrophoresis, for example, by observing
an
electrophoretic pattern associated with the presence of the macromolecular
complex in
the sample. Alternatively, the HBT may serve as a bioreceptor of a biosensor,
and the
biosensor may be used to detect the presence of an Stx in a sample. A
biosensor
comprising an HBT as the bioreceptor and a transducer is also provided.
Methods for isolating and purifying Star-binding polyphenols are also
provided.
For example, a mixture comprising an Stx-binding polyphenolic compound
isolated
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from Humulus lupulus may be contacted with Stx to form a macromolecular
complex
between the compound and Stx. The macromolecular complex may be isolated, and
then the polyphenolic compound may be liberated from the macromolecular
complex to
obtain a purified sample of the polyphenolic compounds) that bind the Stx. In
particular embodiments, the Stx is coupled to an activated chromatographic
matrix or a
biosensor.
Methods for prophylatic or post-exposure treatment of a condition caused by
inhalation of an Stx are also provided. For example, a therapeutically
effective amount
of HBT may be administered intranasally to a subject to protect the subject
from nasal
inhalation of the Stx.
The disclosed methods of neutralizing bacterial pathogenicity differ
significantly
from conventional therapeutic approaches. For example, a vaccine against
0157:H7
would not be effective against other STEC serotypes such as 026 and Ol 11.
However,
methods that utilize HBT (or the components thereof) as Stx-neutralizing
agents are
effective against diseases caused by all STEC serotypes. HBT may work to
prevent
intoxication by intraluminal neutralization and elimination of Stx from the
body. In
contrast, currently available synthetic inhibitors work to block Stx binding
to Gb3,
leaving the toxin in the body, and therefore available to do damage when the
inhibitor
concentration drops.
HBT may be derived from abundant natural sources at reduced cost. HBT also
exhibits reduced absorption and entrance into the circulatory system. Thus,
HBT is
more likely to be tolerated by patients, since the effects of HBT would be
limited to the
alimentary system. Furthermore, since HBT has no effect on bacterial growth,
it may
be used in combination with other therapeutic modalities, such as antibiotics
or
transfusion. Continued growth of an organism in the presence of HBT might lead
to
immunity from extended infection, while the clinical symptoms of intoxication
are
prevented by the HBT.
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Example 1- Hop Bract Tannin (HBT)
Hop (Humulus luplus L.) cone is a well-known ingredient in beer, while the hop
bract is typically discarded. Hop bract is enriched in highly-condensed
catechins (about
50% in polyphenolic fractions). As a by-product of beer brewing, it is
available in
abundance. Hop bract tannin (HBT) compounds in the high-molecular weight
fraction
include highly condensed (from about 10-mer to about 30-rner) catechins. Hop
bract
tannin (HBT) refers to the high molecular weight fraction ( >_ 5 kDa) of a hop
bract
extract, a polyphenolic component thereof, and mixtures of such polyphenolic
components, such as subfractions of the high molecular weight fraction of a
hop bract
extract comprising one or more such components.
Hop bract samples used for the experiments described in the Examples that
follow were prepared by the method of Tagashira et al. (Tagashira et al.,
"Inhibition by
hop bract polyphenols on cellular adherence and water-insoluble glucan
synthesis of
mutans streptococci," Biosei. Biotech. Biochem. 61: 332-335, 1997). In brief,
an
EtOH/H20 solvent was used to extract the polyphenolic constituents from hop
bracts.
Other solvent systems (e.g. solvent systems comprising other alcohols (for
example,
methanol or isopropyl alcohol), ethers (for example, diethyl ether), ketones
(e.g. methyl
ethyl ketone), acetonitrile, and mixtures thereof) that extract polyphenolic
compounds
from hop bracts may be employed to provide HBE. The 13C-NMR spectrum of the
EtOH/Ha0 extract was in good agreement with that of the synthetic catechin-
polymer
(See, Yoneda et al., "Synthesis of high molecular mass condensed tannin by
cationic
polymerization of flavan 3,4-carbonate," J. Chem. Soc., Perkin Trahs. 1: 1025-
1030,
1997).
Low- (HBE-LMW) and high-molecular weight fractions (HBT) of HBE were
separated by ultrafiltration using a 5,000 MW cutoff filter (Amicon Ultra,
Millipore,
Bedford MA). The lower molecular weight limit of the high-molecular weight
fraction
may be determined by the choice of the filter cutoff and may be anywhere
between
about 5 kDa and 30 kDa. The higher molecular weight limit of the high
molecular
weight fraction is typically determined by the molecular weight limit of the
components
of the HBE itself, but may be lowered by ultrafiltration of the fraction with
a second,
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higher molecular weight cutoff filter and retaining the resulting filtrate as
the high
molecular weight fraction.
By spectrometric analysis, acidic degradation of HBT in an alcohol solvent
yielded only cyanidine; no gallic acid or delphinidine was detected. Gel
permeation
chromatography (GPC) experiments showed that the HBT had a weight-average
molecular mass (Mw of 6280, a number-average molecular mass (Mn of 2260, and
Mw/Mn=2.8. The TOF-MS spectrum showed regular interval peaks at M/z=288
(native
HBT) or M/z=498 (acetylated HBT).
