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WO2004011556A1 - Marquage specifique de proteines en fonction du site au moyen de colorants de signalisation a base de cyanine - Google Patents

Marquage specifique de proteines en fonction du site au moyen de colorants de signalisation a base de cyanine Download PDF

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Publication number
WO2004011556A1
WO2004011556A1 PCT/GB2003/003196 GB0303196W WO2004011556A1 WO 2004011556 A1 WO2004011556 A1 WO 2004011556A1 GB 0303196 W GB0303196 W GB 0303196W WO 2004011556 A1 WO2004011556 A1 WO 2004011556A1
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WO
WIPO (PCT)
Prior art keywords
group
groups
alkyl
atoms
protein
Prior art date
Application number
PCT/GB2003/003196
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English (en)
Inventor
Graham Cotton
Original Assignee
Amersham Biosciences Uk Limited
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
Priority claimed from GBGB0217556.0A external-priority patent/GB0217556D0/en
Application filed by Amersham Biosciences Uk Limited filed Critical Amersham Biosciences Uk Limited
Priority to CA002493309A priority Critical patent/CA2493309A1/fr
Priority to JP2004523938A priority patent/JP2005534739A/ja
Priority to EP03771163A priority patent/EP1525266A1/fr
Priority to US10/522,675 priority patent/US20050239144A1/en
Priority to AU2003246957A priority patent/AU2003246957A1/en
Publication of WO2004011556A1 publication Critical patent/WO2004011556A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • B is an affinity tag
  • L 1 and L 2 are independently selected from the group:
  • L' is a bond or is a group containing from 1 - 30 linked atoms selected from carbon atoms and optionally one or more groups selected from -NH-, -O- and -CO-NH-; and R" is Ci - C 4 alkyl, C 6 - C 10 aryl, or C 7 - C i5 aralkyl, which may be optionally substituted with sulphonate; or is the group -(CH 2 ) 2 -CONH 2 .
  • the target bonding group F is attached directly to group M.
  • the present invention provides fluorescent labelling reagents comprising a cyanine dye or derivative thereof, that are modified by incorporating a target bonding group and an affinity tag into the molecule.
  • the target bonding group may be selected from a carboxylic acid thioester group or a 1 ,2-aminothiol group. Where the target bonding group is a thioester group, it is selectively reactive with a 1 ,2-aminothiol group on a target molecule, suitably a protein or peptide, or a derivative thereof.
  • the cyanine dye may contain a 1 ,2-aminothiol group for reaction with a thioester group on the target.
  • labelling of peptides and proteins is site-specific, irrespective of the composition of the primary sequence.
  • site-specific labelling can be achieved directly, by incubating the target with the appropriate derivative of the cyanine dye, suitably, the thioester and 1,2- aminothiol derivatives respectively.
  • inclusion of an affinity tag in the labelling reagent allows subsequent purification of the fluorescent dye labelled protein or peptide.
  • R 3 , R 4 , R 5 and R 6 are independently selected from the group consisting of hydrogen, halogen, amide, cyano, nitro, mono- or di-Ci - C 6 alkyl-substituted amino, carbonyl, carboxyl, C ⁇ - C 6 alkyl, C ⁇ - C 6 alkoxy, aryl, heteroaryl, aralkyl and the group -(CH 2 ) m -Y where Y is selected from sulphonate, sulphate, phosphonate, phosphate and quaternary ammonium and m is zero or an integer from 1 to 6; remaining groups R 8 , R 9 and R 10 are independently C ⁇ - CQ alkyl; and remaining groups R 1 and R 2 are independently selected from hydrogen, Ci
  • the compound has the formula (III):
  • A is selected from O and NR 16 where R 16 is the substituted amino radical:
  • remaining groups R 11 , R 12 , R 13 , R 14 and R 15 are independently selected from the group consisting of hydrogen, halogen, amide, cyano, nitro, amino, mono- or di-Ci - C ⁇ alkyl-substituted amino, carbonyl, carboxyl, Ci - C ⁇ alkyl, Ci - C ⁇ alkoxy, aryl, heteroaryl, aralkyl and the group -(CH 2 ) m -Y where Y is selected from sulphonate, sulphate, phosphonate, phosphate and quaternary ammonium and m is zero or an integer from 1 to 6; remaining groups R 8 , R 9 and R 10 are independently Ci - C ⁇ alkyl; remaining group R 17 is selected from hydrogen, Ci - C 4 alkyl and aryl; and remaining group R 18 is selected from Ci - C 6 alkyl, aryl, heteroaryl,
  • Z 1 and Z 2 may be selected independently from the group consisting of phenyl, pyridinyl, naphthyl, anthranyl, indenyl, fluorenyl, quinolinyl, indolyl, benzothiophenyl, benzofuranyl and benzimidazolyl moieties. Additional one, or two fused ring systems will be readily apparent to the skilled person.
  • Z 1 and Z 2 are selected from the group consisting of phenyl, pyridinyl, naphthyl, quinolinyl and indolyl moieties. Particularly preferred Z 1 and Z 2 are phenyl and naphthyl moieties.
