WO1993008209A1 - Diagnosis and treatment of bacterial dysentery - Google Patents

Diagnosis and treatment of bacterial dysentery Download PDF

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Publication number
WO1993008209A1
WO1993008209A1 PCT/US1992/008929 US9208929W WO9308209A1 WO 1993008209 A1 WO1993008209 A1 WO 1993008209A1 US 9208929 W US9208929 W US 9208929W WO 9308209 A1 WO9308209 A1 WO 9308209A1
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method
slt
αgal
βgal
subunit
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PCT/US1992/008929
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French (fr)
Inventor
Glen D. Armstrong
R. Murray Ratcliffe
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Chembiomed Ltd.
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Priority to US07/778,732 priority
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Publication of WO1993008209A1 publication Critical patent/WO1993008209A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245Escherichia (G)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • G01N33/56916Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00Immunoassays for the detection of microorganisms
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/46Medical treatment of waterborne diseases characterized by the agent
    • Y02A50/468The waterborne disease being caused by a bacteria
    • Y02A50/473The waterborne disease being caused by a bacteria the bacteria being Escherichia coli, i.e. E. coli Infection
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/46Medical treatment of waterborne diseases characterized by the agent
    • Y02A50/468The waterborne disease being caused by a bacteria
    • Y02A50/475The waterborne disease being caused by a bacteria of the genus Shigella, i.e. Dysentery

Abstract

Diagnostic and therapeutic compositions which comprise the αGal(1-4)βGal subunit are described. These compositions permit the rapid diagnosis and treatment of enteric infections caused by E. coli that produce shiga-like toxins (SLT).

Description

DIAGNOSIS AND TREATMENT OF BACTERIAL DYSENTERY

Technical Field

The invention relates to diagnosis and treatment of diarrhea caused by bacterial infection.

More specifically, the invention concerns detection and neutralization of shiga-like toxins (SLT) associated with enteric bacterial infection.

Background Art

Diarrhea caused by strains of pathogenic E. coli has been found to be associated with the production of a variety of enterotoxins. Some pathogenic E. coli enterohemorrhagic produce enterotoxins that are closely related to the shiga toxin associated with Shigella-caused dysentery. The first member of the family of shiga-like toxins (SLT) to be isolated was cytotoxic for African Green Monkey (Vero) cells and was originally called verotoxin. Since its structural similarity to shiga toxin has been established by sequencing of the relevant genes, this toxin is now more commonly called shiga-like toxin I (SLTI) . See, for example, Calderwood, S.B., et al., Proc Natl Acad Sci USA

(1987) 84.:4364-4368; Jackson, M.P., et al., Microb Pathoσ (1987) 2:147-153; Strockbine, N.A. , et al., J Bacteriol

(1988) 170:1116-1122.

Additional members of the SLT family have subsequently been isolated that can be distinguished serologically, on the basis of gene sequence or host specificity (Gannon, V.P.J., et al., J Gen Microbiol (1990) 136:1125-1135; Weinstein, D.L. , et al. , J Bacteriol (1988) 170:4223-4230; Ito, H. , et al. , Microb Pathoor (1990) .8:47-60; Head, S.C., et al., FEMS Microbio Lett (1988) 51:211-216; Schmitt, C.K. , et al. , Infect Immun (1991) £59.:1065-1073; Scotland, S.M., et al. Lancet (1985) ii:885-886; Oku, Y. , et al. , Microb Pathoor (1989) 6.:113-122. Various types of SLTII have been described and have been assigned various designations depending on the strain of E. coli from which they are isolated and the hosts they inhabit. Thus, variants have been designated SLTII; vtx2ha; SLTIIvh; vtx2hb; SLTIIc; SLTIIvp and so forth.