In addition to fractions, individual HBT polyphenolic compounds or mixtures
thereof may be isolated from HBE and used as HBT in the disclosed methods. For
example, affinity chromatography using an endotoxin derivatized chromatography
matrix (See Example 9) may be employed to isolate individual components of
HBE.
Alternatively, individual components of HBT may be separated and purified
using size
exclusion HPLC (e.g. Zorbax GF-250 or GF-450 column, Mac-Mod Corp., Chadds
Ford, PA).
HBT polyphenolic compounds may be described by Formulas 1 or 2 below,
where n = 8 to 28. In addition, polyphenolic compounds having any combination
of the
linkages shown in Formulas 1 and 2 may be isolated (i.e. polyphenolic
compounds
having a mixture of 4-~8 linkages as in Formula 1 and 4-~6 linkages as in
Formula 2).
Furthermore, one or more of the OH groups in these structures may be
derivatized to
form ester and/or ether groups. Esters include, but are not limited to
carboxylate (e.g.
acetate and propionate), phosphate and sulfate esters. Ether groups include
alkoxy
groups such as methoxy and ethoxy groups.
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OH
Formula 1
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OH
n
~H
OH
Formula 2
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Alternatively, fractions of compounds,falling within particular molecular
weight
ranges may be isolated from hop bract extract (e.g. by ultrafiltration or size
exclusion
chromatography) and used in the disclosed methods. For ultrafiltration the
range of
molecular weights depends upon the molecular weight cut-off of the
membrane(s~) used.
For example, fractions containing compounds having weight-average molecular
masses
in ranges such as 5 kDa-30kDa, SkDa-lOkDa, SkDa-8kDa, 8kDa-30kDa, BkDa-lOkDa
and lOld~a-30kDa may be isolated from HBE using commercially available
ultrafiltration membranes (e.g. Millipore, Bedford MA and Vivascience, Acton
MA).
For size exclusion chromatography, collecting the appropriate fractions as
they elute
from the column may be used to isolate a fraction having any arbitrary range
of
molecular weights.
Example 2 - Exotoxins
The HBT fractions and HBT polyphenols disclosed herein may effectively
neutralize a variety of exotoxins, including enterotoxins, such as Shiga
toxins and
cholera toxins. Cholera toxins are described, for example, in Burrows,
"Cholera
toxins," Anfau. Rev. Microbiol., 22:245-268, 1968, and include cholera toxins
A and B.
As used herein, the terms "Shiga toxin" and "Stx" refer to toxins in the Shiga
toxin family that may be neutralized by administration of HBT. The Shiga toxin
family
contains two types of toxins called Stxl (verotoxin 1: VT1 or Shiga-like toxin
1: SLTl)
and Stx2 (VT2, SLT2), both of which are encoded by bacteriophages. Stxl
resembles
the Shiga toxin produced by Shigella dysenteriae type I. Stx2 is
heterogeneous. These
toxins inhibit protein synthesis in eukaryotic cells, and play a role in
hemorrhagic
colitis, and hemolytic uremic syndrome. They also have been found to damage
endothelial cells in both the kidney and the brain, causing renal failure and
neurological
complications. (See, for example, Riley et al., New E~cgl. J. Med., 308: 681-
685, 1983
and Ashkenazi, Afznu. Rev. Med., 44: 11-18, 1993)
Although many variants exist, all Stx have an A-B structure, where the A
subunit possesses N-glycosidase activity and the B subunit binds to a membrane-
bound
glycolipid, globotriasoylceramide. The A-polypeptide N-glycosidase activity
cleaves
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an adenine from the 28S rRNA of the 60S cytoplasmic ribosome. This activity
renders
the 28S rRNA unable to interact with the elongation factors EF-1 and EF-2,
thus
inhibiting protein synthesis. The B polypeptide forms a pentamer that binds to
the
eukaryotic cell receptor globotriaosylceramide (Gb3). Shiga toxins enter cells
by
receptor-mediated endocytosis. Both Stxl and Stx2 have both been shown to
induce
apoptosis in several different cell types.
Stxs have many interesting effects at the cellular level. Once these toxins
have
been endocytosed, they are transported in a retrograde manner through the
Golgi
apparatus to the rough endoplasmic reticulum where they effectively target the
ribosomes. In addition to inhibiting protein synthesis, Shiga toxins induce
production
of cytokines such as interleukin-1, interleukin-6, and interleukin-8. They
have also
been shown to induce expression of tumor necrosis factor (TNF), induce F-
actin
depolymerization, and activate a src family kinase.
Stxl is a major virulence factor in the enterohemorrhagic diarrhea caused by
Stx-producing Escheriehia coli (STEC), such as 0157:H7, 89020097 and 0157:NM
(non-motile). Following administration of antibiotics, E. coli 0157:H7 often
releases
massive amounts of Stxl, resulting in further worsening of symptoms. Other
STEC
include E. coli within serogroups 026, 0103, 0111, 0113 and 0157. Stx2 is also
found in STEC. For example, a variant designated Stx(2f) is found in E. coli
0128
(See, Schmidt et al., Appl. Environ. Microbiol., 66:1205-08, 2000).
