EP1194529A1 - Composition destinee a la conservation d'adenovirus recombinants infectieux - Google Patents

Composition destinee a la conservation d'adenovirus recombinants infectieux

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Publication number
EP1194529A1
EP1194529A1 EP00917147A EP00917147A EP1194529A1 EP 1194529 A1 EP1194529 A1 EP 1194529A1 EP 00917147 A EP00917147 A EP 00917147A EP 00917147 A EP00917147 A EP 00917147A EP 1194529 A1 EP1194529 A1 EP 1194529A1
Authority
EP
European Patent Office
Prior art keywords
acid
liquid
tris
composition
buffer solution
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00917147A
Other languages
German (de)
English (en)
French (fr)
Inventor
Francis Blanche
Shian-Jiun Shih
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centelion SAS
Original Assignee
Aventis Pharma SA
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 FR9904443A external-priority patent/FR2791999B1/fr
Application filed by Aventis Pharma SA filed Critical Aventis Pharma SA
Publication of EP1194529A1 publication Critical patent/EP1194529A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10351—Methods of production or purification of viral material

Definitions

  • the present invention relates to the storage of adenovirus in a stabilized and storable form and the liquid or frozen compositions intended for this storage.
  • adenovirus in stable compositions, undergoes two types of alteration simultaneously and rapidly over time: on the one hand, an aggregation (coagulation) of the particles in the form of clusters or filaments, which is an extremely serious because it is irreversible, and on the other hand a disintegration of the icosahedral structure of the capsids themselves (by a lysis of the capsids and release of the capsomers in the medium).
  • sucrose does not systematically provide stabilization at + 4 ° C, and depending on the case, stabilization at this temperature never exceeds a period of 2 weeks, or at best less than 2 months.
  • Tris buffer and glycerol In Human Gene Therapy, 7, 1693-99 (1996) is also used a storage formulation based on Tris buffer and glycerol, and comprising magnesium chloride. This formulation is stored at -80 ° C. More generally, the Tris-based formulations described in the literature to date systematically contain magnesium chloride (1 mM in general) or / and a salt at physiological concentration (generally NaCl at 150 mM).
  • the virus is stored systematically at -70 ° C / -80 ° C.
  • the infectious recombinant adenoviruses could be stored in an aqueous medium, at temperatures between +4 and + 20 ° C., in the form of a suspension. in a buffer solution capable of fixing the pH of the medium at slightly alkaline values, added with glycerol and without the addition of divalent metal cations or alkaline cations. More specifically, the pH of the medium is fixed between 8.0 and 9.6.
  • Liquid or frozen compositions containing the adenoviral particles in a buffer solution supplemented with glycerol fall within the scope of the present invention. These compositions have the considerable advantage of good stability, but also of being particularly suitable for the storage for a long time of high concentrations of viral particles.
  • the buffer solution capable of fixing the pH of the medium between 8.0 and 9.6 consists either of an acid / base system comprising Tris [tris (hydroxymethyl) aminomethane], or lysine and an acid chosen from a strong acid (hydrochloric acid for example) or a weak acid (maleic acid, malic acid, or acetic acid for example), or from an acid / base system comprising Hepes [acid 2- ( 4- (2-hydroxyethyl piperazine) -1-yl) ethanesulfonic] and a strong base (soda for example).
  • an acid / base system comprising Tris [tris (hydroxymethyl) aminomethane], or lysine and an acid chosen from a strong acid (hydrochloric acid for example) or a weak acid (maleic acid, malic acid, or acetic acid for example), or from an acid / base system comprising Hepes [acid 2- ( 4- (2-hydroxyethyl piperazine) -1-yl) ethan
  • the pH is fixed between 8.4 and 8.8 and even more particularly at 8.4 (value measured at 25 ° C).
  • the concentration of the buffer solution is determined to exert the buffer effect within a limit and a volume or the pH value is not affected.