All of the SLT's are multimeric proteins composed of an enzymatic (A) subunit and multiple (B) subunits responsible for toxin binding to receptors on host tissues. The binding B oligomers of SLTI, SLTII and SLTIIvh recognize host cell globoseries glycolipid receptors containing at minimum the disaccharide subunit αGal(l-4)BGal at the non-reducing terminus; SLTIIvp has been shown to bind to the receptors containing this subunit but not necessarily at the non-reducing end (Samuel, J.E., et al. , Infect Immun (1990) 58_:611-618; Boyd, B., et al. , Nephron (1989) 51:207-210; DeGrandis, S . , et al., J Biol Chem (1989) 2J54:12520-12525; Waddell, T. , et al. , Biochem Biophys Res Comm (1988) 152.:674-679; Lingwood, CA. , et al. , J Biol Chem (1987) 262:8834-8839; Waddell, T., et al. , Proc Natl Acad Sci USA (1990) 82:7898-7901; Cohen, A., et al. , J Biol Chem (1987) 162.:17088-17091; Jacewicz, M. , et al. , J EXP Med (1986) 163:1391-1404: Lindberg, A.A. , et al., J Biol Chem (1987) 262:1779-1785.

SLT activity has been detected in stool samples of symptomatic patients (Karmali, M.A. , et al., J Clin Microbiol (1988) 22.:614-619; Maniar, A.C. , et al., J Clin Microbiol (1990) 28.:134-135) . However, there is difficulty in detecting the presence of SLTs clinically since these are very potent toxins present in low concentrations. In order to assure detection, the SLT present in the sample must be concentrated to enhance the reliability of the assay. Present diagnostic procedures are technically demanding, time consuming and of limited practical use in the clinical setting. Thus there is a clear need for improved diagnostic clinically practical and rapid procedures. Also, antibiotics are not recommended for treatment of enterohemorrhagic E. coli infection (Robson, W.L.M., et al., J Pediatr (1990) 117:675-676) and the use of antimotility drugs also appears to be counterproductive (Cimolai, N. , et al., J Pediatr (1990) 117:676. There is, therefore, also a clear need for new and effective therapeutic agents.

It has now been found that artificial substrates containing the αGal(l-4)βGal (P^ disaccharide) subunit and more preferably the αGal(l-4)βGal(l-4)βGlcNAc (Pχ trisaccharide) or αGal(l-4)βGal(l-4)βGlc (Pk trisaccharide) subunit are effective in detecting and neutralizing members of the SLT family under conditions necessary to effect recovery of the patient and as such represent novel therapeutic and diagnostic tools in the treatment of E. coli-mediated dysentery.

Disclosure of the Invention

The invention provides compositions and methods that permit practical and effective diagnosis of E. coli- caused enterotoxic bacterial infections that present clinically as severe diarrhea, hemorrhagic colitis, hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. The invention also provides compositions useful in the therapy of these and related conditions. Thus, in one aspect, the invention is directed to a method to simply and rapidly bind to a support shiga-like toxins (SLT) from a biological sample at physiological conditions for diagnostic use, which method comprises contacting said sample with an effective amount of an affinity support wherein the affinity ligand comprises an αGal(l-4)j8Gal subunit under conditions wherein said SLT is adsorbed to the affinity support; and detecting any SLT bound to the support. In another aspect, the invention is directed to methods to detect the presence of SLT in a biological sample which method comprises contacting said sample with a composition comprising the αGal(l-4)/3Gal subunit under conditions wherein said subunit is complexed to any SLT present in the sample and detecting the presence of any complex formed.

In a third aspect, the invention is directed to a method to treat enteric infections caused by microorganisms that produce one or more SLTs, which method comprises administering to a subject in need of such treatment an effective amount of a composition comprising the αGal(l-4)/3Gal subunit.

In still other aspects, the invention is directed to pharmaceutical compositions which comprise the αGal(l-4)j8Gal subunit.

Brief Description of the Drawings

Figure 1 A and B demonstrate the toxicity of bacterial extracts obtained using polymixin-B with respect to their ability to kill Vero cells in the presence and absence of various SYNSORBs.

Figure 2 C and D demonstrate the toxicity of bacterial extracts obtained using lysozyme with respect to their ability to kill Vero cells in the presence and absence of various SYNSORBs. Figure 3 A demonstrates that as little as 10 mg of Pk trisaccharide SYNSORB removes >90% of SLT toxins from bacterial extracts.

B demonstrates that the binding of the SLT toxins occurred within 5 minutes of mixing extracts with the Pk SYNSORB.

Figure 4 demonstrates the difficulty in eluting the bound I125 labelled SLTI from various SYNSORBs utilizing a variety of eluants. Figure 5 demonstrates that >90% SLTI, SLTII/IIc and SLTII activity was neutralized by co-incubation of Vero cells and SLT extracts for three days, with as little as 10 mg of Pk trisaccharide SYNSORB.