Stxl used to demonstrate HBT neutralization of Stx was purified from E. coli
MC1061, using pigeon egg ovomucoid-affinity column chromatography according to
the method described by Miyake et al. (Miyake et al., "Binding of avian
ovomucoid to
Shiga-like toxin type 1 and its utilization for receptor analog affinity
chromatography,"
Anal. Biocherrr. 281: 202-208, 2000). Purified StxA was obtained by the method
of
Brigotti et al. (Brigotti et al., "The RNA-N-glycosidase activity of Shiga-
like toxin
l :kinetic parameters of the native and activated toxin," Toxico>z 35: 1431-
1437, 1997).
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Example 3 - Hop Bract Tannins Inhibit RNA N glycosidase Activity of Stx1
This example demonstrates that hop bract tannin (HBT) inhibits the RNA N
glycosidase activity of Stxl . RNA N glycosidase activity was assayed in a
cell-free
rabbit reticulocyte system according methods described by Miyake et al. and
Sargiacomo et al. (See, Miyake et al., "Binding of avian ovomucoid to Shiga-
like toxin
type l and its utilization for receptor analog affinity chromatography," Ahal.
Biochem.
281: 202-208, 2000 and Sargiacomo et al., "Cytotoxicity acquired by ribosome-
inactivating proteins carried by reconstituted Sendai virus envelopes," FEBS
Lett. 157:
150-154, 1983). Rabbit reticulocyte lysate was prepared from female rabbits
(New
Zealand White, 3 kg, Japan SLC, Japan).
Rabbit reticulocyte lysate and samples (Stxl/HBT) dissolved in PBS were
mixed at 4 °C (total 50 ~.1). After additions of 20 p,l of reaction
mixture (described
below), and incubation at 30 °C for 0-15 min, as indicated, 1 ml of 10%
TCA was added
and samples were boiled in 95 °C water bath for 10 min. Precipitates
were collected on
filters and washed with 3 ml of 10% TCA, before radioassay of [14C]. Reaction
mixtures were prepared from 36.6 ml of rabbit reticulocyte lysate and
contained 15 mM
HEPES (pH 7.5), 1 mM ATP, 0.2 mM GTP, 15 mM phosphocreatine, 150 ug/xnl
creatine kinase, 2 mM magnesium acetate, 66 mM KCI, 6 mM dithiothreitol, 240
ug/xnl
haemin, 0.1 mM of each of 19 amino acids (no leucine), and 6.8 uCi/ml [14C]
leucine
(DuPont NEN Research Products, Boston, MA).
FIG. 1 shows how several different hop bract samples affect the reduction of
protein synthesis caused by added Stxl or StxA. Columns show protein synthesis
in
rabbit reticulocyte lysate without 37°C incubation (cross-hatched,
negative control), at
37°C without Stxl (dots, positive control); and in the presence of
either Stxl or StxA
(vertical lines). Column height shows mean~SD [14C] radioactivity (n=3)
incorporated
by the sample due to protein synthesis.
In the rabbit reticulocyte lysate system, natural source hop bract extract
(HBE)
inhibited the RNA N glycosidase activity of Stxl in a dose dependent fashion.
Addition
of HBE increased protein synthesis by counteracting the effects of Stxl, and,
at a
concentration of 200 p,g/mL, returned protein synthesis to levels similar to
that seen
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without Stxl (FIG. la). The high-molecular weight fraction of HBE (HBT) alone
was
also a potent inhibitor of Stx1 activities (FIG. lb) and restored protein
synthesis in a
dose-dependent fashion. To the contrary, the low-molecular weight fraction
(HBE-
LMW) had little inhibitory effect on Stxl activity (FIG. 1e). HBT inhibited
both Stx
(Fig. lb) and purified StxA (FIG. lc~, suggesting that HBT binds directly to
the A-
subunit of Stxl . When EDTA inhibited total protein synthesis, HBT did not
increase
radioactivity on the filter (FIG. 1 e), indicating that the increase in the
presence of HBT
(FIGS. lb, d) was not caused by non-specific capture of [14C] leucine by HBT.
Example 4 - HBT Inhibits Cytotoxicity of Stxl Toward Vero cells.
This example demonstrates that HBT is effective for reducing the toxic effects
of Stxl on kidney cells. Vero cells were seeded in a 96-well microtitre plate
(2 x 104
cells in 100 ~,1 per well) and grown in minimum essential medium (MEM, Sigma-
Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS, JRH
Biosciences,
Lenexa, KS), at 37 °C in a 5% COa atmosphere. Confluent cell monolayers
were used
for the assays. Vero cells were seeded approximately 2 x 105 cells (in 1 ml)
in each
well of a 24-well microtitre plate and cultured for 48 h. The plate was cooled
on ice for
10 min and then the medium was replaced with 0.5 ml of MEM-10%FBS, containing
0.9 uCilml of [14C] leucine. After addition of Stxl and/or HBT (in 50 p,l),
the plate was
incubated at 37 °C for 40 min on a water bath. Protein synthesis was
stopped by
addition of 0.25 ml of 30% TCA. Cells were washed three times with 1 ml of 10%
TCA and lysed in 0.25 ml of 0.5 N KOH for 10 min at 37 °C. The
lysate was
neutralized with 0.25 ml of 0.5 N acetate and protein synthesis was quantified
by
radioassay of [14C].
