WO1996041185B1

WO1996041185B1 - Methods and compositions for binding endotoxins

Info

Publication number: WO1996041185B1
Application number: PCT/US1996/007475
Authority: WO
Filing date: 1996-05-22
Publication date: 1997-01-16
Anticipated expiration: 1997-12-07

Abstract

Disclosed are methods and compositions for binding endotoxins useful for the removal or detection of endotoxins, involving amidine moieties. The amidine moieties are preferably provided by True Blue or 4,6-Diamidino-2-phenylindole (DAPI). A preferred support is POROSR. The compositions may be used in treatment of conditions such as septic shock.

Claims

-24-

AMENDED CLAIMS

[received by the International Bureau on 5 December 1996 (05.12.96); original claims 1-46 replaced by amended claims 1-45 (6 pages)]

1 A method for removing popolysacchaπdes, if present, from a sample comprising the steps of

(a) immobilizing a diamidine containing moiety to a solid support such that it retains its ability

to bind to hpopoly saccharides, (b) introducing a sample to the solid support under conditions sufficient to allow the lipopolysacchaπdes, if present, to bind to the diamidine moiety

2 The method of claim 1 wherein the solid support is selected from the group consisting of

controlled pore glass, membranes, magnetic beads, magnetic particles porous polystyrene

based supports, silica, silica gel, glass fibre frits and paper filters

3 The method of claim 1 wherein the diamidine moiety is l,2-bιs(5-amιdιno-2-benzofuranyl)- ethylene 4 The method of claim 1 wherein the diamidine containing moiety is 4,6-dιamιdιno-2- pheny ndole

5 The method of claim 1 wherein the support is suitable for perfusive chromatography

6 The method of claim 1 wherein the sample comprises recombinant proteins

7 The method of claim 1 wherein the sample comprises fluids for pharmaceutical preparation

8 A method for detecting the absence, presence or concentration of a lipopolysaccharide in a

sample comprising the step of (a) contacting a sample with a detectable, modified diamidine

containing moiety, such that the molecule, if present, will form a complex with the diamidine

containing moiety thereby indicating the absence, presence or concentration of

lipopolysaccharide 9 The method of claim 8 wherein the diamidine containing moiety is 1 ,2-bιs(5-amιdιno-2-

benzofuranyl )-ethylene

10 The method of claim 8 wherein the diamidine moiety is 4,6-dιamιdιno-2-phenylιndole 1 1 The method of claim 8 wherein the sample comprises recombinant proteins 12. The method of claim 8 wherein the sample comprises fluids for pharmaceutical preparations. 13. The method of claim 8 wherein the sample is obtained from a host organism. M. A method for the purification of a sample preparation comprising immobilizing a diamidine

containing moiety to a solid support such that it retains its ability to bind to

lipopolysaccharides; introducing the sample preparation to the support under conditions

sufficient for lipopolysaccharides, if present, to bind to the immobilized diamidine containing moiety; and, eluting the unbound components of the sample. 15. The method of claim 14 comprising the purification of recombinantly produced proteins.

16. An apparatus for the removal or detection of lipopolysaccharides from a sample comprising a

diamidine containing moiety immobilized to a solid support.

17. The apparatus of claim 16 wherein the solid support is selected from the group consisting of

controlled pore glass, chips, membranes, magnetic beads, magnetic particles, porous

polystyrene based supports, silica, silica gel, glass fibre frits and paper filters.

18. The apparatus of claim 17 wherein the support is suitable for perfusive chromatography.

19. The apparatus of claim 16 wherein the diamidine containing moiety is l,2-bis(5-amidino-2- benzofuranyl )-ethylene.

20. The apparatus of claim 16 wherein the diamidine containing moiety is 4,6-diamidino-2-

phenylindole. 21. A kit for the detection or removal of a lipopolysaccharide in a sample comprising: a solid

support having immobilized thereon a diamidine containing moiety capable of binding to

lipopolysaccharide.

22. The kit of claim 21 wherein the solid support is selected from the group consisting of

controlled pore glass, membranes, magnetic beads, magnetic particles, porous polystyrene

based support, silica, silica gel, glass fibre frits and paper filters.

