CA2243118A1 - Use of oligosaccharides for neutralising e. coli toxins - Google Patents

Abstract

This invention is directed to the surprising and unexpected discovery that the clinical incidence of HUS arising from enterohemorrhagic E. coli infection is significantly reduced by the time critical administration of a pharmaceutical composition comprising a pharmaceutically inert affinity support comprising an .alpha.Gal(14).beta.Gal subunit which subunit binds the SLT. Specifically, it has been found that the clinical incidence of HUS arising from enterohemorrhagic E. coli infection is reduced when this pharmaceutical composition is administered within 3 days of presentation of the infection.
Contrarily, administration of this pharmaceutical composition after this time frame or when organs other than the intestine are involved in the infection, substantially reduces the ability of this composition to reduce the incidence of HUS.

Description

USE OF OLIGOSACCHARIDES FOR NEUTRALISING E. COLl TOXINS

Field of the Invention This invention relates to the treatment of fli~rrhP~ caused by pathogenic E. coli infection. More specifically, this invention relates to methods for the neutralization of shiga-like toxins (SLT) associated with enteric E. coli infection which methods inhibit progression of this infection into hemolytic uremic syndrome (HUS).

References The following references are cited in the application as numbers in brackets ([]) at the relevant portion of the application.

1. K~ li, M.A. et al., J. Clin. Microbiol. 22:614-619 (1985).

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

3. Samuel et al., Infect Immunol. 58:611 -618 (1990).

4. Altman,D.G. PracticalStatisticsforMedicalResearch 1sted.
New York, Chapman and Hall: 179-228 (1991).

5. Calderwood, et al., Proc. Natl. Acad. Sci. (USA), 84:4364-4368 (1987)

6. Jackson, et al., Microb. Pathog., 2:147-153 (1987)

7. Strockbine, et al., J. Bacteriol., 170: 1116-1122 (1988)

8. Robson, et al., J. Pediatr., 117:675-676 (1990)

9. Cimolai, et al., J. Pediatr., 117:676 (1990) CA 02243ll8 l998-07-l5

10. Annstrong, et al., International Patent Application Publication No. WO 93/08209, for "DLAG~O~IS Al~ TREATMENT OF
BACTERIAL DYSENTERY, published 29 April 1993

11. Lemieux, R.U., et al., "The properties of a 'synthetic' antigen related to the blood-group Lewis A", J. Am. Chem. Soc., 97:4076-83 (1975).

12. Lemieux, R.U., et al., "Glycoside-Ether-Ester Compounds", U.S. Patent No. 4,137,401, issued January 30, 1979.

13. Lemieux, R.U., et al., "Artificial Oligosaccharide Antigenic Determin~ntc", U.S. Patent No. 4,238,473, issued December 9, 1980.

14. Lemieux, R.U., et al., "Synthesis of 2-Amino-2-Deoxyglycoses and 2-Amino-2-Deoxyglycosides from glycals", U.S. Patent No.
4,362,720, issued December 7, 1982.

15. Cox, D., et al. "A New Synthesis of 4-0-o~-D-Galactopyranosyl-D-Galacto-Pyranose", Carbohy. Res., 62: 245-252 (1978).

16. Dahmén, J., et al., "Synthesis of space a~n, lipid, and ethyl glycosides of the trisaccharide portion [c~-D-Gal-(1-4)-~-D-Gal(1-4)-~-D-Glc] of the blood group pk antigen: preparation of neoglycoproteins", ~arbohydrate Kesearch, 127: 15-25 (1984~.

17. Garegg, P. J., et al., "A Synthesis of 8-Methoxycarbonyloct-1-yl O-(x-D-Galactopyranosyl-(1-3)-0-,B-D-Galactopyranosyl-(1-4)-2-Acet~mi~o-2-Deoxy-~-D-Glucopyranoside", Carbohy. Res., 136:
207-213 (1985).

18. Garegg, P. J., et al., "Synthesis of 6- and 6' -deoxy derivatives of methyl 4-0-(x-D-galactopyranosyl-,~-D-galactopyranoside for studies of inhibition of pyelonephritogenic fimbriated E. coli adhesion to urinary epithelium-cell surfaces", Carbohy. ~es., 137: 270-275 (1985).

19. Jacquinet, J. C., et al., "Synthesis of Blood-group Substances, Part 11. Synthesis of the Trisaccharide O-~x-D-Galactopyranosyl-(1 -3)-0-,~-D-galactopyranosyl-(1 -4)-2-acetz-mi~lo-2-deoxy-D-glucopyranose", J.C.S~. Perkin, I: 326-330 ~1981).
3~ 20. Koilce, K., et al., "Total Synthesis of Globotriaosyl-E and Z-Ceramides and Isoglobotriaosyl-E-Ceramide," Carbohydr. ~es., 163: 189-208 (1987).