Several concentrations of HBT and Stxl were diluted in PBS solution and mixed
(80 p,l final per well) in another 96-well microtitre plate. The plate was
incubated for
lh at 37°C, after which 10 pl from each well were added to wells
containing Vero cells.
The Vero cell plate was incubated for an additional 48 h at 37 °C in a
5% COZ
environment. The viability of Vero cells was measured by Cell Counting Kit
(Dojindo
Laboratories, Kumamoto, Japan), according to the MTT-assay method (Roche
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Diagnostics Corporation, Indianapolis, ll~. The MTT method is based on
spectrophotometric detection of the cleavage of a tetrazolium salt by a
mitochondrial
respiratory chain enzyme, and is a measure of metabolic activity and cell
viability.
Stxl modifies ribosomal RNA irreversibly, thereby inhibiting protein synthesis
and causing cell death. HBT protected Vero cells, in a dose-dependent manner,
from
inhibition of protein synthesis during a 45-min exposure to Stxl (FIG. 2a).
The
columns in FIG. 2a show the mean~SD of [14C] radioactivity (h=3) incorporated
by
Vero cells in the presence of varying amounts of HBT, without 37 °C
incubation (cross-
hatched, negative control), without Stxl (dots, positive control) and in the
presence of
with Stxl (0.7 mg/ml) (vertical lines). These results are consistent with the
results
shown in Example 3 for the rabbit reticulocyte lysate assay.
The effect of HBT and other polyphenol samples on the viability of Vero cells
was also investigated. FIG. 2b shows the mean~SD of MTT-assay data (h=8) for
Vero
cells that were incubated with Stxl (62 pg/ml) at 37 °C for 2 days in
the presence of
HBT (diamonds), HBE-LMW (squares), green tea polyphenol (GTP, triangles), and
oolong tea polyphenol (OTP, circles). FIG. 2b demonstrates that HBE-LMW, GTP
and
OTP fractions did not have protective effects on Vero cells in the presence of
Stxl,
whereas HBT prevented cell death under similar experimental conditions.
The protection afforded by HBT against Stx1 toxicity at varying concentrations
was also investigated. With reference to FIG 2c, cells were treated with Stxl
at three
different concentrations [0.64 ng/ml (diamonds), 107 ng/ml (squares), 227
ng/ml
(triangles)]. For this study the Vero cells were exposed to Stxl and HBT at 4
°C (on
ice) for 30 min, washed with PBS for twice and incubated at 37 °C for 2
days in MEM-
10°/~FBS. The results show that the protective effect (increased cell
viability) of HBT
depends on Stxl concentration and time of exposure of Vero cells to Stx1
(FIGS. 2).
HBT was more effective in neutralizing Stxl activity during short exposure
times. For
longer incubation times, residual free Stxl may bind to Vero cells.
FIG. 2d presents data similar to that shown in FIG. 2c and further
demonstrates
the protective effect of HBT. Specifically, FIG. 2d shows the effect of HBT on
cell
viability in the presence of Stxl at two concentrations under the experimental
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conditions used to generate FIG. 2d. With reference to FIG 2d, the relative
cell viability
in the presence of Stxl alone (cross-hatches) and in the presence of both Stx-
1 and HBT
at 3.1 ~,g/mL (dots) or 25 ~,g/mL is shown.
Example 5 - HBT Inhibits Stxl-induced Fluid Accumulation in Rabbit Ileal Loops
This example illustrates how HBT neutralizes Stxl action on cells in the
mammalian intestine and demonstrates the utility of HBT for preventing Stxl-
induced
diarrhea. Fluid accumulation in rabbit ileal loops induced by Stxl was
evaluated using
the methods described by St. Hilaire et al. (St. Hilaire et al., "Interaction
of Shiga-like
toxin type 1 B-subunit with its carbohydrate receptor," Bioehemistry, 33:
14452-14463,
1994). Male rabbits (Japanese white, 2 kg purchased from Japan SLC) were
starved for
48 hr before operation, although water was available ad libitum. Rabbits were
anesthetized with thiopental sodium and the intestine was exteriorized through
a
midline incision. In each rabbit, 6-10 segments (about 6-8 cm in length) were
isolated
and 100 ng of Stxl and/or HBT sample (total volume 1 ml) were simultaneously
injected into each loop. Rabbits were sacrificed 24 hr. later, and the loops
excised. The
ratio of the volume of accumulated fluid within the loop per the length of the
loop
(ml/cm) is the measure of Stxl toxic activity.
HBT showed potent, dose-dependent inhibition of fluid accumulation induced
by Stxl (FIGS. 3a and 3b). As can be clearly seen in FIG. 3a, severe swelling
of the
intestine is induced by Stxl. Co-administration of HBT and Stxl leads to
reduced
amounts of swelling, and the appearance of the intestinal segment receiving
100 ~,g
HBT is similar to the control segment receiving only phosphate buffered saline
(PBS).