The subunit is bound preferably through a linking arm such as that described by Lemieux, R.U. , et al., J Am Chem Soc. (1975) £2:4076-4083; to a solid, inert support that can be easily eliminated from the gastrointestinal system. An inert silica matrix embodiments of which are commercially available as "SYNSORB™" are preferred.

Modes of Carrying Out the Invention

The compositions useful in the conduct of the methods of the invention include a αGal(l-4)βGal disaccharide subunit, preferably the aGal(l-4)BGal (l-4)βGlcNAc trisaccharide subunit or αGal(l-4)βGal (l-4)βGlc trisaccharide subunit, preferably at the non- reducing terminus of an oligosaccharide. The di- and trisaccharides may be provided as a portion of a larger oligosaccharide coupled to a solid support or coupled directly, preferably through a linking arm such as that described by Lemieux, R.U. , et al., J Am Chem Soc (1975) £2:4076-4083. The di- and trisaccharide subunits may also be coupled directly to pharmaceutically acceptable carriers or constitute a portion of an oligosaccharide — o ,—

coupled to such carriers. Depending on the application for which the compositions of the invention are suggested, the composition is designed to accommodate the di- or trisaccharide subunits so as advantageously to be employed in these applications.

As used herein, "shiga-like toxin" or "SLT" refers to group of toxins produced by enterohemorrhagic E. coli that resemble the Shigella-produced shiga toxins as is commonly understood in the art. These toxins comprise an enzymatically active A subunit and a multimeric receptor binding B subunit. Such SLTs include SLTI and the various grouped toxins designated in the art SLTII.

Rapid, tight binding of SLT's to P-^ disaccharide, P-^ trisaccharide or Pk trisaccharide is demonstrated by the verocytoxicity neutralization and I125 binding assays contained herein. SYNSORB™ bearing haptens, e.g., the GM1 ganglioside Neu5Ac(2-3)βGal(l- 4)βGlc and heat labile toxin from enterotoxigenic E. coli would be expected to behave similarly. A single SYNSORB™ bearing several haptens with specificity for the different binding subunits of several diff rent gastrointestinal infections should now be possible. Such universal SYNSORB™s would provide rapid, simple simultaneous diagnosis of a variety of gastrointestinal disorders.

The SYNSORB™s employed were obtained from Chembiomed (Edmonton, Canada) . In each case the 8-methoxycarbonyloctyl glycoside of the respective hapten is activated and ligated to a silylaminated solid support, wherein the matrix is comprised of Si02, followed by acetylation of the remaining amine groups on the solid support. These formulations are sold commercially as "SYNSORB™"s: "P-^-di," which contains 0.60 μmole/g αGal(l- 4)βGal disaccharide;

"P-L-tri," which contains 0.91 μmole/g Gal(l- 4)βGal(1-4)BGlcNAc trisaccharide; "Pk-tri," which contains 0.74 μmole/g αGal(l-

4)βGal(l-4)βGlc trisaccharide;

"Linear B like tri," which contains 0.47 μmole/g αGal(1-3)βGal(1-4)BGlcNAc trisaccharide;

"Linear B like di," which contains 0.66 μmole/g αGal(1-3)βGal disaccharide;

"Glucose mono," which contains 1.0 μmol B- glucose; 8-methoxycarbonyoctanol activated and ligated to the silylated solid support.

"ASA" which contains only the acetylated silylaminated (ASA) hydrophobic 8-methoxycarbonyloctyl linkage arm.