CA 02243118 1998-07-l~

21. Sch~--h~ , R., et al., "Tumor-Associated Antigen Synthesis:
Synthesis of the Gal-a-(1-3)-Gal-~-(14)-GlcNAc Epitope. A
specific Dete~min~nt for Metastatic Progression?", Liebigs Ann.
Chem., 607-614 (1991).
22. Ratcliffe, R.M., et al., "Sialic Acid Glycosides, Antigens, ~mmlmoadsorbents, and Methods for Their Preparation", U.S.
Patent No. 5,079,353, issued January 7, 1992.
23. Okamoto, K., et al., "Glycosidation of Sialic Acid,"
Tetrahedron, 47: 5835-5857 (1990).
24. Abbas, S.A., et al., "Tumor-Associated Oligosaccharides I:
Synthesis of Sialyl-LewisA Antigenic Determin~nt", Sialic Acids, Proc. Japan-German Symp. Berlin 22-23 (1988).
25. Paulsen, "Advances in Selective Chemical Syntheses of Complex Oligosaccharides", Angew. Chem. Int. Ed. Eng., 21:155-173 (1982).
26. Schmidt, "New Methods for the Synthesis of Glycosides and Oligosaccharides - Are There Alternatives to the Koenigs-Knorr Method?", Angew. Chenz. Int. Ed. Eng., 25:212-235 (1986).
27. Fugedi, P., et al., "Thioglycosides as Glycosylating Agents in Oligosaccharide Synthesis", Glycoconjugate J., 4:97-108 (1987).
28. Kameyama, A., et al., "Total synthesis of sialyl Lewis X", Carbohydrate Res., 209: c1-c4 (1991).
29. Ekborg, G., et al., "Synthesis of Three Disaccharides for the Preparation of Tmmlln~gens bearing Immunodeterminzlnts Known to Occur on Glycoproteins", Carbohydrate Research, 110: 55-67 (1982).
30. Dahmén, J., et al., "2-Bromoethyl glycosides: applications in the synthesis of spacer-arm glycosides", ~arbohydrate Research, 118: 292-301 (1983).
31. Rana, S. S., et al., "Synthesis of Phenyl 2-Acet~mi-lo-2-Deoxy-3-O-o~-L-Fucopyranosyl-~-D-Glucopyranoside and Related Compounds", ~arbohydrate Research, 91: 149-157 (1981).
32. Amvam-Zollo, P., et al., "Streptococcus pneumoniae Type XIV
Polysaccharide: Synthesis of a Repeating Branched Tetr~c~rrll~ride with Dioxa-Type Spacer-Arms", Carbohydrate Research, 150:199-212 (1986).
33. Paulsen, H., "Synthese von oligosaccharid~ termin~ntPn mit amid-spacer vom typ des T-antigens", Carbohydr. Res., 104:195-219 (1982).
34. Chernyak, A. Y., et al., "A New Type of Carbohydrate-Cont~inin~ Synthetic Antigen: Synthesis of Carbohydrate-Cont~ining Polyacrylamide Copolymers having the Speci~lcity of 0:3 and 0:4 Factors of Salmonella", Carbohydrate Research, 128:269-282 (1984).
35. Fernandez-Santana, V., et al., "Glycosides of Monoallyl Diethylene Glycol. A New type of Spacer group for Synthetic Oligosaccharides", J. Carbohydrate Chemistry, 8(3):531-537 (1989).
1536. Lee, R.T., et al., "Synthesis of 3-(2-Aminoethylthio) PropylGlycosides ", Carbohydrate Research, 37: 193-201 (1974).
37. Gannon, et al., J. Gen. Microbiol., 136: 1125-1135 (1990) 38. Weinstein, et al., J. Bacteriol., 170:4223-4230 (1988) 39. Ito, et al., Microb. Pathog., 8:47-60 (1990) 2040. ~ead, et al., FEMSMicrobiol. Lett., 51:211-216 (1988) 41. Schmitt, et al., Infect. Immun., 59:1065-1073 (1991) 42. Scotland, et al., Lancet, ii:885-886 (1991) 43. Oku, et al., Microb. Pathog., 6:113-122 (1989) 44. Boyd, et al., Nephron, 51:207-210 (1989) 2545. DeGrandis, et al., J. Biol. Chem., 264: 12520-12525 (1989) 46. Waddell, et al., Biochem. Biophys. Res. Comm., 152:674-679 (1988) 47. Lingwood, et al., J. Biol. Chem., 262:8834-8839 (1987) 48. Waddell, et al., Proc. Natl. Acad. Sci. (IISA), 87:7898-7901 ~1990) CA 02243ll8 l998-07-l~

4~. Cohen, et al., J. Biol. Chem., 262:17088-17091 (1987) 50. Jacewicz, et al., J. Exp. Med., 163:1391-1404 (1986) 51. Lindberg, et al., J. Biol. Chem., 262:1779-1785 (1987) 52. Armstrong, G.D. et al., Infect. Immunol., 55:1294-1299 (1987) 553. Armstrong, G.D. et al., J. Infecl. Dis. 164:1160-1167 (1991) The disclosure of the above publications, patents and patent application are herein incorporated by reference in their entirety to the same extent as if the language of each individual publication, patent and patent application were specifically and individually included herein.
State of the 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 produce enterotoxins that are closely related to the shiga toxin associatedwith 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) [5,6,7].

Additional members of the SLT family have subsequently been isolated that can be distinguished serologically, on the basis of gene sequence, or on host specificity [37-43]. Various types of SLTII have been described and have been ~ ign~d various designations depending on the strain of E. coli from which they are isolated and the host affected. Thus variants have been designated SLTII; vtx2ha; SLTIIvh; vtx2hb; SLTIIc; SLTIIvp and so forth.

All of the SLT's are ml-ltimt-ric proteins composed of an enzymatic (A) CA 02243118 1998-07-1~

subunit and multiple (B) subunits. The B oligomer is the binding portion of the toxin that allows it to bind to host cell receptors. The B subunits of SLTI, SLTII and SLTIIvh recognize host cell globoseries glycolipid receptors cont,7inin~ at m7inimllm the disaccharide subunit ~Gal(1-4),~Gal at the non-S re-lucing terminus; SLTIIvp has been shown to bind to the receptors cont~ining this subunit but not n~ces.c7rily to the non-reducing end ~2, 44-51]. The A
subunit has an enzymatic activity (N-glycosidase~ that depurinates 28S
ribosomal RNA in m~mm,71i~n cells. This enz,ymatic activity abolishes the ability of the toxin-infected cell to perforrn protein synthesis.

The site for SLT action is endothelial cells found in the kidneys and mesenteric v~culslhlre, and SLTs may cause damage that can result in renal failure and hemoglobin in the urine. SLTs are the causative agent in the hemolytic-uremic syndrome. SLTs may also be partially involved in the pathogenesis of hemorrhagic colitis (bloody diarrhea).

The hemolytic uremic syndrome (HUS) is the leading cause of acute renal failure in childhood and affects approximately 7 - 10% of children in the 5 - 10 days following infection with E. coli 0157:H7 and other verotoxin/shiga-like toxin producing E. coli (VTEC).

Recent attention regarding such pathogenic E. coli has focussed on the known correlation between E. coli c~-nt~min,7tion of certain meats and subsequent infection in hum~n~ after ingestion of this meat. The problem is particularly acute with regard to hamburger meat where ingestion of undercooked meat has been found to be the causative factor in the infection.
This problem is compounded by the fact that the rapid progression of the pathogenic E. coli infection into HUS via the expression of the SLTs suggests the hypothesis that initial colonization of the intestinal tract is followed by endothelial injury and subsequent kidney involvement via the transmembrane delivery of the SLT toxin into the blood stream of the infected individual.

CA 02243ll8 l998-07-l~

As a complicating factor, the art suggests against the use of antibiotics in the treatment of enterohemorrhagic E. coli infection [8]. The use of antimotility drugs also appears to be count~ oductive [9].

One reported method for the treatment of such infections is to orally ~ mini~t~r a ph~rm~elltic~lly inert affinity support comprising an ~Gal(1~4),~Gal subunit to the infected patient [10]. This support passes into the int~stin~l tract of the patient whereupon the lxGal(1~4)~'Gal subunit binds the Shiga-like toxin. Subsequently, the toxin bound to this solid support is elimin~tP~I from the body as part of the stool. This procedure is one of the first, if only, reported methods for removing such toxins from the body which, in turn, inhibits manire~Lalion of the conditions associated with toxin ~c.c. lm~ 3tion.

Notwith~f~n-ling the signific~nt advances made by this reported method, further advances in the treatment of enterohemorrhagic E. coli infections are needed in order to reduce the occurrence of HUS and the high mortality levels associated therewith.