These results are quantified in FIG. 3b and it is evident that even low doses
of HBT,
such as 0.8 p.g per loop, provide a measurable reduction in Stxl induced
swelling.
These data demonstrate that HBT may be used as an effective therapeutic agent
against
enterohemorrhagic diarrhea caused by STEC and other exotoxin producing
organisms.
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Example 6 - HBT Interferes With Binding of Stxl to rRNA.
To elucidate the mechanism of the toxin-neutralizing action of HBT, a kinetic
analysis of the effect of HBT on Stxl activity was performed by measuring the
rate of
protein synthesis in a rabbit reticulocyte lysate system.
At low concentrations of Stxl (e.g. 0.7 ~g/ml), HBT (3.5 ~,glml) reduced the
magnitude of Stxl inhibition of protein synthesis. FIG. 3a shows the time
course plot
of the increase in [14C] radioactivity due to protein synthesis for rabbit
reticulocyte
lysate without HBT or Stxl ( diamonds), with HBT (3.5 ~,g/ml) and Stxl (0.7
~.g/ml)
(squares); and with Stxl alone (0.7 ~,g/ml). Lineweaver-Burk analysis (Fig.
4b) of the
kinetic data showed that the inhibition was competitive (i.e. the maximal
velocity (Vm)
of the Stxl-catalyzed reaction was not changed by HBT, while the Km was
increased
significantly). Without being bound to a particular theory, these results seem
to show
that HBT binds to StxA first, and that HBT-Stxl complex formation prevents
StxA
binding to ribosomal RNA. In contrast to these findings, some nucleic acid
analogues
have been reported to inhibit ribosome-inactivating proteins (RIP), including
Stxl,
noncompetitively (See, for example, Pallance et al., "Uncompetitive inhibition
by
adenine of the RNA-N glycosidase activity of ribosome-inactivating proteins,"
Biochim.
Biophys. Acta 1384: 277-284, 1998; Brigotti, M. et al. 4-Aminopyrazolo [3,4-d]
pyrimidine (4-APP) as a novel inhibitor of the RNA and I~NA depurination
induced by
Shiga toxin l," Nucleic Acids Res., 28: 2383-2388, 2000; and Brigotti et al.,
"A survey
of adenine and 4-Aminopyrazolo [3,4-d] pyrimidine (4-APP) as inhibitors of
ribosome-
inactivating proteins (RIPs)," Life Sci. 68: 331-336, 2000).
Example 7 - IiST Binds Stxl and Forms a Macromolecular Complex.
In general, polyphenols bind to proteins nonselectively (See, for example,
Haslam, "Natural polyphenols (vegetable tannins) as drugs: possible modes of
action,"
J. Nat. Prod., 59: 205-215, 1996). Surprisingly, however, HBT binds Stxl more
avidly
than other proteins. HBT binding to several proteins (Stxl, bovine serum
albumin,
ovalbmin) was compared. Binding between HBT and the proteins was quantified
using
a Biacore-2000 system (Biacore, Co., Stockholm, Sweden). HBT was non-
covalently
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immobilized on the CAS or SA sensor tip by repetitive flow of HBT at 50 ~,g/ml
(20
p,l/min for 180 sec.). Non-immobilized HBT was washed from the sensor with 0.1
N
NaOH (20 p,l/min for 60 sec) and the sensor tip was washed with running buffer
for 18
h before used for the experiment. Proteins at a concentration of 4.2 nM were
mixed
with the sensor tip and the change of reaction unit (R~ was measured.
Surprisingly, HBT bound Stxl more tightly than other proteins (FIG. Sa).
Furthermore, following incubation for 1 h at 37 °C in PBS, HBT
selectively formed
large aggregates with Stxl. These aggregates could be precipitated by
centrifugation.
Precipitation of HBT-Stxl complexes was monitored by SDS-PAGE. HBT and 4.2 nM
of each of the proteins (i.e, Stxl, BSA, ovalbumin) were mixed (total volume
60 pl) and
incubated at 37 °C for 60 min. After centrifugation (60 min, 10,500 g),
the supernatant
was collected and the tube was gently washed twice with PBS (60 p,l). The
proteins in
the supernatant and the precipitate were visualized after SDS-PAGE by silver-
staining
(FIG. Sb). A possible explanation is that HBT, with its elongated, bulky
structure
(shown in FIG. Sc), binds to StxA, leading to formation of large HBT-Stxl
complexes
as depicted in (FIG. 5 d).
Example 8 - Inhibition of Stxl Adherence to Cells
This example demonstrates not only that HBT forms complexes with Stx, it also
inhibits Stx binding to Vero cell surfaces. Stxl was fluorescent-labeled
according to the
protocol given with the FluoroLink-Ab Cy-3 labeling kit PA 33000 (Amersham
Pharmacia Biotech, Uppsala, Sweden). Vero cells were grown on a poly-L-lysine-
coated on cover glasses (18 x 18 mm, Iwaki glass Co., Japan) in a small
culture dish.