A major aspect of the invention is the rapid efficient binding of physiological concentrations of any SLT present in biological samples, thus permitting assay of quantities of these toxins. Typically, in view of the conditions with which these toxins are associated, the biological sample will be a stool sample. The sample is extracted and prepared using standard extraction techniques and the extract is contacted with a solid support derivatized to an affinity ligand, wherein the affinity ligand comprise the αGal(1-4)βGal disaccharide subunit, preferably the αGal(1-4)βGal(1-4)BGlcNAc trisaccharide subunit or αGal(1-4)βGal(1-4)BGlc trisaccharide subunit. Said contact may be in a batch treatment of the sample extract with the solid support, or the solid support may be supplied as a chromatography column and the sample extract applied to the column under conditions wherein any SLT present in a sample is absorbed. SLT may be measured directly on the surface of the SYNSORB™ using any suitable detection system. In one approach, monoclonal or polyclonal antibodies specific for SLT can be utilized to quantify the amount of SLT bound directly to SYNSORB™, labeled, for example, by radioactive, biotinylated, or fluorescent moieties. A wide variety of protocols for detection of formation of specific binding complexes analogous to standard immunoassay techniques is well known in the art. In coupling the relevant subunits to supports, it is preferable that a spacer arm be used so that the affinity ligand is permitted ready access to the toxin to be coupled. A particularly preferred spacer arm is the 8-methoxycarbonyloctyl-activated (MCO) form of the oligosaccharide to be coupled. This spacing appears to be particularly advantageous, and thus alternative chemistries which provide the same spacing may also used. Any spacer arm which is of a corresponding length and is provided functional groups at each terminus for reaction with the disaccharide, trisaccharide or oligosaccharide on the one hand and the solid support on the other may be used; such spacer arms "correspond" to MCO. The nature of these functional groups is understood by those of ordinary skill in the art to depend on the nature t>f the oligosaccharide and the nature of the solid support used.

Compositions containing the αGal(1-4)βGal disaccharide subunit, preferably the αGal(1-4)βGal (l-4)BGlcNAc trisaccharide subunit or αGal(1-4)βGal (l-4)BGlc trisaccharide subunit, that can also be used as therapeutic agents may be supplied wherein the disaccharide subunit or trisaccharide subunits or an oligomer saccharide containing it is coupled to a nontoxic carrier, such as a liposome, biocompatible polymer, or carrier analogous to the above-referenced SYNSORB™s.

Alternatively, the disaccharide or a larger moiety containing it as a subunit may be formulated in standard pharmaceutical compositions for administration to the patient. Typically, the patient will be afflicte with a diarrhetic condition, such as hemorrhagic colitis due to enterohemorrhagic E. coli infection, and the target SLT will be present in the intestinal tract. Thus, a suitable mode of administration is through oral administration or other means of direct application to the gastrointestinal tract. The correct dosage range will depend on the severity of the infection, the mode of administration, the mode of formulation, and the judgment of the attending practitioner.

As shown in Example 3, pharmaceutical formulations are available that survive the conditions of the digestive tract. The oligosaccharides responsible for toxin binding and coupled to the SYNSORBs through a spacer arm are resistant to the potentially degradative conditions found in the digestive tract. Thus, the ability of the derivatized affinity ligands is unimpaired by incubation under acidic conditions approximating those found in the stomach and in the presence of the degradative enzymes and higher pH conditions found in the small intestine. It is required for successful pharmaceutical compositions that they pass through the stomach and the intestines of prior to encountering the sites of infection. The SYNSORB compositions themselves can be used as pharmaceutical compositions since the solid support is physiologically innocuous. In the commercially available SYNSORB, the solid support is silica, a commonly used drying agent in such foods as dairy creamers. Thus, the the compositions tested, per se, are useful pharmaceutical compositions.

The effect of the compositions of the invention in neutralizing SLTs can be measured by comparing activity of SLT with and without treatment with the compositions. Activity of the SLTs can be assayed by taking advantage of the toxicity of these compounds to Vero cells. Vero cells (ATCC CCL81) can be obtained from the American-Type Culture Collection, Rockville, MD. These are maintained at 37°C/5% C02 in minimal essential medium with Earl's salts (MEM, Gibco BRL, Gaithersburg, MD) containing 3% fetal bovine serum (FBS) . Confluent Vero cell monolayers are disrupted using 0.25% tissue- culture grade trypsin and approximately 105 cells in 200 μl FBS-supplemented MEM are added to each well of a 96- well microtiter plate. The plates are incubated overnight at 37°C/5% C02-

The samples to be tested, and suitable controls are added to the various wells and the plates are incubated for 2-3 days at 37°C/5% C02. Cytotoxic effects are readily visible in successful candidate wells as compared to control wells. The results can be quantitated by aspirating the remaining liquid from each of the wells and fixing the Vero cells which remain viable with 95% methanol and staining with Geimsa stain. The results are recorded using a microtiter plate reader set at a wavelength of 620 n , as described by Samuel, J.E. , Infect Immun (1990) 5_8_:611-618 (supra). The absorbance data are plotted versus the logarithm of the dilution of the candidate test solution. The dilution of samples resulting in 50% destruction (CD5Q) of the monolayers is determined by extrapolation from the resulting Vero cell killing curves.