23. The kit of claim 22 wherein the support is suitable for perfusive chromatography. -26- 24. The kit of claim 22 wherein the solid support is a column packed with particulate

chromatography elements, said column being housed in a cartridge.

25. The kit of claim 24 wherein the cartridge is disposable.

26. The kit of claim 21 wherein the diamidine containing moiety comprises a detectable moiety.

27. The kit of claim 26 wherein the modified diamidine containing moiety is 1 ,2-bis(5-amidino-2- benzofuranyl)-ethylene or 4,6-diamidino-2-phenylindole.

28. A method for the removal of lipopolysaccharides from a solution comprising adding to a

solution a diamidine containing moiety which binds to lipopolysaccharides, if present, with high affinity thereby forming a complex.

29. The method of claim 28 further comprising separating said complex, if formed, from the

unbound components of the solution by filtration, chromatography, sedimentation or

electrophoresis.

30. The method of claim 28 wherein the diamidine containing moiety is l,2-bis(5-amidino-2- benzofuranyl)-ethylene or 4,6-diamidino-2-phenylindole.

31. A method for the detection of a lipopolysaccharide in a sample comprising introducing to the

sample a diamidine containing moiety capable of binding to lipopolysaccharide thereby

forming a detectable complex; and detecting said complex.

32. The method of claim 31 wherein the diamidine containing moiety is l,2-bis(5-amidino-2- benzofuranyl)-ethylene or 4,6-diamidino-2-phenylindole.

33. The method of claim 32 wherein the sample is selected from the group consisting of water,

blood, cerebrospinal fluid, sweat, ascites fluid saliva, semen, amniotic fluid, and vaginal fluid.

34. A method of removing or inactivating endotoxins from a bodily fluid of a host organism

comprising the steps of: (a) removing a fluid from the host; (b) contacting the fluid with a

solid support comprising a diamidine containing moiety which binds to endotoxins with high -27- affinity to form a complex; and, (3) separating the complex from the bodily fluid prior to

reintroduction of the fluid into the host organism.

35. The method of claim 34 wherein the diamidine containing moiety is 1 ,2-bis(5-amidino-2-

benzofuranyl)-ethylene or 4,6-diamidino-2-phenylindole. 36. A method for detoxifying endotoxins in a host organism comprising the introduction into the organism of a therapeutically effective dose of a preparation comprising a diamidine

containing moiety capable of binding to endotoxins in vivo with high affinity, thereby

inactivating the lipid A portion of the endotoxin.

37. The method of claim 36 wherein the diamidine containing moiety is 1 ,2-bis(5-amidino-2-

benzofuranyl)-ethylene or 4,6-diamidino-2-phenylindole.

38. A method of treatment for animals having an immunogenic reaction to a bacterial infection comprising the step of administering to the animal a therapeutically effective dose of a

composition comprising a diamidine containing moiety capable of inactivating the lipid A

region of the endotoxins.

39. A pharmaceutical preparation for the treatment of organisms with endotoxin contaminants

comprising a therapeutically effective amount of diamidine containing moieties capable of

binding to the endotoxins thereby rendering the endotoxins inactive.

40. A pharmaceutical preparation substantially pure of endotoxin contamination wherein said

preparation was purified by contact with an diamidine containing moiety immobilized to a

solid support.

41. The method of claim 40 wherein the modified diamidine containing moiety is l,2-bis(5-

amidino-2-benzofuranyl)-ethylene or 4,6-diamidino-2-phenylindole.

42. A method for the prevention of septic shock in animals caused by the presence of

lipopolysaccharides, said method comprising administering to the animal a therapeutically

AMENDED SHEET (ARTICLE 1SJ -28- effective dose of a composition comprising a diamidine containing moiety capable of

inactivating the lipopolysaccharide by binding to the lipopolysaccharide with high affinity.

43. A method for removing a phosphate-containing molecule from a sample comprising

immobilizing a diamidine containing moiety to a solid support; and introducing the sample to the support under conditions suitable for phosphate-containing molecules, if present, to bind

to said diamidine containing moiety thereby forming a complex.

44. The method of claim 43 wherein the phosphate-containing molecule is a nucleic acid or

phosphoprotein.

-29- 45. The method of claim 1 wherein the diamidine moiety is immobilized by a non-amidine containing linkage.