WO2004024070A2 - Factors that bind intestinal toxins - Google Patents
Factors that bind intestinal toxins Download PDFInfo
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- WO2004024070A2 WO2004024070A2 PCT/US2003/028282 US0328282W WO2004024070A2 WO 2004024070 A2 WO2004024070 A2 WO 2004024070A2 US 0328282 W US0328282 W US 0328282W WO 2004024070 A2 WO2004024070 A2 WO 2004024070A2
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- stx
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- tannin
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- 0 *C(*)(C(C(C(c(cc1)cc(O)c1O)Oc1cc(O)c2)O)c1c2O)c(c(O)c(C(C(C(c(cc1O)ccc1O)O1)O)c(c(O)c(CC(C(c(cc2O)ccc2O)O2)O)c2c2)c2O)c1c1)c1O Chemical compound *C(*)(C(C(C(c(cc1)cc(O)c1O)Oc1cc(O)c2)O)c1c2O)c(c(O)c(C(C(C(c(cc1O)ccc1O)O1)O)c(c(O)c(CC(C(c(cc2O)ccc2O)O2)O)c2c2)c2O)c1c1)c1O 0.000 description 4
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- 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.
- enteric diseases caused by bacterial infection
- toxins elaborated by the organism appear to be responsible for the clinical presentation.
- vaccination against the toxic products of the organism may be sufficient for prevention of disease.
- immunization prevents the overt signs of infection.
- 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 Escherichia coli strains
- HUS hemolytic uremic syndrome
- Stxl is the dominant virulence factor in diseases caused by STEC.
- antibiotics are used for STEC infections.
- STEC such as E. coli O157:H7
- massive amounts of Stxl leading to a worsening of the clinical condition.
- 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 vancomycin-resistant enterobacteria (VRE), leaving some conditions untreatable.
- MRSA methicillin- resistant Staphylococcus aureus
- VRE vancomycin-resistant enterobacteria
- Stxl The biological activities of Stxl are well characterized. It is cytotoxic for Nero 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 s bunits, 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 R ⁇ A N-glycosidase activity.
- StxA cleaves an N-glycosidic bond of adenosine at position 4,324 from the 5'-terminus of the 28S ribosomal R ⁇ A [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.
- StxB Stxl B- subunits
- 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.
- 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.
- FIGS, la-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.
- hop bract extract HBE, Fig. la
- hop bract tannin HBT, Fig. lb
- HBE-LMW hop-bract extract low molecular weight fraction
- 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 Nero 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. 5a-c are a graph, a digital image and a pair of diagrams demonstrating and illustrating the formation of specific HBT-Stxl complexes.
- 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. 5b.
- Figs. 5c and 5d show, respectively, a polyphenolic component of HBT and a model that may explain the behavior observed in Figs. 5a and 5b.
- FIG. 6 is a series of light micrographs (top panels) and fluorescent micrographs (bottom panels) showing binding of fluorescent-labeled Stxl to a Nero cell surface in the absence of HBT and showing no binding of the labeled Stxl to Nero cell surfaces in the presence of HBT.
- HBT HBE-HM
- hop bract tannin hop bract extract, high molecular weight fraction (Mw > 5 kDa) or a polyphenolic component or subtraction thereof.
- 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.
- enteric administration refers to delivery of an agent to at least a part of the gastrointestinal tract.
- 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.
- theranostic refers to a treatment having both a diagnostic and therapeutic component.
- Theranostic 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.
- exotoxin refers to a toxin produced by a microorganism and the term “enterotoxin” refers to a toxin that shows toxicity toward intestinal cells.
- Hop bract tannin 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 portion of the toxin-producing organisms. The HBT may be administered enterically, such as intraluminally, to block the action of the toxin.
- HBT Hop bract tannin
- 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.
- 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).
- 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.
- the polycatechin may have the formula
- the polycatechin may have the formula
- n 8 to 28.
- 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 maybe anywhere from 1 to 28 linkages of one type, and anywhere from 28 to 1 linkages of the other type.
- 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.
- HBE hop bract extract
- the Stx-producing organism causing an infection may be an Stxl- or Stx2- producing organism.
- the Stx-producing organism is a Shiga toxin-producing Eschericia coli (STEC).
- STC Shiga toxin-producing Eschericia coli
- theranostic methods of treating a subject having an infection caused by an Stx-producing organism are selected and then administered to the subject enterically, such as infraluminally, in an amount effective to alleviate a clinical presentation of the infection.
- selection is used to identify 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.
- 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 may 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.