SUMMARY OF THE INVENTION
This invention is directed to the surprising and unexpected discovery that the clinical incidence of HUS arising from enterohemorrhagic E. coli infection is signifi~ntly reduced by the time critical ~fimini~tration of a ph~rm~celltical composition comprising a pharm~rel-tiç~lly inert affinity support comprising an o~Gal(1~4)~Gal subunit which subunit binds the SLT. Specifically, it has been found that the clinical incidence of HUS arising from enterohemorrhagic E. coli infection is reduced when this pharmace ltir~l composition is a~1minictered within 3 days of presentation of the infection. Contrarily, ~mini.~tration of this pharmaceutical composition after this time frame or when organs other than the intestine are involved in the infection, substantially reduces the ability of this composition to reduce the incidence of HUS.

CA 02243118 1998-07-1~
W 0971~9431 PCT/C~97100436 Accordingly, in one of its method aspects, this invention is directed to a method for inhibiting the development of hemolytic uremic syndrome in a patient arising from enterohemorrhagic E. coli infection mP~ te~l by shiga-like toxins which method comprises ~lmini~tering to said patient an effective S arnount of a ph~ cellt~ 1 composition co,-".li~hlg a ph~ e~ltir~lly inertaffinity support comprising an o~Gal(1 4),BGal subunit which is bound to said support through a non-peptidyl linker arm, wherein said subunit binds SLT
toxin and wherein this pharmaceutical composition is ~lmini~tered within about 3 days of presentation of the infection. In a ~lc;Ç~lled embodiment the ph~ relltic~l composition is ~lmini~tered to the patient prior to organ involvement other than involvement of the intestin-?.

For the purposes of this invention, the L"esenl~tion of the infection is deterrnined after the identifil~tion of at least one condition associated with an SLT m~ tçcl E. coli infection. Such conditions include, for example, patients with diarrhea and one of the following: abdominal ~;,anlpillg, blood in the stool, rectal prolapse, detection of a verotoxin-producing E. coli in the patient's stool;
ingestion of food suspected of cont~ining a verotoxin-producing E. coli; or close contact with an individual known to have an SLT me~ te~l infection.
Preferably, the presentation of the infection is manifested by bloody diarrhea.
In a particularly preferred embodiment, the initial clinical evaluation that theindividual is afflicted with an SLT m~Ai~teri E. coli infection is con~lrmPfl via diagnostic evaluation of the stool. One diagnostic tool commercially available for detecting SLT m~ te-l E. coli infection is sold by Meridian Diagnostic, Inc., Cincinnati, Ohio, USA 4~244 under the name Premier EHEC.

In a further aspect, the invention provides a method for inhibiting the development of hemolytic uremic syndrome in a patient presenting an enterohemorrhagic E. coli infection me~ te-l by shiga-like toxin, which method comprises ~1mini~ ring to the patient an effective amount of a ph~ ceutical composition comprising a pharm~reutiç~lly inert affinity support comprising an CA 02243ll8 l998-07-l~
W O 97/49431 PCT/CAg7/00436 oligosaccharide selected from the group consisting of o~Gal(1~4),~Gal, o~Gal(1~4),l3Gal(l ~4),BGlcNAc and o~Gal(1~4)~Gal(l~)~Glc which oligosaccharide is bound to the support through a non-peptidyl linker arm, wherein this pharm~ellti~ ~l composition is ~ ini~tered within about 3 days of S presentation of the infection. In a l)refell~d embodiment the I)h~ ellti~
composition is ~lmini~tered to the patient prior to organ involvement other thanthe intestin~.

BRIEF DESCRIPIION OF THE DRAWINGS
Figure lA and B demonstrate the toxicity of bacterial extracts obtained using polymyxin-B with respect to their ability to kill Vero cells in the presence and absence of various SYNSORBs.

Figure 2A and B demonstrate the toxicity of bacterial extracts obtained using Iysozyme with respect to their ability to kill Vero cells in the presence and absence of various SYNSORBs.

Figure 3A demonstrates that as little as 10 mg of Pk trisaccharide SYNSORB removes >90% of SLT toxins from bacterial extracts.

Figure 3B demonstrates that the binding of the SLT toxins occurred within 5 minutes of mixing extracts with the Pk SYNSORB.

Figure 4 demonstrates that difficulty in eluting the bound Il~5 labelled SLTI from various SYNSORBs lltili7.ing a variety of eluants.

Figure 5 demonstrates that >90% S~TI, 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.

DESCRIPTION OF T~E PREFFRR~,l- EMBODl~[ENTS
As noted above, this invention relates to methods for the neutralization of shiga-like toxins (SLT) associated with enteric ~. coli infection which methods inhibit progression of this infection into hemolytic uremic syndrome (HUS). However, prior to ~ c~.C.cin~ this invention in further detail, the following terms will first be defined:

A. Definitions As used herein the following terrns have the following mt~nings:

The term "shiga-like toxin" or "SLT" or "verotoxin" refers to a group of toxins produced by enterohemorrhagic E. coli that resemble the Shigella-produced shiga toxins as is cornmonly understood in the art. These toxins comprise an enzym~ti~ y active A subunit and a multimeric receptor binding B
subunit. Such SLTs include SLTI and the various grouped toxins (le~ign~ted in the art as SLTII.

Rapid tight binding of SLTs to Pl disaccharide, Pl trisaccharide, or Pl.
trisaccharide is demonstrated by the verocytotoxicity neutralization assays contained herein.

The term "organ involvement" refers to clinically defined organ involvement mt~ t~1 by SLTs which correlates to the natural progression of the disease. Organs other than the intestines include, by way of example, the kidney, heart, elements of t~e central nervous system ~"CNS") ~i.e., brain, spinal cord, etc.), liver, and the like. Conventional blood chemistry tests can evaluate liver, heart, and kidney involvement whereas clinical symptoms including dementia, convulsions, disorientation, etc. are employed to determine CNS involvement.

"Hemolytic urernic syndrome" is considered to be present if there is .~
-CA 02243118 1998-07-l~
W O 97/49431 PCT/CA97/0~436 renal inJury and either hemolysis or thrombocytopenia. Renal injury requires an elevation in the serum cre~tinin~ concentration ( > 50 ~umol/L for those lessthan 5 years, or > 60 ,umol/L for those 5-6 years) or a difference in the recorded cre~tinin~ values during the acute phase of the illness of more than 50% or at least 10 red blood cells per high power field on urine microscopy.
Hemolysis is judged to be present if the hemoglobin concentration is C 105g/L
or if there are red blood cell fragments on the smear or if a red blood cell transfusion was ~(lmini~tf~red before the hemoglobin dropped to c 105g/L.
Thrombocytopenia is defined as a platelet concentration of less than 150 X
109/L.

The term "biocompatible" refers to chemical inertness with respect to animal or human tissues or body fluids. Biocompatible materials are non-se~ .i "g .