  • solubilising groups may be attached by means of a Ci to C 6 alkyl linker chain to said aromatic ring structures and may be selected from the group -(CH 2 )m-Y where Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and m is hereinbefore defined.
  • Alternative solubilising groups may be carbohydrate residues, for example, monosaccharides, or polyethylene glycol derivatives.
  • water solubilising constituents include Ci - C 6 alkyl sulphonates, such as -(CH 2 )3-SO 3 ⁇ and -(CH 2 ) 4 -SO 3 " ⁇
  • Ci - C 6 alkyl sulphonates such as -(CH 2 )3-SO 3 ⁇ and -(CH 2 ) 4 -SO 3 " ⁇
  • one or more sulphonate or sulphonic acid groups attached directly to the aromatic ring structures of a dye of formula (II) or (III) are particularly preferred. Water solubility may be advantageous when labelling proteins.
  • the compound of formula (I) is a fluorescent reporter molecule.
  • none of the substituent groups R in the compounds of formula (II) and (III) contains a nitro group.
  • the compound of formula (I) is non-fluorescent or substantially non-fluorescent dye wherein at least one of the groups R attached to the aromatic ring structures of the compounds of formula (II) and (III) comprises at least one nitro group.
  • the at least one nitro group may be attached directly to the Z 1 and/or Z 2 ring structures.
  • a mono- or di-nitro-substituted benzyl group may be attached to the Z 1 and/or Z 2 ring structures which optionally may be further substituted with one or more nitro groups.
  • non-fluorescent or substantially non-fluorescent cyanine dye or cyanine dye derivatives according to the invention may be used to label one component of a fluorescent donor/acceptor pair in assays involving the detection of binding and/or cleavage events in reactions involving biological molecules, as described in EP 1086179 B1 (Amersham Biosciences UK Limited).
  • the compounds according to the present invention are useful for covalently labelling target biological materials in a site specific manner for applications in biological detection systems.
  • Suitable target materials include proteins, post-translationally modified proteins, peptides, antibodies, antigens, and protein-nucleic acids (PNAs).
  • the reporter moiety may also be conjugated to species which can direct the path of the reporter within or aid entry to or exit from cells (live or dead); such as for example, long alkyl residues to allow permeation of lipophilic membranes, or intercalating species to localise a reporter in a nucleus or other cellular enclave containing double-stranded DNA.
  • a method for labelling a protein of interest wherein said protein contains or is derivatised to contain an N- terminal cysteine comprising: i) adding to a liquid containing said protein a compound of formula (I):
  • D is a dye selected from a cyanine dye or a derivative thereof;
  • B is an affinity tag;
  • F comprises a target bonding group selected from a carboxylic acid thioester group and a 1 ,2-aminothiol group;
  • M is a group adapted for attaching to F
  • L 1 and L 2 are independently selected from the group:
  • R' is hydrogen or Ci - C 4 alkyl, p is 0 - 5, r is 1 - 5 and s is 1 or 2.
  • Q is selected from: -CHR'-, -O- and -CO-NH-, where R' is hereinbefore defined.
  • Covalent labelling using compounds of the present invention may be accomplished with a target having at least one carboxylic acid thioester group or 1 ,2-aminothiol group as hereinbefore defined.
  • the target may be incubated with an amount of a compound of the present invention having at least one group F as hereinbefore defined that can covalently bind with the complementary group of the target material.
  • the target material and the compound of the present invention are incubated under conditions and for a period of time sufficient to permit the target material to covalently bond to the compound of the present invention.
  • the thioester group F may be reacted and form a covalent bond with any of the above target materials that contains, or has been derivatised to contain, a 1 ,2-amino thiol group.
  • the protein of interest may be selected from the group consisting of antibody, antigen, protein, peptide, microbial materials, cells and cell membranes.
  • a method of separating and/or purifying the dye-labelled protein of interest by affinity chromatography utilising the affinity of the affinity tag moiety for an immobilised ligand (or specific binding partner) attached to a support material.
  • Affinity chromatography provides a quick and convenient method to enable the separation of labelled and unlabelled protein molecules under physiological conditions.
  • Proteins labelled with an affinity tag can be selectively bound to an affinity column and any unreacted protein removed by washing the column.
  • Suitable specific binding moieties include avidin or streptavidin (for a biotin tag); immobilised metal ions, for example, Cu(ll), Ni(ll), Fe(ll) and Fe(lll) (for His-tag or iminodiacetic acid).