The following examples are intended to illustrate but not to limit the invention. _1 _

Example 1 SYNSORB - Verocytotoxicitv Neutralization Assays

E . coli strains 0157:H-(E32511) , which produce SLTII/SLTIIc and 026:H11(H19) which produces SLTI only, or strain C600(933W), which produces SLTII only, were grown overnight at 37°C on tryptic soy broth (Difco, Detroit, MI) agar plates. Polymyxin and lysozyme extracts were prepared as described previously [Karmali, M.A., et al., J Clin Microbiol (1985) 22.:614-619 and

Head, S., et al., Infect Immunol (1990) 58:1532-1537) 1.

The first neutralization assay was designed to test the ability of SYNSORBs to absorb SLT activity from the E . coli extracts. The assay involved incubating 1 m of the E. coli extracts for 30 min. at room temperature in 1.5 mL microcentrifuge tubes (Fisher) with 2 to 50 mg SYNSORB on an end-over-end rotator. The tubes were then removed from the apparatus and after the SYNSORB had settled to the bottom (a few seconds) , serial five-fold dilutions of the absorbed extracts were prepared in unsupplemented MEM. Twenty (20) μL of each dilution was added to the appropriate wells in 96 well microtiter plates containing Vero cells. Bacterial extracts to which no SYNSORB was added served as controls. Once cytotoxic effects became apparent (2 to 3 days in the incubator) the growth medium was aspirated from each of the wells and Vero cells which remained viable were fixe with 95% methanol and stained with Giemsa stain (Fisher) . The results were then recorded using a microtiter plate reader set at a wavelength of 620 nm as described previously [Samuel et al., Infect Immun. 5_8_:611-618 (1990)]. The absorbance data were then plotted versus the logarithm of the extract dilution. The dilution of the extracts resulting in 50% destruction (CD50) of the monolayers was determined by extrapolation from the resulting Vero cell killing curves. Individual experiments were always performed in duplicate and, unless otherwise indicated, repeated at least two times. The percentage of neutralization was computed from the equation: 100-(100[CD50 oligosaccharide SYNSORB-treated extracted + CD50 acetylated silyl-aminated (ASA) SYNSORB- treated extract]) . The non-parametric Mann-Whitney test using the two-tailed statistic T was employed to compute the significance level of difference between groups of independent observations [Altman, D.G., Practical statistics for medical research, 1st ed. New York, Chapman and Hall: 179-228 (1991)].

The second neutralization assay (co-incubation assay) was designed to test the ability of Pk trisaccharide SYNSORB to protect Vero cells from SLT activity over 3 days at 37°C. This assay involved incubating 180 μL of serial five-fold dilutions of polymyxin extracts in ethylene oxide-sterilized 1.5 mL microcentrifuge tubes each containing 2, 5 or 10 mg of Pk trisaccharide SYNSORB. After 1 h incubation with

SYNSORB, the entire contents of each microcentrifuge tube were added to Vero cells monolayers in microtiter plates prepared as described above. The microtiter plates prepared as described above. The microtiter plates were then incubated at 37°C for 3 days and the results of the experiment were recorded as described above (Figures 1 and 2) .