The term "compatible linker arm" refers to a moiety which serves to space the oligosaccharide structure from the biocompatible solid support and which is bifunctional wherein one functional group is capable of covalently binding to a reciprocal functional group of the support and the other functionalgroup is capable of binding to a reciprocal functional group of the oligosaccharide structure. (~ompatible linker arms preferred in the present invention are non-peptidyl linker arms. That is to say that the linker arms do not employ a peptide group to link the oligosaccharide structure to the solid support.

The term "solid support" refers to an inert~ solid material to which the oligosaccharide sequences is bound via a compatible linker arm. Where use is in vivo, the solid support will be biocompatible.

The solid supports to which the oligosaccharide structures of the present invention are bound may be in the form of particles. A large variety of CA 02243ll8 l998-07-l~
W O 97t49431 PCT/CA97/00436 biocompatible solid support materials are known in the art. Examples thereof are silica, synthetic .~ilic~tes such as porous glass, biogenic .~ilir:ltes such as diatomaceous earth, silicate-cont~ining minerals such as kaolinite, and synthetic polymers such as poly~,Ly~ e, polypropylene, and polysaccharides. So1id supports made of inorganic materials are preferred. Preferably the solid supports have a particle size of from about l0 to 500 microns for in vivo use.
In particular, particle sizes of l00 to 200 microns are preferred.

The terrn "SYNSORB" refers to synthetic 8-methoxycarbonyloctyl oligosaccharide structures covalently coupled to CHROMOSORB pTM (Manville 10 ~ Corp., Denver, C~olorado) Ill], which is a derivatized silica particle.

Synthetic oligosaccharide sequences covalently attached to a biocr~mp~tihle solid support, e.g., CHROMOSORB pTM (SYNSORB) may be used to bind SLT toxins or verotoxins. These compositions are useful to prevent HUS and associated conditions. SYNSORB is particularly l,lefelled for these compositions because it is non-toxic and resistant to mechanical and chemical decomposition. SYNSORBs have been found to pass unaffected through the rat gastrointestinal tract. They were found to be eiimin~t~i completely and rapidly (99% elimin~t~cl in 72 hours) following oral ~,rimini~tration. Additionally, the high density of oligosaccharide moieties on SYNSORB is particularly useful for binding verotoxin.

For the purpose of this application, all sugars are referenced using conventional three letter nomenclature. All sugars are ~nm~fl to be in the D-form unless otherwise noted, except for fucose, which is in the L-form.
Further, all sugars are in the pyranose form.

T.inking moieties that are used in indirect bonding are preferably organic bifunctional molecules of appropriate length (at least one carbon atom) which serve simply to distance the oligosaccharide structure from the surface of the CA 02243118 1998-07-l~

solid support.

The compositions of this invention are preferably represented by the formula:
(OLIGOSACCHARIDE-Y-R)~,- SOLID SUPPORT
S where OLIGOSACCHARIDE comprises the o~Gal(1~4)~Gal subunit and contains at least two 2 sugar units and preferably no more than 6 sugar units which oligosaccharide binds to the shiga-like toxin, Y is oxygen, sulfur or nitrogen, R is an aglycon linking arm of at least 1 carbon atom, SOLID
SUPPORT is as defined above, and n is greater than or e~ual to 1. Preferred aglycons are from 1 to about 10 carbon atoms. Oligosaccharide sequences cont~ining about 2 to 10 saccharide units may be used. Sequences with about 2 to 3 saccharide units are preferred. Preferably, n is such that the composition contains about 0.25 to 2.50 micromoles oligosaccharide per gram of composition.

Numerous aglycon linking arms are known in the art. For example, a linking arm comprising a para-nitrophenyl group (i.e., -OC6H4pNO2) has been disclosed [29]. At the applv~liate time during synthesis, the nitro group is reduced to an amino group which can be protected as N-trifluoroacet~mido.
Prior to coupIing to a support, the trifluoro~cet~mido group is removed thereby llnm~king the amino group.

A linking arm cont~ining sulfur has been disclosed [30]. Specifically, the linking arm is derived from a 2-bromoethyl group which, in a substitution reaction with thionucleophiles, has been shown to lead to linking arms possessing a variety of terminal functional groups such as -OCH2CH2SCH2CO2CH3 and -OCH2CH2SC6H4-pNH~. These terminal ffinctional groups permit reaction to complementary functional groups on the solid support, thereby forming a covalent linkage to the solid support. Such reactions are well known in the art.

W O 97/49431 PCT/~ 97/00436 A 6-trifluo~ lo-hexyl linking arm (-O-(CH2)~NHCOCF3) has been disclosed [31] in which the trifluoro~c e~ O ~L)r~te~ g group can be removed, llnm~Qlrin~ the ~ lal ~ amino group used for coupling.

Other exemp1ifir~t;nne of known linking arms include the 7-mPth~cycarbonyl-3,6,dioxaheptyl linking arm t32]
(-OCH2-CH2)20CH2CO2CH3); the 2-(4-met'noxycall,onyll~u~ncall,oxamido)ethyl t33~ (-OCH2CH2NHC(O)(CH2)4CO2CH3); the allyl linking arm r34]
(-OCH2CH = CH2) which, by radical co-polylllcl ~tion with an a~LupliaLe monomer, leads to co-polymers; other allyl linking arms [35] are known (-O(CH2CH2O)2CH2CH=CH2). Additionally, allyl linking arms can be derivatized in the presence of 2-aminoeth~n~hiol [36] to provide for a linking arm -OCH2CH2CH2SCH2CH2NH2. Other suitable linking arms have also been disclosed [12-14, 16, 171-Preferably, the aglycon linking arm is a hydrophobic group and most preferably, the aglycon linking arm is a hydrophobic group selecte~1 from the group consisting of-(CH2)8C(O)-. -(CH2)5OCH2CH2CH2- and -(CH2)8CH2O-.
Non-peptidyl linking arms are p~c~fell~d for use as the c~ palil)le linking armsof the present invention.

The use of glycopeptides is not desirable because glycopeptides contain several, often different, oligos~e~h~ri-les linked to the same protein.
Glycopeptides are also difficult to obtain in large amounts and require expensive and tedious purification. Likewise, the use of BSA or HSA
conjugates is not desirable, for example, due to questionable stability in the gastroi..~ l tract when given orally.

2~ The compositions useful in the conduct of the methods of the invention comprise the aGal(1-4),BGal ~1ic~rrh~ride subunit which subunit can be used alone or in co~lju~ ion with a higher oligosaccharide, e.g., the CA 02243118 1998-07-1~

~Gal(1-4)~Gal~1-4~GlcNAc tri~cchzlride or the ~Gal(1-4)~Gal(1-4),~Glc trisaccharide. The ~xGal(1-4)~BGal disaccharide subunit is preferably found at the non-reducing terminus of an oligos~l~ch~ride.

The oligosaccharide is coupled to a solid support or coupled directly, preferably through a linking arm such as that described by Lemieux et al. [11].
The di and trisaccharide units may also be coupled directly to ph~ cellti~lly acceptable carriers or constitute a portion of an oligosaccharide coupled to such carriers.