  • a target peptide or protein containing an N-terminal cysteine residue is agitated with an excess of a cyanine dye thioester derivative, e.g. Cy5-MESNA (Cy5-mercaptoethanesulphonic acid ester), in phosphate buffer (typically 200 mM NaCI, 200 mM sodium phosphate) at ⁇ pH 7.3 - 7.4 containing ⁇ 1.5% MESNA.
  • concentration of the target polypeptide in the labelling reaction is generally between 100 ⁇ M to 10 mM, whilst the Cy5-MESNA is generally present in excess, for example 1.5 to 3-fold molar excess.
  • Cy5-MESNA concentration of Cy5-MESNA
  • concentration of Cy5-MESNA is usually maintained at or above 1 mM.
  • a solution of Cy5-MESNA and MESNA cofactor is directly added to the lyophilised target.
  • the target is first exchanged into an appropriate buffer, which is known not to affect the labelling reaction.
  • An equal volume of a solution of Cy5-MESNA and MESNA thiol cofactor in ligation buffer is then added to the protein to give the desired final concentration of the reactants.
  • the reaction mixture is agitated overnight at room temperature.
  • the reaction time may be lowered to less than one hour for high reactant concentrations or, if the stability of the target polypeptide is an issue, the labelling reaction may be performed efficiently at 4°C.
  • dithiothreitol (DTT) is added to a final concentration of -50 mM and the desired material isolated by affinity chromatography.
  • reagents may be utilised in the labelling reaction to increase product yield if necessary. Examples include, but are not limited to guanidinium chloride, urea, dimethylformamide, dimethylsulphoxide, acetonitrile, triton X-100, octyl glucoside, 1 ,6-hexanediol and glycerol.
  • the ligation reaction using the derivatised cyanine dye according to the presen itt iinnvveennttiioonn mmaayy be optimally performed at between pH 7.0 and pH 8.0 and at temperatures varying between 4°C and 37°C. It is envisaged that such a range of conditions are compatible to the site-specific labelling reaction described herein.
  • the advantage of the present method is that it enables the introduction of an extrinsic label into a proteinacious substrate in a regioselective and specific manner, thus minimising any detrimental effects that labelling may have on the biological function of the protein.
  • the importance of controlling stoichiometry of labelling is important where dye overload may interfere with biological activity.
  • this controlled labelling stoichiometry is directed towards a single terminal site, rather than towards an internal site, this may have the benefit of further maintaining the biological viability of the labelled species.
  • Figure 1 illustrates the products from the labelling reaction of an N-terminal cysteine derivative of the Grb2SH2 domain with the thioester derivative, ⁇ -D- desthiobiotin- ⁇ -Cy5-L-lysine-MESNA according to Examples 3 and 4.
  • the activated dye solution was then added to a stirred solution of 2-mercaptoethanesulphonic acid, sodium salt (MESNA, 40mg, 0.243mmol) in DMF (2mls) and DIEA (30 ⁇ l, 0.1724mmol) under a dry nitrogen atmosphere.
  • MESNA 2-mercaptoethanesulphonic acid, sodium salt
  • H-Cys(Trt)-Gly-Leu-Asp(OtBu)-Lys(Boc)-Arg(Pmc)-Gly-Cys(Trt)-Gly- rink amide resin was synthesised using a commercially available Applied Biosystems Model 433A automated peptide synthesiser using FastMocTM chemistry, following the instrument manufacturer's recommended procedures throughout.
  • the peptide was synthesised on a 0.25 millimolar scale employing O-(benzotriazol-1 -yl)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate ( ⁇ BTU) as the activating agent.
  • the reduced reaction mixture was then diluted 1 :5 with 0.1%TFA in water and purified by reverse phase HPLC [Phenomenex Jupiter C18, eluent A: 0.1 %TF A/water, eluent B: 0.1%TFA acetonitrile, 5-50% B over 30mins @ 1ml /min, UV at 214nm, 650nm].
  • the two components of the reaction mixture were identified as : Cy5- Cys-Gly-Leu-OH, mono-isotopic mass: C 44 H 60 N5O11S3 requires 930.3451.
  • reaction mixture was poured into diethyl ether to give a brown gum.
  • the supernatant was decanted off and the gum again treated with diethyl ether. No solid formed.
  • the gum was dried under reduced pressure and the product, D-desthiobiotinamidocaproate N-hydroxy succinimidyl ester was used directly in the next dye coupling reaction, assuming a theoretical yield of 62mg.
  • ⁇ -D-Desthiobiotin- ⁇ -Cy ⁇ -L-lysine-OH 10mg, ⁇ . ⁇ mol
  • PyAOP 10mg, 19.2 ⁇ mol
  • MESNA ⁇ mg, 0.30mmol
  • DIEA 10 ⁇ l, 0.06mmol
  • N-Cys-Grb2SH2 N-terminal cysteine Grb2SH2 (N-Cys-Grb2SH2) (200 ⁇ M in PBS buffer pH 7.2) (200 ⁇ l) was added ⁇ -D-desthiobiotin- ⁇ -Cy5-L-lysine-MESNA (2mM in reaction buffer) (200 ⁇ l).
  • N-Cys-Grb2SH2 was prepared using recombinant techniques.
  • the reaction buffer consisted of phosphate buffer (200mM), pH 7.2 containing sodium chloride (200mM) and 4% MESNA.
  • the reaction mixture was left at RT for 12 hrs, wrapped in foil to protect from light. The reaction was then quenched with dl-dithiothreitol (final concentration 60mM).

