WO1996041185A1 - Procedes et compositions liant des endotoxines - Google Patents

Procedes et compositions liant des endotoxines Download PDF

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Publication number
WO1996041185A1
WO1996041185A1 PCT/US1996/007475 US9607475W WO9641185A1 WO 1996041185 A1 WO1996041185 A1 WO 1996041185A1 US 9607475 W US9607475 W US 9607475W WO 9641185 A1 WO9641185 A1 WO 9641185A1
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WO
WIPO (PCT)
Prior art keywords
amidine
containing moiety
sample
lipopolysaccharide
amidine containing
Prior art date
Application number
PCT/US1996/007475
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English (en)
Other versions
WO1996041185A9 (fr
WO1996041185B1 (fr
Inventor
David Evans
Satish Jindal
Original Assignee
Perseptive Biosystems, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Perseptive Biosystems, Inc. filed Critical Perseptive Biosystems, Inc.
Publication of WO1996041185A1 publication Critical patent/WO1996041185A1/fr
Publication of WO1996041185B1 publication Critical patent/WO1996041185B1/fr
Publication of WO1996041185A9 publication Critical patent/WO1996041185A9/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

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. Production and release of lipopolysaccharide occurs in patients infected with gram negative
  • Septic shock leads to a cascade of host responses to lipopolysaccharide, importantly, the elaboration of proinflammatory cytokines including tumor necrosis factor and various interleukins by monocytes, ultimately manifesting in the shock syndrome.
  • proinflammatory cytokines including tumor necrosis factor and various interleukins by monocytes
  • Endotoxins are especially problematic in the pharmaceutical industry where federal regulatory agencies require that the production and purification of biologically active proteins be monitored to confirm that processes are functioning within established boundaries. Endotoxins often are introduced into a process from contaminated raw materials or from source material. For
  • endotoxins may be introduced from buffers, salts, chromatographic media, cell culture, fermentation additives, and/or water. Contamination by endotoxins is particularly common when expressing recombinant proteins in bacteria. 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
  • 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, then the ability of the purification process to remove and detect endotoxins must have sufficient capacity to accommodate variability. Because of the wide size distribution of endotoxins, methods that rely on size will be unreliable unless all of the factors that influence size distribution are well controlled.
  • 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 inhibit the response to LPS in monocytes and macrophages, it has been postulated that the 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 from both the hydrophobic region of synthetic peptides together with some cationic character were required for optimal activity in the binding of these peptides to Lipid A.
  • the use of PmxB is
  • PmxB is only able to recognize certain serotypes of endotoxins with significant affinity.
  • 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 to the limitations and disadvantages of the related art.
  • 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
  • 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 detectable moiety.
  • 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 property by which said complex can be separated or identified.
  • 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.
  • 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
  • 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 apparatus for diagnostic or therapeutic applications in specimens having phosphate containing molecules.
  • the invention will be discussed herein as it relates to lipopolysaccharides.
  • Figure 1 A 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
  • Figure 3 depicts the removal of LPS in the presence of various proteins on POROSTM- True Blue columns.
  • 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 the support, and, any lipopolysaccharides present will bind to the amidine containing moiety, thus forming a complex immobilized on the support.
  • the modified amidine containing moieties of the invention are modified for attachment to a support while maintaining their ability to bind lipopolysaccharide or phosphate containing
  • modified amidine containing moieties are modified pentamidines such as True Blue and DAPI.
  • True Blue and DAPI are commercially
  • True Blue 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 specificity than PmxB for the different serotypes of LPS. Additionally, True Blue is flourescent, thereby facilitating detection and quantitation in a suitable assay. Finally, True Blue contains a
  • 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
  • Samples which may be utilized in the claimed invention include water, pharmaceutical preparations, body fluids, including, but not limited to, blood, serum, plasma, urine cerebrospinal
  • claimed invention may be used in the pharmaceutical industry for e.g. removing
  • the invention is particularly useful for removing endotoxins from preparations derived from recombinant proteins.
  • the claimed invention is suitable for removing endotoxins from solutions. For example, on a small scale, a sample solution may simply be exposed to the modified pentamidine moieties of the invention immobilized on a solid support thereby removing endotoxins as the solution
  • the endotoxins if present, will bind to the amidine containing moiety and be retained on the support.
  • amidine containing moiety it is not necessary that the amidine containing moiety be
  • 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 may be gavaged with the support, which would then remove or inactivate the lipopolysaccharide in the gut, which is the major site of absorption This may reduce the level of lipopolysaccharide load in the host system prior to reperfusion and hence provide increased chances for survival.
  • the methods of the invention are to be used to detect the absence
  • a detectable moiety is any moiety capable of detection, suitable for use in the claimed invention, including, but
  • 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 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
  • 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
  • the invention encompass pharmaceutical or diagnostic preparations.
  • a pharmaceutical preparation comprising an amidine containing
  • amidine moieties capable of binding to endotoxins, may be introduced into a host organism.
  • the amidine moieties may then bind to the endotoxins thereby rendering them non-toxic.
  • amidine containing moieties may be used as part of a dialysis type apparatus to
  • Such devices may be any suitable biosensor, such as a biosensor, thereby allowing the capture of lipopolysaccharides.
  • a biosensor such as a biosensor, thereby allowing the capture of lipopolysaccharides.
  • Such devices may be any suitable biosensor, such as a biosensor, thereby allowing the capture of lipopolysaccharides.
  • lipopolysaccharides 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 purposes, or for detection of pyrogenic quantities of LPS in preparations for administration to humans or other animals. It is also contemplated that the
  • support be used in the form of a cartridge or chip. Additionally, one may, for example, by loading
  • kits for detecting the presence, absence or concentration of lipopolysaccharides may be configured to detect clinically relevant lipopolysaccharides' in biological samples.
  • FTTC-lipopolysaccharide was coincubated with the proteins indicated in Fig. 3. Material was injected and run in 50mM Tris, pH 7.0. Material bound to the column was eluted using IN
  • 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 (prior to NaOH elution of bound material).
  • 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 have the advantage that the True Blue should not leach off with use, the columns can be run at higher pressures and flow rates and can be sanitized by treatment with IN NaOH without significant loss of activity. 2.
  • lipopolysaccharide incubated in the absence of resin was included as a control.
  • Support in the absence of the ligand exhibited only marginal removal of lipopolysaccharide when compared to a control sample which was incubated in the absence of
  • 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-mediated color
  • a tandem column set up consisting of an anion exchange plumbed above a reversed-phase
  • the time and dilution factor should be constant under similar conditions; the loss of ligand bound to its target under these conditions should thus be directly related to its dissociation rate (k_ ⁇ ).
  • Pentamidine ⁇ PmxB ⁇ 9-mer ⁇ 10-mer Empirically, the relative off reaction constants obtained by the tandem column method based upon recovery of ligands from the lipopolysaccharide-ligand complex after varying the wash time correlates well with the rank order of the published values for their affinities for this target.
  • 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). Intermediate concentrations of PmxB produced a graded decline in the 9-mer peak. Amounts of the 9-mer and
  • PmxB bound to lipopolysaccharide when plotted against the concentration of PmxB (Fig. 3B), indicated that PmxB competes with binding of the 9-mer in a concentration-dependent fashion
  • Fig. 4 concentration of lipopolysaccharide revealed saturable binding
  • Fig. 4 Hill plots of the data obtained from these curves (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.