- 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 Six-binding polyphenols are also provided.
- a mixture comprising an Stx-binding polyphenolic compound isolated 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 compound(s) that bind the Stx.
- the Stx is coupled to an activated chromatographic matrix or a biosensor.
- a therapeutically effective amount of HBT may be administered intranasally to a subject to protect the subject from nasal inhalation of the Stx.
- HBT Stx-neutralizing agents
- 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.
- 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.
- 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.
- HBT Hop Bract Tannin
- 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-mer) 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 subtractions 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,” Biosci. Biotech. Biochem. 61: 332-335, 1997).
- an EtOH/H O 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.
- High- (HBE-LM ) 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, higher molecular weight cutoff filter and retaining the resulting filtrate as the high molecular weight fraction.
- HBT polyphenolic compounds or mixtures thereof may be isolated from HBE and used as HBT in the disclosed methods.
- affinity chromatography using an endotoxin derivatized chromatography matrix See Example 9 may be employed to isolate individual components of HBE.
- HBT HBT-specific kinase
- HPLC size exclusion HPLC
- GF-250 or GF-450 column e.g. Zorbax GF-250 or GF-450 column, Mac-Mod Corp., Chadds Ford, PA.
- 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).
- 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.
- 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.
- ultrafiltration the range of molecular weights depends upon the molecular weight cut-off of the membrane(s') used.
- fractions containing compounds having weight-average molecular masses in ranges such as 5 kDa-30kDa, 5kDa-10kDa, 5kDa-8kDa, 8kDa-30kDa, 8kDa-10kDa and 10kDa-30kDa may be isolated from HBE using commercially available ultrafiltration membranes (e.g. Millipore, Bedford MA and Nivascience, Acton MA).
- 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.
- HBT fractions and HBT polyphenols disclosed herein may effectively neutralize a variety of exotoxins, including enterotoxins, such as Shiga toxins and cholera toxins.
- enterotoxins such as Shiga toxins and cholera toxins.
- Cholera toxins are described, for example, in Burrows, "Cholera toxins,” Annu. Rev. Microbiol, 22:245-268, 1968, and include cholera toxins A and B.
- 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: NTl or Shiga-like toxin 1: SLTl) and Stx2 (NT2, 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.
- Stx have an A-B structure, where the A- subunit possesses ⁇ -glycosidase activity and the B subunit binds to a membrane-bound glycolipid, globotriasoylceramide.
- the A-polypeptide ⁇ -glycosidase activity cleaves 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 (Gb 3 ). 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 mterleukin-1, mterleukin-6, and interleukin-8. They have also been shown to induce expression of rumor necrosis factor (TNF), induce F- actin depolymerization, and activate a src family kinase.
- TNF rumor necrosis factor
- Stxl is a major virulence factor in the enterohemorrhagic diarrhea caused by Stx-producing Escherichia coli (STEC), such as O157:H7, 89020097 and O157:NM (non-motile). Following administration of antibiotics, E. coli O157:H7 often releases massive amounts of Stx 1 , 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 MCI 061, 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. Biochem. 281: 202-208, 2000). Purified StxA was obtained by the method of Brigotti et al.
- hop bract tannin inhibits the R ⁇ A N- glycosidase activity of Stxl .
- R ⁇ A 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 1 and its utilization for receptor analog affinity chromatography," Anal. Biochem.
- Rabbit reticulocyte lysate was prepared from female rabbits (New Zealand White, 3 kg, Japan SLC, Japan).
- 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/ml creatine kinase, 2 mM magnesium acetate, 66 mM KC1, 6 mM dithiothreitol, 240 ug/ml haemin, 0.1 mM of each of 19 amino acids (no leucine), and 6.8 uCi ml [ 1 C] 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).
- HBE natural source hop bract extract
- HBT inhibited both Stx (Fig. lb) and purified StxA (FIG. Id), suggesting that HBT binds directly to the A- subunit of Stxl .
- EDTA inhibited total protein synthesis
- HBT did not increase radioactivity on the filter (FIG. le), indicating that the increase in the presence of HBT (FIGS, lb, d) was not caused by non-specific capture of [ 14 C] leucine by HBT.
- Example 4 - HBT Inhibits Cytotoxicity of Stxl Toward Vero cells.
- Nero cells were seeded in a 96-well microtitre plate (2 x 10 4 cells in 100 ⁇ l 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% CO atmosphere. Confluent cell monolayers were used for the assays. Nero cells were seeded approximately 2 x 10 5 cells (in 1 ml) in each well of a 24-well microtitre plate and cultured for 48 h.