The foregoing determination was repeated using varying amounts of Pk-tri and various times of incubation, with the results shown in Figures 3A and 3B. As shown in Figure 3A, as little as 5 mg SYNSORB was capable of neutralizing the activity of the extracts of both E32511 and H19 strains; similarly, as shown in Figure 3B, only about 5 min incubation was required to achieve this result in either extract. Example 2 Iodinated SLTI Binding Assay Purified SLTI was iodinated in 12 x 75 mm acid washed glass culture tubes coated with 40 μg of lodo Gen (Pierce Chemical Co., Rockford, IL) . About 6 μg of purified SLTI was incubated for 1 min with 20 MBq 125-ι labeled sodium iodide in 100 μl PBS. The reaction mixture was passed through a glass wool-plugged Pasteur pipette into 200 μl PBS containing a solution of cystein (1 mg/ml) in PBS as described by Armstrong, G.D., et al.. Infect Immunol (1987) 55:1294-1299. After 1 min, 200 μl of PBS containing 1% BSA was added to the mixture and th iodinated SLTI was purified by passing the solution through a 1 cm x 30 cm Sephadex-G 25 gel filtration column with 0.1% BSA in PBS. The efficiency of the iodination reaction was determined by measuring the number of counts that were incorporated into trichloroacetic acid precipitated protein. Aliquots of the iodinated SLTI were stored at -90°C. The assays were performed in PBS containing

0.1% BSA to reduce nonspecific binding. 2 mg of the various SYNSORBs were incubated for 30 min on an end- over-end rotator with approximately 20,000 dpm of the iodinated SLTI prepared in Preparation B above (specific activity, 2.2 x 107 dp /μg, CD5Q in the Verocytotoxicity assay, 0.4 pg/ml) , in 0.5 ml PBS/BSA) . The SYNSORBs were then washed with 3 x 1 ml portions of PBS BSA to remove unbound counts. The derivatized SYNSORBs were counted in an LKB Rackgamma model 1270 Gamma Counter. The results are shown in Table 1.

Figure imgf000016_0001

The SLT bound to Pk-tri SYNSORB could be partially released using 0.1 M acetic acid, 6 M guanidine HC1, or by heating in boiling water bath for 30 min in 10% SDS. However, neither 0.5 M lactose, 0.5 M galactose, or 0.2 M EDTA could displace the bound SLTI (Figure 4) . Subsequent experiments showed that 2 mg of

Pk-tri neutralized approximately 90% of the activity in E. coli H19 (SLTI) but about 10 mg Pk-tri SYNSORB was required to neutralize the activity of the E. coli 32511 (SLTII/SLTIIc) or E. coli C600/933W (SLTII) to a similar extent (Figure 5) .

Example 3 Performance Under Digestive Tract Conditions

Pk Trisaccharide SYNSORB was incubated for the various times at 37°C in 0.01 M HC1 to simulate conditions in the stomach, then washed extensively in PBS to remove the HCl. The HCl-treated SYNSORB was then tested for SLTI and SLTII neutralizing activity in the Vero cytotoxicity assay as described hereinabove,and by Armstrong, G.D. et al . , J Infect Pis (1991) 164:1160- 1167.

Briefly, E. coli 026:H11 (SLTI) or 0157:H~ (SLTII) were grown overnight at 37°C on Tryptic Soy Agar (TSA) . The bacteria from 5 plates were harvested in 3.0 —1.5 _—

ml PBS containing 0.1 mg/ l Polymyxin B sulfate. The resulting suspension was then clarified by centrifugation.

Five mg of Pk trisaccharide SYNSORB was mixed with approximately 1 ml of polymyxin extract of the bacterial strains listed in the table below. Data represent the average of two independent determinations, each performed in duplicate. Values in brackets give th range for each value.

Table 2

Ability of HCl-Treated Pk Trisaccharide SYNSORB to

Neutralize SLT Activity in the Vero Cell Assay

HC1 Percent SLT Activity Neutralized

Figure imgf000017_0001

As shown in Table 2, incubation with HC1 does not appreciably diminish neutralizing activity. To simulate intestinal conditions, various SYNSORBs were incubated for 2 hours at 37°C in buffer or in rat intestinal sacs. The incubated SYNSORBs were assayed for neutralizing activity against SLTI and SLTII, generally as described above. Briefly, SYNSORBs recovered from the rat intestines were sonicated for 30 to 60 seconds in a Branson Model B-220 Ultrasonic Cleaner to disrupt clumps of aggregated material. The sonicated SYNSORBs were then washed 4 times with 5 ml of double distilled, deionized H20 and dried under vacuum. Control SYNSORBs were treated in a similar manner. The polymyxin- extract described above was diluted to 8 ml with PBS. Five mg of SYNSORB was added to 0.9 ml portions of the diluted polymyxin extract. These were then incubated at room temperature for 1 hour on an end-over-end rotator. The resulting supernatant solutions were analyzed for SLTI or SLTII activity. Percent neutralization was calculated relative to the CD5Qs of polymyxin extracts incubated with the ASA control SYNSORB. These results are shown in Tables 3-6.

