WO1996041185A9 - Procedes et compositions liant des endotoxines - Google Patents

Procedes et compositions liant des endotoxines

Info

Publication number
WO1996041185A9
WO1996041185A9 PCT/US1996/007475 US9607475W WO9641185A9 WO 1996041185 A9 WO1996041185 A9 WO 1996041185A9 US 9607475 W US9607475 W US 9607475W WO 9641185 A9 WO9641185 A9 WO 9641185A9
Authority
WO
WIPO (PCT)
Prior art keywords
containing moiety
sample
diamidine
amidine
lipopolysaccharide
Prior art date
Application number
PCT/US1996/007475
Other languages
English (en)
Other versions
WO1996041185B1 (fr
WO1996041185A1 (fr
Filing date
Publication date
Application filed filed Critical
Publication of WO1996041185A1 publication Critical patent/WO1996041185A1/fr
Publication of WO1996041185B1 publication Critical patent/WO1996041185B1/fr
Publication of WO1996041185A9 publication Critical patent/WO1996041185A9/fr

Links

Definitions

  • Lipopolysaccharide also known as endotoxin, (the terms are used interchangeably herein) is expressed in the outer membrane of Gram negative bacteria.
  • the molecule consists of a hydrophobic Lipid A region linked to a core oligosaccharide and an outer polysaccharide chain.
  • Septic shock leads to a cascade of host responses to lipopolysaccharide, importantly, the elaboration of proinflammatory cytokines including tumor necrosis factor and
  • endotoxins may be introduced from buffers, salts, chromatographic media, cell culture, fermentation additives, and/or water. Contamination by endotoxins is particularly common when
  • Endotoxins vary in size depending on whether
  • divalent ions and surface active agents are present.
  • the location of a product in the hosts and the mechanism of cell disruption can have a significant impact on the amount of endotoxin released with a product. If cell disruption methods change, as they often do during scale-up fermentation,
  • Lipid A The amphiphilic lipid portion of lipopolysaccharide, termed Lipid A, elicits most of the toxic effects of lipopolysaccharide, and therefore represents the toxic center of the endotoxin.
  • the antibiotic polymyxin B (PmxB) binds with relatively high affinity to the Lipid A portion of lipopolysaccharide and results in the inability of lipopolysaccharide to invoke the inflammatory response in macrophages and monocytes, its primary site of action. Since Lipid A partial structures can act in a similar fashion to PmxB and
  • Lipid A region would be an appropriate therapeutic target for drug development.
  • Rustici et al. examined the ability of synthetic peptides to mimic the action of PmxB in binding to Lipid A, thus acting as inhibitors of the inflammatory response; they found that a contribution
  • the present invention is directed to affinity supports and methods for the detection or removal of endotoxins which substantially obviate one or more of the problems due
  • the invention relates to molecules capable of binding to endotoxins of various serotypes and methods for detecting and/or removing endotoxins from a sample.
  • the invention relates to a method for removing lipopolysaccharides from a sample by immobilizing an amidine containing moiety to a solid support such that it retains its ability to bind to lipopolysaccharides, and introducing the sample to the support under conditions sufficient for
  • the lipopolysaccharides if present, to form a complex with the amidine containing moiety.
  • the invention relates to detecting the absence, presence or concentration of a lipopolysaccharide in a sample by contacting the sample with a detectable modified amidine containing moiety. If lipopolysaccharide is present, it will form a complex with
  • the methods are both quantitative and qualitative, and may, in various embodiments, be incorporated into diagnostic or therapeutic applications, such as to remove or detect lipopolysaccharide in a sample.
  • the invention further relates to an apparatus and a kit including an amidine containing
  • modified amidine moieties may be used in solution, provided that the complex formed has at least one physical
  • amidine containing moieties which bind to endotoxins may also have a bacteriocidal
  • the invention encompasses pharmaceutical compositions containing an amidine containing moiety which binds to endotoxins with a high affinity, as well as any desired fillers, adjuvants or stabilizers.
  • the invention further contemplates methods for the treatment or diagnosis of endotoxin contamination using amidine containing moieties which bind to endotoxins with a high affinity.
  • compositions containing modified amidine containing moieties may be introduced to a host in a therapeutically effective dose, or used to detect or remove endotoxins from fluids removed from the animal.
  • the claimed invention relates to methods for purifying a sample preparation, for example, recombinantly produced proteins.
  • the claimed invention may be used in methods, apparatus, kits and compositions for the detection or removal of any phosphate-containing molecule, as well methods, compositions and
  • Figure IA depicts the removal of endotoxins on various columns having True Blue immobilized on a POROSTM support.
  • Figure IB depicts an SDS-PAGE analysis of unbound globulins to resin.
  • Figure 2 depicts the recovery of LPS (labelled with FITC) on a POROSTM-True Blue
  • FIG. 3 depicts the removal of LPS in the presence of various proteins on POROSTM-
  • FIG. 4 depicts the Frontal analysis of POROSTM-True Blue
  • the method of removing lipopolysaccharides from a sample solution initially involves immobilizing an amidine containing moiety to a solid support such that the moiety retains its ability to bind to lipopolysaccharides.
  • the sample solution is then introduced to
  • any lipopolysaccharides present will bind to the amidine containing moiety, thus
  • modified amidine containing moieties of the invention are modified for attachment to a support while maintaining their ability to bind lipopolysaccharide or phosphate containing compounds such as DNA or RNA.
  • Prefererred modified amidine containing moieties are
  • True Blue and DAPI modified pentamidines such as True Blue and DAPI.
  • True Blue and DAPI are commercially available through Molecular Probes. (True Blue and DAPI); SIGMA CHEMICAL CO. (TB and DAPI).
  • True Blue (TB) has the formula:
  • DAPI is a compound having one of the following the chemical formulae:
  • True Blue is particularly useful for practice of this invention because it has a higher affinity than pentamidine for binding endotoxins and has a broader
  • True Blue contains a double bond which can be utilized for immobilization to a solid support in a chemically stable manner. This is particularly advantageous because detoxification requires conditions which would
  • DAPI diamidino-2-phenylindole
  • amidine moieties preferably bind to the phosphate structure in the
  • amidine containing moieties include, but are not limited to, controlled
  • the support may be porous or non-porous depending upon the desired application of the invention.
  • the support is suitable for perfusion chromatography, such as POROSTM, available from PerSeptive Biosystems, Inc.