B. Synthesis Chemical methods for the synthesis of oligosaccharide structures can be accomplished by methods known in the art. These materials are generally assembled using suitably protected individual monosaccharides.

The specific methods employed are generally adapted and optimized for each individual structure to be synthesized. In general, the chemical synthesis of all or part of the oligosaccharide glycosides first involves formation of a glycosidic linkage on the anomeric carbon atom of the reducing sugar or monosaccharide. Specifically, an approp-iately protected form of a naturally occurring or of a chemically modified saccharide structure (the glycosyl donor) is selectively modified at the anomeric center of the reducing unit so as to introduce a leaving group comprising halides, trichloroacetimic1~t~, acetyl, thioglycoside, etc. The donor is then reacted under catalytic conditions well known in the art with an aglycon or an ap~ ,pliate form of a carbohydrate acceptor which possesses one free hydroxyl group at the position where the glycosidic linkage is to be established. A large variety of aglycon moieties areknown in the art and can be attached with the proper configuration to the - anomeric center of the reducing unit.

Appropriate use of compatible blocking groups, well known in the art of CA 02243118 1998-07-1~
W 097149431 PCTIC~97/00436 ~ 16 carbohydrate synthesis, will allow selective modification of the synth~i7~1 structures or the further ~tt~r.hment of additional sugar units or sugar blocks to the acceptor structures.

After formation of the glycosidic linkage, the saccharide glycoside can S be used to effect coupling of additional s~c ch~ride unit(s) or çh-olnir~lly modified at selected positions or, after conventional deprotection, used in an enzymatic synthesis. In general, chemical coupling of a naturally occurring or chemically modified saccharide unit to the saccharide glycoside is accomplished by employing established chemistry well documented in the literature [12-28~.

The oligosaccharide structure(s) is covalently bound or noncovalently (passively) adsorbed onto the solid support. The covalent bonding may be via reaction between functional groups on the support and the compatible linker arm of the oligosaccharide structure.

The inert affinity supports comprising an ~Gal(1 4),BGal subunit which is bound to said support through a non-peptidyl linker arm used in the methods of this invention are made by methods known in the art [11, 12] as follows. In each case the 8-methoxycarbonyloctyl glycoside of the respective hapten is activated and ligated to a silyl,.min~ted solid support, wherein the matrix is comprised of SiO2, followed by the acetylation of the rem,.inin~ amine groups on the solid support. These formulations are:
Pl-di, which contains at least 0.60 ,umol/g o~Gal(14),~Gal disaccharide;
Pl-tri, which contains at least 0.91 ,umol/g c~Gal(I-4),~Gal(1-4),~GlcNAc trisaccharide; and Pk-tri, which contains at least 0.74 ,bmol/g o~Gal(I~),~Gal(14),~Glc trisaccharide.

CA 02243ll8 l998-07-l~

C. Pharmaceutical Compositions The methods of this invention are achieved by using ph~ relltic~l compositions comprising one or more oligosaccharide structures, which bind SLT toxin and/or verotoxin, ~tt~rh~l to a solid support.

S When used for oral ~lmini.~tration, which is preferred, these compositions may be fonmll~t~o~l in a variety of ways. It will preferably be in liquid or semisolid form. Compositions including a liquid pharm~relltir~lly inert carrier such as water may be considered for oral aflmini~tration. Other ph~Trnzlcelltic~lly compatible liquids or semisolids, may also be used. The use of such liquids and semisolids is well known to those of skill in the art.

Compositions which may be mixed with semisolid foods such as applesauce, ice cream or pudding may also be preferred. Formulations, such as SYNSORBs, which do not have a disagreeable taste or aftertaste are preferred.
A nasogastric tube may also be used to deliver the compositions directly into the stomach.

Solid compositions may also be used, and may optionally and conveniently be used in formulations cont~ining a pharm~re~ltically inert carrier, including conventional solid carriers such as lactose, starch, dextrin or magnesium stearate, which are conveniently presented in tablet or capsule form.
The SYNSORB itself may also be used without the addition of inert ph~ reutical carriers, particularly for use in capsule forrn. When a ph~ re~lticzllly inert carrier is employed the carrier is typically employed in the amount ranging from about 1 to about 99 percent based on the total weight of the composition and more preferably from about 75 to about 95 weight percent.
-Doses are selected to provide neutralization and elimin~tion of SLT
toxin and/or elimin~tion of E. coli found in the gut of the affected patient.

CA 02243ll8 l998-07-l~
W 097/49431 PC~/CA97/00436 Preferred doses are from about 0.25 to 1.25 micromoles of oligosaccharide/kg body weight/day, more preferably about 0.5 to 1.0 micromoles of oligosaccharide/kg body weightlday. Using the SYNSORB compositions described above, this means about 0.5 to 1.0 gram SYNSORB/kg body weight/day, which gives a concentration of SYNSORB in the gut of about 20 mg/ml. A~imini~tr;~tion is expected to be 2 to ~ times daily, preferably for a period of one week. The specific dose level and sch~ 71e of ~-1mini~tration will, of course, vary for each individual depending on factors such as the particular oligos~cchzlride structure employed, the age and condition of the subject, the extent of the disease condition, all of which are well within the skill of the art.

A-lmini.~tration of the oligosaccharide-cont~inin~ compositions of the present invention during a period of up to seven days will be useful in treatingSLT-associated diarrhea and associated conditions.

As ~li.cc~ ed previously, oral ~lmini~tration is preferred, but formulations may also be considered for other means of a~lmini.ctration such as per rectum. The usefulness of these formulations may depend on the particular composition used and the particular subject receiving the treatment. These formulations may contain a liquid carrier that may be oily, aqueous, emulsified or contain certain solvents suitable to the mode of ~lmini~tration.

Compositions may be form~ ted in unit dose form, or in multiple or subunit doses. For the expected doses set forth previously, orally ~-imini.~t~red liquid compositions should preferably contain about 1 micromole oligosaccharide/ml .

CA 02243ll8 l998-07-l~

D. Methodolo~y SLT toxin may be neutralized by oligosaccharide sequences comprising the ~Gal(1~4),~'Gal subunits which sequences bind the toxin. In particular, such oligosaccharide sequences covalently ,.tt~-~hf'(l to solid supports via non-peptidyl compatible linker arms have been found to neutralize SLT toxin effectively. Examples of such compositions are certain SYNSORBs, which bind and neutralize SLT toxin activity.

The ability of several oligosaccharide seque~ es attached to Chromosorb P via an 8-methoxylcarbonyloctyl (MCO) spacer arrn to neutralize SLT toxin has been tested.