  • the molar concentration of acid + base can vary from 10 to 500 mM, preferably from 20 to 100 mM, and more particularly it is fixed at 20 mM.
  • the Tris / HCl buffer solution at a concentration of 20 mM gives particularly satisfactory results.
  • the composition contains 10 to 50% of glycerol, and preferably between 20 and 25% (volumes to volumes).
  • compositions according to the invention can also optionally contain other adjuvants.
  • the latter can be chosen from polymers (polyethylene glycols, for example PEG 400, PEG 8000), pluronics (Pluronic F68 for example) in particular at a rate of approximately 1 to 20% by weight, polysorbates (in particular Tween-20 , for example at 0.01 to 1% by weight), or chosen from sugars such as for example sucrose, mannitol, or dextrose (in particular at a rate of approximately 5 to 10%) or also among alcohols (in particular ethanol) at a rate of 1 to 10% by volume.
  • polymers polyethylene glycols, for example PEG 400, PEG 8000
  • pluronics Pluronic F68 for example
  • polysorbates in particular Tween-20 , for example at 0.01 to 1% by weight
  • sugars such as for example sucrose, mannitol, or dextrose (in particular at a rate of approximately 5 to 10%) or also among alcohols (in
  • compositions according to the invention are particularly suitable for the storage of high concentration adenoviral preparations. Indeed, the compositions thus stabilized make it possible to maintain the viral particles in liquid aqueous suspension (in particular between +4 and + 20 ° C.) while preserving the infectious power of the virus and this even at very high viral concentrations (from values of 1E8 pv / ml up to 1E12 pv / ml or up to 1E13 pv / ml and possibly even up to 5E13 pv / ml).
  • the virus can also be frozen at -20 ° C., thus stored for several months or longer, then thawed in this formulation without damage either to its structure or to its infectious power.
  • compositions can be prepared by suspending in the buffer solution, adenoviral particles initially obtained in aqueous solution, then purified, followed by the addition of glycerol and optionally by the addition of an adjuvant such as cited above.
  • the infectious recombinant adeno virus can be obtained according to the usual methods of production in cells of transcomplementing packaging lines, for example cells of line 293 or of line PER-C6.
  • the viral particles can then be purified by centrifugation in a cesium chloride gradient as described for example in Journal of General Virology, 34, 19-35 (1977). More preferably, the viral particles are purified by liquid chromatography in anion exchange mode, gel filtration, hydrophobic mode or by metal chelation. Purification by anion exchange is particularly advantageous since it makes it possible to obtain, in a single chromatography step, a pure viral preparation, free of proteins, nucleic acids, and other impurities and metabolites originating from the producer cell, and free of compounds provided by the culture medium.
  • adenoviral particles are then formulated in the selected preservation buffer, using in particular dialysis, diafiltration, or gel filtration chromatography methods.
  • the composition thus obtained can optionally be frozen and stored at a desired storage temperature (for example -20 ° C.), but this operation is however not essential for long-term storage, the compositions being stable in the state liquid between +4 and + 20 ° C.
  • compositions according to the invention are stable, without significant degradation [physical and biological stability (infectious power)] for a period of at least 12 months at + 4 ° C or at least 5 months at + 20 ° C.
  • physically stable solution is more particularly understood to mean a solution which does not show the appearance of coagulation, of sedimentation of particles or of precipitate (by visual estimation, by measurement of optical density, by analysis by electron microscopy, or by l analysis of particle size distribution) after the considered storage period.
  • the present invention relates more particularly to the storage of infectious adenovirus in a stabilized form and the liquid or frozen compositions intended for this storage.
  • the formulation according to the invention has the advantage of being the first composition produced allowing the storage of adenoviruses in liquid form at temperatures of +4 to + 20 ° C. while having the possibility of having a high viral concentration.
  • the invention is particularly interesting in its application to recombinant adenoviruses, but it can be applied in general to all adenoviruses (wild or recombinant).