- MEM minimum essential medium
- FBS fetal bovine serum
- 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 uCi ml of [ 14 C] leucine. After addition of Stxl and or HBT (in 50 ⁇ 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 ⁇ KOH for 10 min at 37 °C. The lysate was neutralized with 0.25 ml of 0.5 ⁇ acetate and protein synthesis was quantified by radioassay of [ 14 C]. Several concentrations of HBT and Stxl were diluted in PBS solution and mixed
- the plate was incubated for lh at 37°C, after which 10 ⁇ l from each well were added to wells containing Nero cells.
- the Nero cell plate was incubated for an additional 48 h at 37 °C in a 5% CO 2 environment.
- the viability of Nero cells was measured by Cell Counting Kit (Dojindo Laboratories, Kumamoto, Japan), according to the MTT-assay method (Roche Diagnostics Corporation, Indianapolis, IN).
- 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 Nero cells, in a dose-dependent manner, from inhibition of protein synthesis during a 45-min exposure to Stxl (FIG. 2a).
- FIG. 2b demonstrates that HBE-LMW, GTP and OTP fractions did not have protective effects on Nero cells in the presence of Stxl, whereas HBT prevented cell death under similar experimental conditions.
- HBT The protection afforded by HBT against Stxl toxicity at varying concentrations was also investigated.
- FIG 2c cells were treated with Stxl at three different concentrations [0.64 ng/ml (diamonds), 107 ng/ml (squares), 227 ng/ml (triangles)].
- the Nero 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 Nero cells to Stxl (FIGS. 2). HBT was more effective in neutralizing Stxl activity during short exposure times. For longer incubation times, residual free Stxl may bind to Nero 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 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.
- 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).
- 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).
- PBS phosphate buffered saline
- HBT may be used as an effective therapeutic agent against enterohemorrhagic diarrhea caused by STEC and other exotoxin producing organisms.
- FIG. 3a shows the time course plot of the increase in [ 14 C] 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 (Nm) of the Stxl -catalyzed reaction was not changed by HBT, while the Km was increased significantly).
- 4-Aminopyrazolo [2>,4-d pyrimidine (4-APP) as a novel inhibitor of the R ⁇ A and D ⁇ A depurination induced by Shiga toxin 1," 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)," / /e Sct. 68: 331-336, 2000).
- Example 7 - HBT Binds Stxl and Forms a Macromolecular Complex.
- polyphenols bind to proteins nonselectively (See, for example,
- HBT 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., Sweden). HBT was non-covalently immobilized on the CA5 or SA sensor tip by repetitive flow of HBT at 50 ⁇ g/ml (20 ⁇ l/min for 180 sec).
- Non-immobilized HBT was washed from the sensor with 0.1 N NaOH (20 ⁇ 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 (RU) was measured.
- HBT bound Stxl more tightly than other proteins (FIG. 5a). 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 ⁇ l) 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 ⁇ l).
- the proteins i.e, Stxl, BSA, ovalbumin
- Stxl was fluorescent-labeled according to the protocol given with the FluoroLink-Ab Cy-3 labeling kit PA 33000 (Amersham Pharmacia Biotech, Uppsala, Sweden). Nero 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 photographs in upper row of FIG. 6 show Nero cells visualized by phase microscopy. In the lower row of FIG. 6, the photographs show Nero cells visualized with fluorescent pigment (Cy-3) labeled Stxl from the same view.
- Example 9 - HBT Has No Antibiotic Activity on O157: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 ⁇ l).
- HBT may be administered in combination with antibiotics to provide a treatment directed both toward the organisms themselves and the toxins they produce.
- 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.
- 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 Stxl 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 nd Ed., Benjamin/Cummings Publishing Co, Redwood City, CA, 1993.
- 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.
- 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.
- crude HBE is passed through an affinity column containing an Stxl functionalized matrix and the components of the HBE that specifically bind to the matrix are eluted to provide a purified sample of HBT that may be administered to a subject to aggregate Stxl 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).
- biological samples e.g. blood, urine, feces, or tissue.
- 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.
- 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 No-Dinh and Cullum (No-Dinh and Cullum, "Biosensors and biochips: advances in biological and medical diagnostics,” Frsenius J. Anal. Chem., 366:540-551, 2000).
- a microarray of biosensors is provided.
- 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).
- 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.
- the strongest binding polyphenolic compound may be administered to a subject.
- a subject 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.
- 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.
- the presence of Stxl in the sample is indicated by the presence of a precipitate.