The oligosaccharide sequences ~tt~',hf~-l to solid supports useful in the present invention include those which bind SLT toxin. The binding affinity of an oligosaccharide to SLT toxin is readily detectable by a simple in vitro test,as for example, set forth in Example 1 below. For the purposes of this invention, oligosaccharide sequences attached to solid supports which bind SLT
toxin means those compositions which reduce endpoint titers from cytotoxic activity in vero cell assays by at least 50% and preferably by at least 95%, using the assay set forth in tne Examples section.

Other oligosaccharide sequences attached to solid supports useful in the present invention are those which can bind SLT toxin significantly better (p<O.OS, using ~ liate standard st,-ti~tic~ll methods, such as the Wilcoxon or Student's T-test) than a control support that does not contain any attached oligosaccharide sequences (e.g., CHROMOSORB P).

The effect of the compositions of the invention in neutralizing SLTs can - 25 be measured by comparing activity of the SLT with and without treatment with the compositions. Activity of the SLTs can be assayed by ta'king advantage of the toxicity of these compounds to Vero cells. Vero cells (ATCC CCL81) can CA 02243118 1998-07-l~
W O 97/49~31 PCT/CA97/00436 be obtained from the American Type Culture Collection, Rockville MD.

In the methods of this invention, it has been found that the clinical incidence of HUS arising from enterohemorrhagic E. colt infection is reduced when the ph~rrn~f entical compositions described above are ~llm;nictered within 3 days of ~l~senlation of the infection and prior to organ involvement other - than intPst;n~l involvement. Contrarily, ~1mini.ctration of this pharrn~re-ltic~l composition after this time frame when organs other than the intestine are involved in the infection subst~nti~lly reduces the ability of this composition to reduce the incidence of HUS.

Preferably, the initial clinical evaluation that the individual is afflicted with an SLT me~ t~l E. coli infection is confirmed via diagnostic evaluation of the stool. One diagnostic tool commercially available for detecting SLT
me~ ted E. coli infection is sold by Meridian Diagnostic, Inc., Cinci~ ali, Ohio, USA 45244 under the name Premier EHEC.

As can be appreciated from the disclosure above, the present invention has a wide variety of applications. Accordingly, the following examples are offered by way of illustration and not by way of limitation.

EXAMPI,ES
In the examples below, all temperatures are given in degrees Celsius and the following abbreviations have the following me~nin~.c. If not defined below, then the abbreviations have their art recognized mf~nin~.c.

ASA = acetylated silyl~min~te~l hydrophobic 8-methoxycarbonyloctyl linkage arm BSA = bovine serum albumin cm = centimeter dpm = decays per minute EDTA = ethylene ~ min~ tetraacetic acid CA 02243118 1998-07-1~

g = gram HUS = hemolytic uremic syndrome kg = kilogram L = liter LPS = lipopolysaccharide M = molar MEM = minim~l Eagles m~ m mg = milligrams ~m~l millilit~rs min. = mimlt~
mm = millimeters nm = nanometers PBS = phosphate buffered saline pg = picogram SDS = sodium dodecyl sulfate SLT = shiga-like toxin ,ug = microgram ,uL = microliter ,umol = micromol In the examples below, Examples 1-3 are from Armstrong, et al. [10]
and are included herein to establish that oligosaccharides comprising a o~Gal(1 ~4),(~Gal subunit possess similar in vitro properties. Example 4 below illustrates in vivo results for the Pk trisaccharide and demonstrates the affect of timing of ~lmini~tration of the composition in reducing the incidence of HUS.

Example 1. SYNSORB - Verotoxicitv Neutralization Assays.
E. coli strains 0157:H-(E32511), which produces SLTII/SLTIIc and 026:H11(H19) which produces SLTI only or stain C600(933W), which produces SLTII only, were grown overnight at 37~C on tryptic soy broth (Difco, Detroit, MI) agar plates. Polymyxin and Iysozyme extracts were prepared as described previously [1,2].

The first neutralization assay was designed to test the ability of SYNSORBs to absorb SLT activity from the E. coli extracts for 30 min. at room temperature in 1.5 mL microcentrifuge tubes (Fisher) with 2 to 50 mg CA 02243118 1998-07-l~
W 097/49431 PCT/CA97/00~36 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 p~ dled in unsupplemented MEM. Twenty (20) ~L of each dilution was added to the a~lo~.iate wells in 96 well microtiter plates cont~inin~ Vero cells. Bacterial - extracts to which no SYNSORB was added served as controls. Once cytotoxic effects became al3~al~,nt (2 to 3 days in the incubator) the growth medium was aspirated from each of the wells and Vero cells which rem~in~(l viable were fixed 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 [3]. The absorbance data were then plotted versus the logarithrn of the extract dilution. The dilution of the extracts rçslllting in 50% destruction (CD50) of the monolayers was determined by extrapolation from the rçsn1ting Vero cell killing curves. Individual experiments were always performed in duplicate and unless otherwise in-1icated, repeated at least two times. The percentage of neutralization was computed from the equation: 100-(lOO[CD50 oligosaccharide SYNSORB-treated extracted + CD5n acetylated silyl-~min:~ted (ASA) SYNSOR13-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 14~.
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 ~bL of serial five-fold dilutions of polymyxin extracts in ethylene oxide-sterilized 1.5 mL
microcentrifuge tubes each cont~ining 2, 5 or 10 mg of P,~ trisaccharide SYNSORB. After 1 hour incubation with the SYNSORB, the entire contents of each microcenlliruge tube were added to Vero cells monolayers in 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 30 ~ above (3~igures 1 and 2).

.

CA 02243118 1998-07-l~
W O 97/49~31 PCT/CA97/00436 The foregoing determination was repeated using varying amounts of Pk-tri and various times of infllh~tion, 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;
S similarly, as shown in figure 3B, only about 5 min. incubation was required to achieve this result in either extract.

Example 2 Iodinated SLTI Bindin~ Assav Purified SLTI was iodinated in 12 x 75 mm acid-washed glass culture tubes coated with 40 ,ug of Iodo Gen (Pierce Chemical Co., Rockford, Illinois, USA). About 6 ,ug of purified SLTI was in/~ub:~tPfl for 1 min. with 20 MBq l25-I labeled sodium iodide in 100 ,uL PBS. The reaction mixture was passed through a glass wool-plugged Pasteur pipette into 200 ,uL PBS cont~inin~ a solution of cysteine (1 mg/mL) in PBS as described by Armstrong, G.D. et al.
r52] After 1 min., 200 ,~L of PBS cont~ining 1% BSA was added to the mixture and the 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 was stored at -90~C.

The assays were performed in PBS cont~ining 0.15% BSA to reduce nonspecific binding. 2 mg of the SYNSORB were incubated for 30 min. on an end-over-end rotator with approximately 20,000 dpm of the io-lin~t~l SLTI
prepared above (specific activity, 2.2 x 107 dpm/llg, CD50 in the Verocytotoxicity assay, 0.4 pg/mL), in 0.5 mL PBS/BSA). The SYNSORB
was then washed with 3 x 1 mL portions of PBS/BSA to remove unbound - counts. The derivatized SYNSORB were counted in an LKB Rackg~mm~
model 1270 (~mmz~ C~ounter. Similarly, ASA was also employed to determine the effect of using this material.