Table 3 shows the results of neutralization of SLTI activity by SYNSORB incubated in buffer; Table 4 shows neutralization of SLTI activity by SYNSORB incubated in intestinal sacs; Table 5 shows neutralization of SLTII activity by SYNSORB incubated in buffer; and Table 6 shows neutralization of SLTII activity by SYNSORB incubated in rat intestinal sacs.

Table 3

Percent SLTI SYNSORB Activity Neutralizeda

ASA SYNSORBb 0 Pχ Disaccharide 93 (88 - 97)

P^ Trisaccharide 98 (96 - 100)

a. Average of duplicate determinations. Range in brackets. b. Control SYNSORB containing only the acetylated silylaminated (ASA) hydrophobic 8- methoxycarbonyloctyl linkage arm. Table 4

Percent SLTI

SYNSORB Activity Neutralizeda

ASA SYNSORB 0

Ε> _ Disaccharide 82 ± 6

P^ Trisaccharide 98 ± 2

Average of triplicate determinations ± standar deviation of the mean.

Table 5 n SYNSORB Percent SLTII Activity Neutralizeda

ASA SYNSORB 0

P- Disaccharide 84 P^ Trisaccharide 98

a. Results of one determination.

Table 6

Percent SLTII

SYNSORB Activity Neutralizeda

ASA SYNSORB

P- Disaccharide 65 ± 9' P^ Trisaccharide 96' a"! Average of triplicate determinations ± standard deviation of the mean, b. Results of one determination. As shown above, neither the conditions of the stomach nor those of the small intestine are detrimental to the activity of the derivatized SYNSORBs in neutralizing SLTI or SLTII.

Claims

Claims
1. A method to rapidly bind and remove shiga- like toxins (SLT) from a biological sample which method comprises contacting said sample with an affinity suppor having an affinity ligand comprising the disaccharide subunit αGal(1-4)βGal, under conditions wherein said SLT is adsorbed to the affinity support; separating the support containing the adsorbed SLT from the sample.
2. The method of claim 1 wherein the αGal (1-4)βGal subunit is at the non-reducing terminus of an oligosaccharide.
3. The method of claim 2 wherein the oligosaccharide is the αGal(1-4)βGal(1-4)BGlcNAc trisaccharide or the αGal(1-4)βGal(1-4)βGlc trisaccharide.
4. The method of claim 3 wherein said affinity ligand is bound to said affinity support through a spacer arm.
5. The method of claim 4 wherein said spacer arm corresponds to 8-methoxycarbonyloctyl (MCO) .
6. A method to rapidly diagnose enteric infection mediated by SLT which method comprises contacting a sample from a subject suspected of harboring such an infection with a composition which comprises the disaccharide subunit αGal(1-4)βGal under conditions wherein any SLT contained in said sample binds said composition; and detecting the SLT bound to said composition as an indication of said infection.
7. The method of claim 6 wherein said composition comprises the αGal(1-4)βGal(1-4JβGlcNAc or αGal(1-4)βGal(1-4)BGlc trisaccharides.
8. The method of claim 6 wherein said detecting of bound SLT comprises contacting said bound SLT with a radioactive, biotinylated or similarly fluorescently labeled monoclonal or polyclonal antibody.
9. The method of claim 7 wherein said detecting of bound SLT comprises contacting said bound SLT with a radioactive, biotinylated or similarly fluorescently labeled monoclonal or polyclonal antibody.
10. A method to prevent or ameliorate an enteric infection mediated by SLT in a subject which method comprises administering to a subject in need of such treatment an effective amount of a composition comprising the αGal(1-4)βGal subunit.
11. The method of claim 10 wherein said composition comprises the αGal(1-4)βGal(1-4JβGlcNAc subunit or αGal(1-4)βGal(1- )βGlc subunit.
12. The method of claim 10 wherein said subunit is coupled to a pharmaceutically acceptable support.
13. The method of claim 12 wherein said pharmaceutically acceptable support comprises a matrix.
14. The method of claim 12 wherein said subunit is coupled to said pharmaceutically acceptable support through a spacer arm.
15. The method of claim 14 wherein said spacer arm corresponds to MCO.
16. A pharmaceutical composition useful in treating enteric infections mediated by SLT which composition comprises as active ingredient a moiety comprising the disaccharide subunit αGal(1-4)βGal in admixture with a pharmaceutically acceptable excipient.
17. The pharmaceutical composition of claim 16 wherein said moiety comprises the trisaccharide subunit αGal(1-4)βGal(1-4)BGlcNAc or the trisaccharide subunit αGal(1-4)βGal(1-4)BGlC.
18. The composition of claim 17 wherein said active ingredient is coupled to a pharmaceutically acceptable support.
19. The composition of claim 18 wherein said pharmaceutically acceptable support comprises a silica matrix.
20. The composition of claim 18 wherein said coupling is through a spacer arm.
21. The composition claim 20 wherein said spacer arm corresponds to MCO.
PCT/US1992/008929 1991-10-18 1992-10-19 Diagnosis and treatment of bacterial dysentery WO1993008209A1 (en)