- 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.
- the sensors described in Example 10 above may also be used to detect the presence of microbial toxins in an environment.
- 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.
- 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).
- 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 subject.
- compositions 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, ciprofloxacin, co-trimoxazole, norfloxacin, ofloxacin, fosfomycin and kanamycin and combinations thereof.
- one or more other biologically active ingredients such as cefixime, tetracycline, ciprofloxacin, co-trimoxazole, norfloxacin, ofloxacin, fosfomycin and kanamycin and combinations thereof.
- compositions may include, for example, an amount of a toxin-neutralizing agent such that the subject receives a dosage of between about O.OOOlg/kg and lOOg/kg.
- 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.
- 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, alumina, 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.
- 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
- 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 elixirs, or can be presented as a dry product for reconstitution with water or other suitable vehicle before use.
- 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.
- 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.
- 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.OOOlgkg and lOOg/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
- Base salts include, but are not limited to, ammonium salts, alkali metal salts (such as sodium and potassium salts), 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 Cl-8 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.
Abstract
Description
Claims
Priority Applications (4)
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EP03749554A EP1545209A4 (en) | 2002-09-10 | 2003-09-09 | Factors that bind intestinal toxins |
AU2003267079A AU2003267079B2 (en) | 2002-09-10 | 2003-09-09 | Factors that bind intestinal toxins |
CA002498225A CA2498225A1 (en) | 2002-09-10 | 2003-09-09 | Factors that bind intestinal toxins |
JP2004536403A JP2006508924A (en) | 2002-09-10 | 2003-09-09 | Factors that bind bacterial toxins |
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US40974202P | 2002-09-10 | 2002-09-10 | |
US60/409,742 | 2002-09-10 |
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WO2004024070A3 WO2004024070A3 (en) | 2004-08-05 |
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PCT/US2003/028282 WO2004024070A2 (en) | 2002-09-10 | 2003-09-09 | Factors that bind intestinal toxins |
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EP (1) | EP1545209A4 (en) |
JP (1) | JP2006508924A (en) |
AU (1) | AU2003267079B2 (en) |
CA (1) | CA2498225A1 (en) |
WO (1) | WO2004024070A2 (en) |
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JP2012153659A (en) * | 2011-01-27 | 2012-08-16 | Akita Univ | Humulus lupulus leaf extract and method for producing the same |
JP6232177B2 (en) * | 2011-11-28 | 2017-11-15 | 花王株式会社 | Verotoxin deactivator |
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JP2986805B2 (en) * | 1989-05-19 | 1999-12-06 | 三井農林株式会社 | Antitoxin for bacterial exotoxin |
US6251461B1 (en) * | 1997-10-10 | 2001-06-26 | S. S. Steiner, Inc. | Antimicrobial activity of hops extract against Clostridium botulinum, Clostridium difficile and Helicobacter pylori |
CN1348368A (en) * | 1999-02-15 | 2002-05-08 | 日加威士己株式会社 | ADP-ribosylation inhibitors and remedies for endotoxin bacterial enteric infection containing proanthocyanidin as the active ingredient |
JP2000342192A (en) * | 1999-06-08 | 2000-12-12 | Natl Fedelation Of Agricult Coop Assoc | Feed as countermeasure for edema |
JP2001249134A (en) * | 1999-12-28 | 2001-09-14 | Matsushita Electric Ind Co Ltd | Reagent for measuring protein concentration, method for measuring protein concentration using it, and urinalysis |
JP4521703B2 (en) * | 2000-05-17 | 2010-08-11 | アサヒビール株式会社 | Lipase inhibitor obtained from hops |
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2003
- 2003-09-09 WO PCT/US2003/028282 patent/WO2004024070A2/en active Application Filing
- 2003-09-09 CA CA002498225A patent/CA2498225A1/en not_active Abandoned
- 2003-09-09 AU AU2003267079A patent/AU2003267079B2/en not_active Ceased
- 2003-09-09 EP EP03749554A patent/EP1545209A4/en not_active Withdrawn
- 2003-09-09 JP JP2004536403A patent/JP2006508924A/en active Pending
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Also Published As
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CA2498225A1 (en) | 2004-03-25 |
EP1545209A2 (en) | 2005-06-29 |
AU2003267079A1 (en) | 2004-04-30 |
AU2003267079B2 (en) | 2008-08-14 |
EP1545209A4 (en) | 2009-07-29 |
JP2006508924A (en) | 2006-03-16 |
WO2004024070A3 (en) | 2004-08-05 |
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