  • the amidine containing moieties are immobilized to the support by double bond linkages, rather than amidine linkages.
  • Samples which may be utilized in the claimed invention include water, pharmaceutical, and
  • preparations body fluids, including, but not limited to, blood, serum, plasma, urine cerebrospinal
  • the claimed invention may be used in the pharmaceutical industry for e.g. removing
  • the invention is a composition of lipopolysaccharide from water or other fluids used in pharmaceutical processing.
  • the invention is a
  • the claimed invention is suitable for removing endotoxins from solutions.
  • the claimed invention is suitable for removing endotoxins from solutions.
  • the support passes by the support.
  • the endotoxins if present, will bind to the amidine containing moiety
  • amidine containing moiety it is not necessary that the amidine containing moiety be immobilized on a support.
  • amidine moieties having a high affinity for lipopolysaccharide may also be useful to inactivate lipopolysaccharides.
  • amidine containing moieties may have bacteriocidal properties and may
  • the invention may be useful as a means to remove endotoxin from solutions.
  • Supports according to the invention may be used in any format, for example, a chromatography column, discs, chips (i.e. for surface plasma resonance detection).
  • affinity supports may have application as a potential therapeutic in cases of reperfusion ischemia injury.
  • Patients expressing symptoms of endotoxic shock (septicemia) may be gavaged with the support, which would then remove or inactivate the lipopolysaccharide in the gut, which is the major site of abso ⁇ tion This may reduce the level of lipopolysaccharide load in the host system prior to reperfusion and hence provide increased chances for survival.
  • endotoxic shock septicemia
  • the methods of the invention are to be used to detect the absence
  • the sample is contacted with a detectable, amidine containing moiety, such that, if lipopolysaccharides are
  • a detectable moiety is any moiety capable of detection, suitable for use in the claimed invention, including, but not limited to: enzymes, fluorophores, chromophores, radioisotopes, electrochemical moieties and
  • detectable moieties can be readily conjugated to the amidine containing moieties using any technique know in the art, however, should not interfere with the ability of the modified pentamidine to bind to the lipopolysaccharides.
  • the invention contemplates that the detectable moiety may be intrinsic to the moiety.
  • the claimed invention may be embodied in an individual apparatus or kit which can be easily used in a wide range of diagnostic tests.
  • the kit may optionally be disposable.
  • the invention contemplates an apparatus or kit useful to remove lipopolysaccharides from a solution.
  • the invention can detect the presence of endotoxins in solution, and can be used as a component of a quality assurance system for any solution. For example, one may wish to periodically sample production batches to test for impurities such as endotoxins.
  • the invention may be incorporated into the upstream processing not only to test for the absence, presence or concentration of endotoxins, but also to actually remove contaminants at any stage of the processing.
  • a pharmaceutical preparation comprising an amidine containing
  • amidine a moiety capable of binding to endotoxins, may be introduced into a host organism.
  • the amidine containing moieties may then bind to the endotoxins thereby rendering them non-toxic.
  • the amidine containing moieties may be used as part of a dialysis type apparatus to
  • Such devices may be used to either remove lipopolysaccharides from a sample, or to detect or quantitate lipopolysaccharide levels in solution, e.g. in serum from patients suspected of having sepsis, in pharmaceutical analyses for validation pu ⁇ oses, or for detection of pyrogenic quantities of LPS in preparations for administration to humans or other animals.
  • the support be used in the form of a cartridge or chip. Additionally, one may, for example, by loading a portion of a hypodermic needle with detectable modified pentamidine moieties, quickly and
  • lipopolysaccharides may be configured to detect clinically relevant lipopolysaccharides' in
  • [3] POROS treated as for [1] and [2] in the absence of TB After washing 30 ⁇ l of a dilute slurry of these resins extensively in lipopolysaccharide-free water, specific binding of lipopolysaccharide was assessed by incubation of these resins with know quantities of lipopolysaccharide. Unbound lipopolysaccharide was measured using the Limulus
  • BSA BSA
  • D Myoglobin
  • E Gamma Globulins
  • lipopolysaccharide to these proteins. Optimization of pH and salt conditions may reduce the levels of unwanted non-specific binding. In this regard we have observed a slight increase in the capacity of the column for lipopolysaccharide if the buffer was changed to 20mM phosphate, pH 7.0 (data not shown). Resin [1] removes> 95% lipopolysaccharide from solution A 2.1mm x 100 mm column was packed with [1]. lipopolysaccharide activity in an aliquot of FITC-lipopolysaccharide was measured prior to injection over the column and in the void
  • lipopolysaccharide containing FTTC-lipopolysaccharide as a marker were made and fractions of
  • the affinity column tested appears to exhibit selectively for endotoxin even when running at fairly high flow rates.
  • the capacity of the column is not quite as high as the "theoretical" value (5mg/ml) for the Affi-prep polymyxin B support sold by BioRad.
  • the column does not involve as high as the "theoretical" value (5mg/ml) for the Affi-prep polymyxin B support sold by BioRad.
  • the column does not quite as high as the "theoretical" value (5mg/ml) for the Affi-prep polymyxin B support sold by BioRad.
  • the columns can be run at higher pressures and flow rates and can be sanitized by treatment with IN NaOH without
  • amidine containing moieties to bind a variety of different serotypes of
  • lipopolysaccharide endotoxin
  • Figure 2 shows the effect of preincubation of the resins with a known quantity of endotoxin on recovery of endotoxin assayed using the LAL assay, lipopolysaccharide incubated in the absence of resin was included as a
  • amidine groups are critical for optimal interaction between these molecules and lipopolysaccharide since none of the amine-containing compounds or amidines tested were capable of inhibiting the lipopolysaccharide- ediated color
  • the time and dilution factor should be constant under similar conditions; the loss of ligand bound
  • Fig. 3a shows only those chromatograms obtained from incubating the lipopolysaccharide and 9-mer in the absence (trace I) and presence (trace II) of PmxB at the highest concentration (5 ⁇ M).
  • Fig. 4 Inset revealed a Hill coefficient (n H ) for pentamidine of 1.2 (correlation 0.99) and PmxB n H ⁇ 1.42 (correlation 0.985) suggesting that there may be more than one binding site for the ligand on each molecule of lipopolysaccharide and only one ligand molecule may bind at a time, possibly due to steric hindrance.