CA 02243ll8 lsgx-07-l5 The results are shown in Table 1.

Table 1 SYNSORB% SLTI Bound Pk-tri 93 The SLT bound to Pk-tri SYNSORB could be partially released using 0.1 M acetic acid, 6 M gll~ni~line HCl, or by heating in boiling water bath for 30 min. in 10% SDS. However, neither 0.5 M lactose, 0.5 M g~l~rtose, or 0.2 M
EDTA could displace the bound SLTT ~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 HCI to simulate conditions in the stomach, then washed extensively in PBS to remove the HCI. The HCl-treated SYNSORB was then tested for SLTI and SLTII neutralizing activity in the Vero cytotoxicity assay as describedhereinabove, and by Armstrong, G.D. et al. ~53].

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 mL PBS cont~ining 0.1 mg/mL Polymyxin B sulfate.
The res~ ;ng suspension was then clarified by centrifugation.

Five mg of P1 trisaccharide SYNSORB were mixed with approximately CA 02243118 1998-07-l~

1 mL of polymyxin extract of the bacterial strains listed in the table below.
Data represent the average of two independent detennin~tions, each performed in duplicate. Values in brackets give the range for each value.
. Table 2 Ability of HCl-Treated Pk Trisaccharide SYNSORB to Neutralize SLT Activity in the Vero Cell Assay HCIPercent SLT Activity Neutralized Incubation Time 026:H11 0157:H7 C600 (Hours at 37~C) (SLTI) (SLTII/IIc) (SLTII) 0 94 (4) 88 (5) 55 (10) 94 (6) 80 (2) 66 (5) 4 96 (5) 91 (8) 73 (2) 18 94 (6) 93 (6) 70 (7) As shown in Table 2, incubation with HCl does not appreciably ~1imini.~h neutralizing activity. To stim~ t~ intestinal conditions, various SYNSORBs were incubated for 2 hours at 37~C in buffer or in rat intestinal sacs. The inrub~fPd 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 CDsos of polymyxin extracts incubated with the ASA control SYNSORB.
These results are shown in Tables 3-6.

CA 02243118 l99X-07-15 Table 3 shows the results of neutralization of SLTI activity by SYNSORB inrl1b~t~d in buffer; Table 4 shows neutralization of SLTI activity by SYNSOE~B incubated in int~stinz~l sacs; Table S shows neutralization of SLTII activity by SYNSORB inr~l~att?d in buffer; and Table 6 shows S neutralization of SLTII activity by SYNSORB incubated in rat i,.~ l sacs.

Table 3 SYNSORB Percent SLTI: Activity Neutralized~
ASA SYNSORBb O
P, Disaccharide 93 (88 - 97) P, Trisaccharide 98 (96 - 100) a. Average of duplicate del~ln~ ations. Range in brackets.
b. Control SYNSORB cont~ining only the acetylated silyl~min~erl (ASA) hydrophobic 8-methoxycarbonyloctyl linkage arm.

Table 4 SYNSORB Percent SLTI Activity Neutralized~
ASA SYNSORBb O
Pl Disaccharide 82 i 6 Pl Trisaccharide 98 i 2 a. Avera~e of triplicate determinations + standard deviation of the mean.
b. ~ontrol SYNSORB cont:-ining only the acetylated silyl~min~te~l (ASA) hydrophobic 8-methoxycarbonyloctyl linkage arm.

Table 5 SYNSORB Percent SLTI Activity Neutralized;

Pl Disaccharide 84 CA 02243118 1998-07-l~

P, Trisaccharide 98 a. Results of one ~let~ n~tion.

Table 6 SYNSORB Percent SLTI Activity Neutralizeda P, Disaccharide 65 ~t ~a Pl Trisaccharide 96b a. Average of triplicate determinations ~t 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 n~ntr~li7ing SLTI or SLTII.

Example 4. Performance in Human Patients Pharrn~celltic~l compositions comprising Pk SYNSORB was ~(lmini.ctered to children at 13 C~7~n~ n sites.

To allow treatment at the earliest possible point in the gastroil-lPs~i.,~l illness, children were eligible for inclusion in this study before the results of stool cultures were known. Children were limited to the ages of from 6 months to 15 years of age. The children were eligible for the study if they had diarrhea (had passed at least 2 loose stools in the prece~ling 24 hours) and one- of the following: abdominal cramping, blood in the stools, rectal prolapse, close contact with an individual with known VTEC infection or HUS, or E. coli 0157:H7 cultured from the stool. Patients were excluded if they had chronic CA 02243118 1998-07-l~
W O 97/49431 PCT/CAg7/00436 renal or hematologic disease or if they had evidence of hemolysis, renal injury or thrombocytopenia on either an acute or chronic basis, if they had chronic bowel disease, if they had an encephalopathy that would preclude ingestion of oral medication or if they were receiving anticonvulsant medications or S pancreatic enzyme supplements. Patients remained in the study unless stool cultures identified a non-VTEC stool pathogen.

A complete blood count, peripheral blood smear, serum urea and cre~tininP urinalysis and serum for anti-0157 antibodies was obtained from all subjects prior to enrollment. Serum samples from enrollment and day 60 after ~0 ~ enrollment were tested for anti-0157 LPS antibodies using a passive hemaggll-~in~tion assay. An anti-0157 LPS titer of > 1:500 was considered consistent with recent infection.

Stool samples from all patients were submitted at entry to the hospital microbiology laboratory and were routinely inoculated onto a modified sorbitol-MacConkey agar. Colonies which failed to ferment sorbitol were identified as . coli by standard biochemical tests, and isolates were serogrouped by agglutination using 0157 antisera. Stool samples from those with negative routine cultures were forwarded to the National Laboratory for Enteric Pathogens at the Center for Disease Control in Ottawa, Canada. These samples were evaluated for the presence of other verotoxin-producing ~. coli.

Eligible subjects were stratified on the basis of age and center and then randomized. Patients received either SYNSORB-P}~ mixed in baby food or an e~ual volume of ground corn meal placebo with similar taste and texture. The primary outcome of interest was the proportion of patients with evidence of HUS at day seven of treatment.

The study drug was ~mini~tered twice daily for seven days. The dose of SYNSORB-Pk was approximately 500 mg/kg/day. The placebo was ground CA 02243ll8 l998-07-l~

corn meal, selected because it had a mildly gritty texture similar to SYNSORB-Pk and like SYNSORB-Pk had no taste. To improve palatability, both SYNSORB-Pk and the corn meal placebo were mixed in a commercially available baby food (fruit). If a dose was vomited within 30 minutes, it was S rea-lmini~tered. Subjects continued to receive the study drug for seven days if their stool culture results identified E. coli 0157 or if no bacterial pathogen was identified. If the stool culture identified an alternate cause for symptoms, such as SalmoneZla, Shigella, Campylobacter or Yersinia species, the subject stopped taking the medication.