The solution of Cy-3 labeled Stxl was added to the Vero cell monolayer at
4°C for 30
min (See, for example, Hitotsubashi et al., "Some properties of purified
Escherichia coli
heat-stable enterotoxin II," Infect. Immun., 60: 4468-4474, 1994). After
incubation at
37°C for a period of time (0-1 h), the cells on the cover glasses were
washed twice with
PBS, and fixed in 3% formaldehyde for 20 min at room temperature. Cells were
inspected using a fluorescence-microscope system (Nikon Co., Tokyo, Japan).
Visible
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and fluorescence micrographs of the Vero cells on the cover glasses following
brief (0
h) and extended exposure (1 h) to Stx with and without HBT are shown in FIG.
6.
The data clearly demonstrate that HBT inhibits Stxl binding to the Vero cell
surface (after a 30-min exposure to Stxl at 4 °C), thereby preventing
Stxl translocation
to the cytosol (after 60 min at 37 °C). The data also support a
conclusion that HBT
forms complexes with Stxl, preventing its toxic effects on cells, perhaps
through its
interaction with the A subunit. The photographs in upper row of FIG. 6 show
Vero
cells visualized by phase microscopy. In the lower row of FIG. 6, the
photographs show
Vero cells visualized with fluorescent pigment (Cy-3) labeled Stxl from the
same view.
The photographs under bar marked 0 h show the cells after 30 min treatment (at
4 °C)
with Cy-3 labeled Stxl, and those under the bar marked 1 h show the cells
after 1.0 h
incubation at 37 °C, following the 4 °C, 30 min exposure. For
both times, only the cells
treated with Stxl alone show the presence of the fluorescent-labeled Stx-
molecules on
the surface of the Vero cells. In contrast, addition of HBT prevented
adherence of the
fluorescent-labeled Stxl molecules to the Vero cells, as evidenced by the dark
fluorescence images.
Example 9 - HBT Has No Antibiotic Activity on
0157:H7 and Does Not Interfere With the Action of Antibiotics
This example demonstrates that HBT acts upon the toxin produced by
enterohemorrhagic bacteria, rather than the bacteria themselves. E. coli
O157:H7,
isolated from a male patient in Chiba prefecture, Japan in 1999 (Dr. F.
Nomura, Chiba
University, Graduate School of Medicine) and cultured in Muller-Hinton Broth
medium
(Gibco BRL, Grand Island, NY, 100 ~.l), were combined with HBT and/or
streptomycin
(Meiji Seika, Co. Ltd., Tokyo, Japan) and dissolved in PBS (total volume 10
~1).
Samples were added to 96-well plates and incubated overnight (16-20 h) at 37
°C.
Growth of 0157:H7 was measured by absorbance at 600 nm.
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Addition of HBT (up to 200 ~g/ml) had no effect on 0157:H7 growth. It also
did not prevent the antibiotic effect of streptomycin (data not shown). These
results
demonstrate that HBT may be administered in combination with antibiotics to
provide a
treatment directed both toward the organisms themselves and the toxins they
produce.
Example 10 - Isolation of HBT Constituents Effective Against Stxl
The specific interaction between HBT constituents and the Stxl protein that
was
demonstrated in Example 7 above may be exploited to isolate polyphenolic
components
from crude hop~bract extract and polyphenolic components from other plant
materials.
For example, affinity chromatographic methods for isolating polyphenolic
compounds
based on the specific HBT/Stx interaction are enabled, as is selective
precipitation of
polyphenolic compounds.
An affinity chromatographic stationary phase is produced by reacting Stx 1
molecules with an activated chromatography matrix. Activated matrices of
several
types are available from Sigma, St Louis, MO. Preparation of affinity
chromatography
matricies is described in Boyer, "Modern Experimental Biochemistry," 2"d Ed.,
Benjamin/Cumrnings Publishing Co, Redwood City, CA, 1993. For example,
cyanogen bromide activated matrices are especially useful for providing Stx-
derivatized
affinity matrices because all ligands containing primary amino groups (e.g.
proteins)
are easily attached to cyanogen bromide under mild conditions.
Once prepared, the affinity matrix is placed in a column according to methods
well known in the art and a sample, presumably containing polyphenolic
compounds
capable of interacting specifically with Stxl, is passed through the column.
The
column is then rinsed to remove weakly bound constituents of the sample. The
strongly
bound constituents are then eluted from the column using, for example, a
solution
containing an Stxl specific antibody or a solution containing a chaotropic
agent such as
urea, or guanidine.
In one embodiment, crude HBE is passed through an affinity column containing
an Stxl functionalized matrix and the components of the HBE that specifically
bind to
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the matrix are eluted to provide a purified sample of HBT that may be
administered to a
subj ect to aggregate Stx 1 intraluminally.