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  • Health & Medical Sciences (AREA)
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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 true WO1996041185A1 (fr) 1996-12-19
WO1996041185B1 WO1996041185B1 (fr) 1997-01-16
WO1996041185A9 WO1996041185A9 (fr) 1997-05-15

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PCT/US1996/007475 WO1996041185A1 (fr) 1995-06-07 1996-05-22 Procedes et compositions liant des endotoxines

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000067900A1 (fr) * 1999-05-10 2000-11-16 Prometic Biosciences Ltd. Nouveaux agents de detoxication a base de triazine et leur utilisation
US6774102B1 (en) 1999-09-29 2004-08-10 Gambro Dialysatoren Gmbh & Co. Kg Extracorporeal endotoxin removal method

Citations (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
EP0097463A2 (fr) * 1982-06-16 1984-01-04 Beecham Group Plc Dérivés amidinés
US4491660A (en) * 1980-01-10 1985-01-01 Abbott Laboratories Matrix polymers for binding endotoxins
EP0221308A1 (fr) * 1985-09-30 1987-05-13 Miles Inc. Immobilisation d'acides nucléiques sur les support du nylon dérivé
EP0376480A2 (fr) * 1988-12-27 1990-07-04 Becton, Dickinson and Company Test pour chlamydia
EP0431593A2 (fr) * 1989-12-07 1991-06-12 Daicel Chemical Industries, Ltd. Procédé et agent de séparation

Patent Citations (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
EP0097463A2 (fr) * 1982-06-16 1984-01-04 Beecham Group Plc Dérivés amidinés
EP0221308A1 (fr) * 1985-09-30 1987-05-13 Miles Inc. Immobilisation d'acides nucléiques sur les support du nylon dérivé
EP0376480A2 (fr) * 1988-12-27 1990-07-04 Becton, Dickinson and Company Test pour chlamydia
EP0431593A2 (fr) * 1989-12-07 1991-06-12 Daicel Chemical Industries, Ltd. Procédé et agent de séparation

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000067900A1 (fr) * 1999-05-10 2000-11-16 Prometic Biosciences Ltd. Nouveaux agents de detoxication a base de triazine et leur utilisation
US6773599B1 (en) 1999-05-10 2004-08-10 Prometic Biosciences Ltd. Triazine-based detoxification agents and their use
US7364656B1 (en) 1999-05-10 2008-04-29 Prometic Biosciences Ltd. Triazine-based detoxification agents and their use
US6774102B1 (en) 1999-09-29 2004-08-10 Gambro Dialysatoren Gmbh & Co. Kg Extracorporeal endotoxin removal method

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