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US07/778,732 1991-10-18

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AT92922817T AT198991T (en) 1991-10-18 1992-10-19 Treatment of bacterial dysentery
EP92922817A EP0610356B1 (en) 1991-10-18 1992-10-19 Treatment of bacterial dysentery
CA002121604A CA2121604C (en) 1991-10-18 1992-10-19 Diagnosis and treatment of bacterial dysentery
DK92922817T DK0610356T3 (en) 1991-10-18 1992-10-19 Treatment of bacterial dysentery
DE69231675T DE69231675T2 (en) 1991-10-18 1992-10-19 Treatment of bacterial dysentery
GR20010400515T GR3035665T3 (en) 1991-10-18 2001-03-29 Diagnosis and treatment of bacterial dysentery.

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EP (2) EP1018342A3 (en)
AT (1) AT198991T (en)
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DE (2) DE1018342T1 (en)
DK (1) DK0610356T3 (en)
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US6121242A (en) * 1996-06-21 2000-09-19 Synsorb Biotech, Inc. Treatment of bacterial dysentery
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US5849714A (en) * 1996-06-21 1998-12-15 Synsorb Biotech Inc. Treatment of bacterial dysentery
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US6013634A (en) * 1996-11-08 2000-01-11 Synsorb Biotech, Inc. Solid support matrices containing a toxin binding oligosaccharide
WO1998030572A1 (en) * 1997-01-10 1998-07-16 Synsorb Biotech, Inc. PROCESSES FOR THE PREPARATION OF αGal(1→4)βGal(1→4)Glc-OR
US5922848A (en) * 1997-05-30 1999-07-13 Sybsorb Biotech, Inc. Method of recovering shiga-like toxins and vaccines comprising inactivated shiga-like toxin
WO1998054215A1 (en) * 1997-05-30 1998-12-03 Synsorb Biotech Inc. Method of recovering shiga-like toxins and vaccines comprising inactivated shiga-like toxin
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US6224891B1 (en) 1998-02-23 2001-05-01 Synsorb Biotech, Inc. Compounds and methods for the treatment of bacterial dysentery using antibiotics and toxin binding oligosaccharide compositions
US5962423A (en) * 1998-08-07 1999-10-05 The Governors Of The University Of Alberta Treatment of bacterial dysentery
US6310043B1 (en) 1998-08-07 2001-10-30 Governors Of The University Of Alberta Treatment of bacterial infections

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GR3035665T3 (en) 2001-06-29
US5955449A (en) 1999-09-21
EP1018342A2 (en) 2000-07-12
ES2154265T3 (en) 2001-04-01
DE69231675D1 (en) 2001-03-08
EP0610356B1 (en) 2001-01-31
EP1018342A3 (en) 2001-04-11
US5620858A (en) 1997-04-15
CA2121604A1 (en) 1993-04-29
DK0610356T3 (en) 2001-02-19
DE1018342T1 (en) 2001-01-11
DE69231675T2 (en) 2001-06-28
ES2150888T1 (en) 2000-12-16
EP0610356A1 (en) 1994-08-17
CA2121604C (en) 2002-01-15
EP0610356A4 (en) 1995-03-29
DK610356T3 (en)

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