Example 11- Detection of Enterohemorrhagic Infection
The toxin specific binding properties of the components of HBT enable
biosensors and methods for detecting the presence of toxins in biological
samples (e.g.
blood, urine, feces, or tissue). For example, HBT, or a polyphenolic compound
isolated
therefrom, is immobilized on a transducer, such as an electrode surface, to
provide a
bacterial toxin specific sensor. A biological sample, presumably containing
the toxin,
may then be contacted with the sensor and a change in a property of the
transducer may
be detected (e.g., a change in the potential or current passing through an
electrode).
Sensor response may be calibrated against standard solutions of the toxin and
used to
quantify the amount of the toxin in the biological sample.
A biosensor includes a biological recognition system (bioreceptor) and a
transducer. The interaction of the analyte with the bioreceptor produces an
effect
measured by the transducer that may be converted, for example, into an
electrical
signal. Transducer types include optical transducers (e.g. luminescence,
absorption,
surface plasmon resonance), electrochemical transducers and mass-sensitive
transducers (e.g. surface acoustic waves, microbalances). Optical transducers
may be
based on different types of spectroscopy (e.g. absorption, fluorescence,
phosphorescence, Raman, SERS, refraction or dispersion) and different
spectrochemical properties may be monitored (e.g. amplitude, energy,
polarization,
decay time and/or phase). Electrochemical transducers include conducting
polymers
(e.g. poly N-methylpyrroles, polyanilines, and poly o-phenylenediamine),
carbon and
metals (e.g. gold and platinum). Mass-sensitive transducers include
piezoelectric
crystals. The bioreceptor may be attached to the transducer either covalently
or non-
covalently. Additional details of biosensor technology are described by Vo-
Dinh and
Cullum (Vo-Dinh and Cullum, "Biosensors and biochips: advances in biological
and
medical diagnostics," Fz°senius J. Anal. Clzezzz., 366:540-551, 2000).
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In a particular embodiment, a microarray of biosensors is provided. These
"biochips" may include particular polyphenolic HBT compounds or fractions
(e.g. 10-
mer through 30-mer polycatechins or fractions having particular mass ranges or
average
masses) deposited on individual transducer elements to form an array of
detectors.
Such biochips are useful for determining the most effective treatment for a
particular
toxin-mediated infection (i.e. theranostic determinations). For example, a
sample of the
toxin produced by a microorganism may be contacted to a biochip having
polyphenolic
compounds as bioreceptors and the polyphenolic compound that most effectively
binds
the toxin is identified by the transduced signal it produces relative to the
other
polyphenolic compounds serving as bioreceptors on the biochip. Once
identified, the
strongest binding polyphenolic compound may be administered to a subj ect. For
example, a subj ect may ingest the compound to intraluminally precipitate the
toxin and
increase its elimination, while simultaneously attenuating the toxin's effects
on
intestinal cells.
The Biacore system used above in Example 7 is an example of a biosensor that
incorporates immobilized HBT compounds as bioreceptors. The Biacore sensor
chip
transducer operates by surface plasmon resonance. If sufficient amounts of
protein can
be recovered from the surface of such chips, it may be possible to identify
ligands using
mass spectrometry. For example, proteins may be vaporized using matrix
assisted laser
desorption directly from the sensor surface or proteins eluted from the sensor
surface
may be measured following electrospray ionization.
In another embodiment, a biological sample is contacted with a solution
containing HBT and any precipitate formed due to the formation of HBT-toxin
macromolecular complexes is separated from the resulting solution by, for
example,
centrifugation. In one embodiment, the presence of Stxl in the sample is
indicated by
the presence of a precipitate. In a more particular embodiment, the amount of
Stxl in
the biological sample is quantified by measuring, such as by gravimetric
analysis, the
amount of precipitate. Calibration standards may be employed.
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Example 11- Detection of Microbial Toxin Aerosols
The sensors described in Example 10 above may also be used to detect the
presence of microbial toxins in an environment. For example, a biosensor
having HBT
components as the bioreceptor may be used to detect the presence of microbial
toxins in
the air or on surfaces. Presence of microbial toxins may be detected by
contacting the
sensor with, for example, a solution prepared from a filtered air sample or a
solution
prepared from a swab sample of a surface. Such sensors may find utility as
early-
warning detectors of attacks with microbial toxins.
Example 12 - Protection from Microbial Toxin Aerosols
Because they effectively form complexes and selectively precipitate microbial
toxins, especially Shiga toxins, HBTs may be administered either
prophylactically or
post-exposure to prevent development of the symptoms of intoxication (e.g. in
an
aerosol, drinking water, or food). In a particular embodiment, HBT, or one or
more
components thereof, are administered intranasally to precipitate and
neutralize Shiga
toxins that have been or might be inhaled by a subj ect.
Example 13 - Pharmaceutical Compositions
Pharmaceutical formulations according to the present invention encompass
formulations that include an amount (for example, a unit dosage) of a toxin
neutralizing
agent together with one or more non-toxic pharmaceutically acceptable
excipients,
including carriers, diluents, and/or adjuvants, and optionally other
biologically active
ingredients such as a therapeutically effective amount of an antibiotic where
the amount
can kill at least a portion of a pathogen population. Standard pharmaceutical
formulation techniques are used, such as those disclosed in Remington's
Pharmaceutical
Sciences, Mack Publishing Co., Easton, PA (19th Edition).