The primary outcome measure for the trial was the proportion of subjects with hemolytic uremic syndrome by the time of laboratory re-evaluation 8 -10 days after enrollment. Hemolytic uremic syndrome was considered to be present if there was renal injury and either hemolysis or thrombocytopenia. Renal injury required an elevation in the serum cre~tinin~-concentration (>50 ,umol/L for those less than 5 years, or >60 ,umol/L for those 5~6 years~ or a difference in the recorded cre~tinine values during the acute phase of tlle illness of more than 50% or at least 10 red blood cells per high power field on urine microscopy. Hemolysis was judged present if the hemoglobin concentration was 5 105g/L or if there were red blood cell fragments on the smear or if a red blood cell transfusion was ~rimini~tered before the hemoglobin dropped to 5 105g/L. All peripheral blood smears were forwarded to the central study laboratory at the Children's Hospital of Eastern Ontario in Ottawa, where they were graded by laboratory technicians blinded to the treatment status of the subject.

- 25 Thrombocytopenia was defined as a platelet concentration of less than 150 X 109/L. Mild HUS was present if the peak elevation in cre~tinine concentration was less than 100 ,umol/L; moderate HUS required a cre~tinin~
concentration of 101-400 ,umol/L and dialysis less than seven days; severe HUS required cre~tinine concentration of above 400 ,umol/L or dialysis for -CA 02243118 l99X-07-l~

seven or more days or death. In addition to these primary outcomes, patients were also classified as having the secondary outcomes of isolated hemolytic ~nf~mi~, isolated thrombocytopenia, or isolated renal injury as defined earlier.Parents recorded in a daily diary the frequency of diarrhea and vomited doses of medication as well as the frequency of possible adverse events.

The results of this evaluation were analyzed to deterrnine the effect of this protocol on those patients with a verified SLT m~Ai~ l pathogenic E. coli infection and who were compliant with the treatment regimen. The analysis of these results were divided into two groups, i.e., those treated within three days of first presenting symptoms of E. coli infection and those treated after three days of first presenting symptoms of E. coli infection. Tables 7 and 8 below set forth the results of this evaluation.
-Table 7. Outcome by Group - Those only with E. coli or stool positive cases and treated ~ 3 days post syndrome SYNSORB PLACl~BO
No. of cases % No. of Cases %
Normal 24 64.9 20 58.8 Isolated thrombo Non-anemic hemolysis l 2.7 2 5.9 Hemolytic anemia 1 2.7 --Renal Insufficiency 5 13.5 2 5.9 Mild HUS 4 10.8 6 17.6 Moderate HUS -- 2 5.9 Severe HUS 1 2.7 2 5.9 Alternate pathogen -- --Adverse event -- --.
Missing lab values l 2.7 --Non-compliant -- --RA error -- --CA 02243118 1998-07-1~

Table 8. Outcome by Group - Those only with E. coli or stool positive cases and treated > 3 days post syndrome SYNSORB PLACEBO
No. of cases % No. of Cases %
Normal 12 50.0 16 66.7 Isolated thrombo -- 1 4.2 Non-anemic hemolysis 1 4.2 1 4.2 Hemolytic anemia 4 16.7 --Renal Insufficiency 3 12.5 2 8.3 Mild HUS 2 8.3 1 4.2 Moderate HUS 1 4.2 2 8.3 Severe HUS 1 4.2 --Alternate pathogen -- --Adverse event -- --Missing lab values -- I 4.2 . Non-compliant -- --RA error -- --The above results demonstrate that when a patient with disease symptoms attributable to an SLT m~ t~l E. coli infection is treated within three days of presentation of these symptoms, the overall incidence of HUS in the treated patients is 13.5% as compared to the overall incidence of 29.4% for placebo treated patients or a relative reduction in risk of 54.1%. The above results further demonstrate that when a patient with disease symptoms attributable to an SLT mediated E. coli infection is treated after three days ofpresentation of these symptoms, there is no relative reduction in risk for CA 02243118 1998-07-l~

development of HUS as compared to placebo.

Accordingly, these results demonstrate the criticality of early - intervention in the SLT merli~tefl E. coli infection via the methods of this invention.

While the present invention has been described with reference to what are considered to be the pl~rellt:d examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims (13)

WHAT IS CLAIMED IS:

1. A method for inhibiting the development of hemolytic uremic syndrome in a patient arising from enterohemorrhagic E. coli infection mediated by shiga-like toxins which method comprises administering to said patient an effective amount of a pharmaceutical composition comprising a pharmaceutically inert affinity support comprising an .alpha.Gal(1~4).beta.Gal subunit which is bound to said support through a non-peptidyl linker arm, wherein said subunit binds SLT toxin wherein this pharmaceutical composition is administered within about 3 days of presentation of the infection.

2. The method of Claim 1 wherein the linker arm comprises from 1 to 10 carbon atoms.

3. The method of Claim 2 wherein the linker arm is -(CH2)8C(O)-.

4. The method of Claim 1 wherein the solid inert affinity support is silica.

5. The method of Claim 1 wherein the pharmaceutical composition is administered to the patient prior to organ involvement other than intestinal involvement.

6. A method for inhibiting the development of hemolytic uremic syndrome in a patient presenting with an enterohemorrhagic E. coli infection mediated by shiga-like toxin, which method comprises administering to the patient an effective amount of a pharmaceutical composition comprising a pharmaceutically inert affinity support comprising an oligosaccharide selected from the group consisting of .alpha.Gal(1~4).beta.Gal, .alpha.Gal(1~4).beta.Gal(1~4).beta.GlcNAc and .alpha.Gal(1~4),.beta.Gal(l~4).beta.Glc which oligosaccharide is bound to the support through a non-peptidyl linker arm, wherein this pharmaceutical composition is administered within about 3 days of presentation of the infection.

7. The method of Claim 6 wherein said oligosaccharide sequence is .alpha.Gal(1~4).beta.Gal.

8. The method of Claim 6 where said oligosaccharide sequence is .alpha.Gal(1~4).beta.Gal(1~4).beta.GlcNAc.

9. The method of Claim 6 where said oligosaccharide sequence is .alpha.Gal(1~4).beta.Gal(1~4).beta.Glc.

10. The method of Claim 6 wherein the linker arm comprises from 1 to 10 carbon atoms.

11. The method of Claim 10 wherein the linker arm is -(CH2)8C(O)-.

12. The method of Claim 6 wherein the solid inert affinity support is silica.

13. The method of Claim 6 wherein the pharmaceutical composition is administered to the patient prior to organ involvement other than intestinal involvement.