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  • Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
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  • Biomedical Technology (AREA)
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  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
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  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Indole Compounds (AREA)

Abstract

L'invention concerne des composés représentés par la formule (I), dans laquelle D est un colorant choisi parmi un colorant cyanine ou un dérivé de celui-ci, B représente un marqueur d'affinité ; F comprend un groupe se liant avec la cible, choisi entre un groupe thioester d'acide carboxylique et un groupe 1,2-aminothiol ; M représente un groupe capable de se lier avec F, et L1 et L2 comprennent chacun indépendamment l'un de l'autre un groupe contenant de 1 à 40 atomes liés choisis dans les atomes de carbone, qui peuvent éventuellement comprendre un ou plusieurs groupes choisis parmi -NR'-, -O-, -CH=CH-, -CO-NH- et les groupes phénylényle, R' étant choisi entre l'hydrogène et l'alkyle C1 - C4. L'invention concerne également des procédés consistant à réaliser une fixation directe du groupe de signalisation formé du colorant cyanine soit sur l'extrémité N-terminale, soit sur l'extrémité C-terminale d'un peptide ou d'une protéine de synthèse ou recombinés, et leur dérivées, selon un mode de liaison spécifique du site, associée à une purification de la molécule marquée résultante.
PCT/GB2003/003196 2002-07-30 2003-07-28 Marquage specifique de proteines en fonction du site au moyen de colorants de signalisation a base de cyanine WO2004011556A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA002493309A CA2493309A1 (fr) 2002-07-30 2003-07-28 Marquage specifique de proteines en fonction du site au moyen de colorants de signalisation a base de cyanine
JP2004523938A JP2005534739A (ja) 2002-07-30 2003-07-28 シアニン染料リポーターを用いたタンパク質の部位特異的標識
EP03771163A EP1525266A1 (fr) 2002-07-30 2003-07-28 Marquage specifique de proteines en fonction du site au moyen de colorants de signalisation a base de cyanine
US10/522,675 US20050239144A1 (en) 2002-07-30 2003-07-28 Site-specific labelling of proteins using cyanine dye reporters
AU2003246957A AU2003246957A1 (en) 2002-07-30 2003-07-28 Site-specific labelling of proteins using cyanine dye reporters