A pharmaceutical formulation according to the invention includes HBT fractions
and/or one or more purified HBT polyphenols, and can also include, for
example, one
or more other biologically active ingredients, such as cefixime, tetracycline,
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ciprofloxacin, co-trimoxazole, norfloxacin, ofloxacin, fosfomycin and
kanamycin and
combinations thereof.
The dosage of the combined biologically active agents is sufficient to achieve
concentrations at the site of action that are similar to those that are shown
to achieve in
vivo protection from microbial toxins. Pharmaceutical formulations may
include, for
example, an amount of a toxin-neutralizing agent such that the subject
receives a dosage
of between about 0.0001 g/kg and 1 OOg/kg.
The compositions can be in the form of tablets, capsules, powders, granules,
lozenges, liquid or gel preparations, such as oral, topical, or solutions or
suspensions
(e.g., eye or ear drops, throat or nasal sprays, etc.) and other forms known
in the art.
Such pharmaceutical compositions can be administered systemically or locally
in any manner appropriate to the treatment of a given condition, including
orally,
rectally, nasally, buccally, by inhalation spray, or via an implanted
reservoir.
Pharmaceutically acceptable carriers include, but are not limited to, ion
exchangers, alunvna, aluminum stearate, lecithin, serum proteins (such as
human serum
albumin), buffers (such as phosphates), glycine, sorbic acid, potassium
sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts or
electrolytes such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,
sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,
polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol, sodium
carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-
block
polymers, polyethylene glycol, and wool fat.
Tablets and capsules for oral administration can be in a form suitable for
unit
dose presentation and can contain conventional pharmaceutically acceptable
excipients.
Examples of these include binding agents such as syrup, acacia, gelatin,
sorbitol,
tragacanth, and polyvinylpyrrolidone; fillers such as lactose, sugar, corn
starch, calcium
phosphate, sorbitol, or glycine; tableting lubricants, such as magnesium
stearate, talc,
polyethylene glycol, or silica; disintegrants, such as potato starch; and
dispersing or
wetting agents, such as sodium lauryl sulfate. Oral liquid preparations can be
in the
form of, for example, aqueous or oily suspensions, solutions, emulsions,
syrups or
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elixirs, or can be presented as a dry product for reconstitution with water or
other
suitable vehicle before use.
The pharmaceutical compositions can also be administered enterally in a
sterile
aqueous or oleaginous medium. The composition can be dissolved or suspended in
a
non-toxic enterally-acceptable diluent or solvent, e.g., as a solution in 1,3-
butanediol.
Commonly used vehicles and solvents include water, physiological saline,
Hank's
solution, Ringer's solution, and sterile, fixed oils, including synthetic mono-
or di-
glycerides, etc. Additives may also be included, e.g., buffers such as sodium
metabisulphite or disodium edeate; preservatives such as bactericidal and
fungicidal
agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or
chlorhexidine, and thickening agents, such as hypromellose.
The dosage unit involved depends, for example, on the condition treated,
nature
of the formulation, nature of the condition, embodiment of the claimed
pharmaceutical
compositions, mode of administration, and condition and weight of the patient.
Dosage
levels are typically sufficient to achieve a tissue concentration at the site
of action that is
at least the same as a concentration that has been shown to neutralize
microbial toxins
in vitro. For example, a dosage of about O.OOOlg/kg and 100g/kg of the active
ingredient may be useful in the treatment of toxin-mediated conditions. The
unit dosage
can also be formulated to include both the HBT and another therapeutic agent,
such as
an anti-infective agent, for example, an antibiotic.
The compounds can be used in the form of salts, preferably derived from
inorganic or organic acids and bases, including, but not limited to: acetate,
adipate,
alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,
camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate,
heptanoate,
hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
oxalate,
pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,
propionate,
succinate, tarixate, thiocyanate, tosylate, and undecanoate. Base salts
include, but are
not limited to, ammonium salts, alkali metal salts (such as sodium and
potassium salts),
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alkaline earth metal salts (such as calcium and magnesium salts), salts with
organic
bases (such as dicyclohexylamine salts), N-methyl-D-glucamine, and salts with
amino
acids (such as arginine, lysine, etc.). Basic nitrogen-containing groups can
be
quaternized, e.g., with such agents as C1-~ alkyl halides (such as methyl,
ethyl, propyl,
and butyl chlorides, bromides, and iodides), dialkyl sulfates (such as
dimethyl, diethyl,
dibutyl, an diamyl sulfates), long-chain halides (such as decyl, lauryl,
myristyl, and
stearyl chlorides, bromides, and iodides), aralkyl halides (such as benzyl and
phenethyl
bromides), etc. Water or oil-soluble or dispersible products are produced
thereby.
Pharmaceutical compositions can be included in a kit accompanied by
instructions for intended use, for example instructions required by a
pharmaceutical
regulatory agency, such as the Food and Drug Administration in the United
States.
It will be apparent that the precise details of the methods or compositions
described may be varied or modified without departing from the spirit of the
described
invention. We claim all such modifications and variations that fall within the
scope and
Spirit of the claims below.