Abstract

L'invention décrit des procédés et des compositions liant des endotoxines, utiles pour l'extraction ou la détection d'endotoxines, qui comprennent notamment des fragments d'amidine. Ces fragments d'amidine sont de préférence de type 'True Blue' ou de 'DAPI' (4,6-Diamidino-2-phénylindole). On utilise de préférence le support POROSR. Cette composition peut servir dans le traitement d'états tels que le choc septique.
PCT/US1996/007475 1995-06-07 1996-05-22 Procedes et compositions liant des endotoxines WO1996041185A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US47478995A 1995-06-07 1995-06-07
US08/474,789 1995-06-07

Publications (3)

Publication Number Publication Date
WO1996041185A1 WO1996041185A1 (fr) 1996-12-19
WO1996041185B1 WO1996041185B1 (fr) 1997-01-16
WO1996041185A9 true WO1996041185A9 (fr) 1997-05-15

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/007475 WO1996041185A1 (fr) 1995-06-07 1996-05-22 Procedes et compositions liant des endotoxines

Country Status (1)

Country Link
WO (1) WO1996041185A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9910807D0 (en) 1999-05-10 1999-07-07 Prometic Biosciences Limited Novel detoxification agents and their use
US6774102B1 (en) 1999-09-29 2004-08-10 Gambro Dialysatoren Gmbh & Co. Kg Extracorporeal endotoxin removal method

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1550819A (en) * 1975-06-04 1979-08-22 Nat Res Dev Polymeric support for biogically active materials
US4276050A (en) * 1980-01-10 1981-06-30 Abbott Laboratories Method for systemic endotoxin detection
US4491660A (en) * 1980-01-10 1985-01-01 Abbott Laboratories Matrix polymers for binding endotoxins
EP0097463A3 (fr) * 1982-06-16 1985-05-15 Beecham Group Plc Dérivés amidinés
US4806546A (en) * 1985-09-30 1989-02-21 Miles Inc. Immobilization of nucleic acids on derivatized nylon supports
US5030561A (en) * 1988-12-27 1991-07-09 Becton, Dickinson And Company Chlamydia assay using amidine modified supports or particles
US5136032A (en) * 1989-12-07 1992-08-04 Daicel Chemical Industries, Ltd. Method for separating phosphopolyol compounds using a separating agent

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