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB0217556.0A GB0217556D0 (en) 2002-07-30 2002-07-30 Site-specific labelling of proteins using cyanine dye reporters
GB0217556.0 2002-07-30
US10/241,333 US20040023408A1 (en) 2002-07-30 2002-09-11 Site-specific labelling of proteins using cyanine dye reporters
US10/241,333 2002-09-11

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WO2004011556A1 true WO2004011556A1 (fr) 2004-02-05

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PCT/GB2003/003196 WO2004011556A1 (fr) 2002-07-30 2003-07-28 Marquage specifique de proteines en fonction du site au moyen de colorants de signalisation a base de cyanine

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EP (1) EP1525266A1 (fr)
JP (1) JP2005534739A (fr)
AU (1) AU2003246957A1 (fr)
CA (1) CA2493309A1 (fr)
WO (1) WO2004011556A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007017602A2 (fr) * 2005-08-11 2007-02-15 Laboratoires Synth-Innove Marqueurs, leur procede de fabrication et leurs applications

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009014177A1 (fr) * 2007-07-25 2009-01-29 Ajinomoto Co., Inc. Procédé d'élimination sélective d'un dérivé dibenzofulvène
JP5982676B2 (ja) * 2011-02-28 2016-08-31 学校法人 いわき明星大学 抗ｐａｆ活性を有するビオチニル化ペプチド化合物
WO2018138139A1 (fr) 2017-01-24 2018-08-02 Gentian As Procédé d'évaluation des récepteurs transmembranaires des cellules sanguines

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5569587A (en) * 1986-04-18 1996-10-29 Carnegie Mellon University Method for labeling and detecting materials employing luminescent arysulfonate cyanine dyes
WO1999064519A1 (fr) * 1998-06-11 1999-12-16 Amersham Biosciences Corp Procede et reactif de test de transfert d'energie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5569587A (en) * 1986-04-18 1996-10-29 Carnegie Mellon University Method for labeling and detecting materials employing luminescent arysulfonate cyanine dyes
WO1999064519A1 (fr) * 1998-06-11 1999-12-16 Amersham Biosciences Corp Procede et reactif de test de transfert d'energie
EP1086179B1 (fr) * 1998-06-11 2003-04-23 Amersham Biosciences UK Limited Procede et reactif de test de transfert d'energie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCHULER BENJAMIN ET AL: "Specific labeling of polypeptides at amino-terminal cysteine residues using Cy5-benzyl thioester", BIOCONJUGATE CHEMISTRY., vol. 13, no. 5, 18 July 2002 (2002-07-18), AMERICAN CHEMICAL SOCIETY, WASHINGTON., US, pages 1039 - 43, XP002259205, ISSN: 1043-1802 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007017602A2 (fr) * 2005-08-11 2007-02-15 Laboratoires Synth-Innove Marqueurs, leur procede de fabrication et leurs applications
FR2889700A1 (fr) * 2005-08-11 2007-02-16 Synthinnove Lab Marqueurs, leur procede de fabrication et leurs applications
WO2007017602A3 (fr) * 2005-08-11 2007-08-02 Synth Innove Lab Marqueurs, leur procede de fabrication et leurs applications
US8034626B2 (en) 2005-08-11 2011-10-11 Laboratoires Synth-Innove Labels, their production process and their uses

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EP1525266A1 (fr) 2005-04-27
CA2493309A1 (fr) 2004-02-05
AU2003246957A1 (en) 2004-02-16
JP2005534739A (ja) 2005-11-17

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