  • adenoviruses can be mentioned, which can advantageously be stored in a composition according to the invention: all of the wild human adenoviruses, belonging to the six known subgroups called A, B, C, D , E, and F, and more particularly all 49 different serotypes of human adenoviruses making up these six subgroups.
  • the invention may be applied to simian wild viruses, bovines, equines, pigs, sheep, or canines belonging to the family of Adenoviridae.
  • the invention may be applied to mutant viruses originating from wild viruses belonging to the family of Adenoviridae.
  • recombinant adenovirus vectors to which the present invention can be applied, there may be mentioned all the modified adenoviruses comprising one or more deletion in the region of the genome called El, or in the region E2, or in the region E3 or in the region E4 , as well as recombinant viruses with multiple deletions combined in the above regions, as well as completely deleted recombinant viruses (called gutless) [FASEB, 11, 615 (1997)].
  • the present invention also applies to recombinant adenoviral vectors further comprising a nucleic acid of interest.
  • the nucleic acid of interest can be inserted at different sites in the adenovirus genome. Advantageously, it is inserted at the level of the region E1, E3, or E4. However, it is clear that other sites can be used. In particular, access to the nucleotide sequence of the genome allows those skilled in the art to identify regions making it possible to insert the nucleic acid of interest.
  • the nucleic acid of interest can be any DNA sequence introduced, in particular any sequence whose transfer and / or expression in the target cell is sought. In particular, it may contain one or more therapeutic genes and / or one or more genes coding for antigenic proteins.
  • the therapeutic genes which can thus be transferred are all the genes whose transcription and possibly translation into the target cell generate products having a therapeutic effect.
  • the therapeutic products mention may more particularly be made of enzymes, blood derivatives, hormones, limphokines: interleukins, interferons, TNF, etc. (WO93 / 19191), growth factors, neuro transmitters or their precursors or enzymes. synthesis, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc., apolipoproteins: ApoAl, ApoIV, ApoE, etc.
  • dystrophin or a minidystrophin WO93 / 062273
  • the genes used to control restenosis GAX, NOS, etc.
  • the tumor suppressor genes p53, Rb, Rapl A, DCC, k-rev , etc
  • the genes coding for factors involved in coagulation Factors VII, VIII, IX, suicide genes: TK, etc.
  • natural or artificial immunoglobulins Fab, ScFv (WO94 / 29446)
  • anti-apoptotic genes AKT, etc.
  • the therapeutic gene can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs.
  • Such sequences can for example be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent application EP 140 308.
  • the present invention applies to recombinant adenoviral vectors allowing the production of retroviruses [Tumor Targetting, 3, 59 (1998)], to adenoviruses comprising one or more modifications in one or more proteins constituting the viral capsid, in particular fiber (protein IV) and hexon protein (protein II), or adenoviruses devoid of fiber [Journal of Virology, 73, 1601 (1999)], each of these modifications having been introduced with the aim of modifying the natural tropism of adenovirus [Current Opinion in Biotechnology, 8, 583 (1997)].
  • compositions according to the invention are particularly advantageous because they can be used for the preparation of a medicament intended for therapeutic or prophylactic treatment by gene therapy.
  • Infectious viruses have many applications among which we can cite their use in the field of vaccination and in the field of gene therapy. In these applications, after transfer of their genetic material (RNA or DNA) into the host cell, the viruses use the cellular machinery of the infected cell to carry out the synthesis of proteins encoded by their own genome, and thus induce the specific biological effect research.
  • infectious recombinant viruses carrying a gene of therapeutic interest are used to transfer this gene into specific cells of the organ of the patient to be treated.
  • a wide variety of therapeutic protocols have been described and are currently under clinical evaluation for transferring and expressing therapeutic genes using viral vectors.
  • non-replicative adenoviral vectors have been widely developed in recent years for the transfer of genes coding for therapeutic proteins.
  • these genes mention may be made of the p53 tumor suppressor gene, which is involved in the control of cell proliferation.
  • Various protocols for clinical trials of transfer of the p53 gene using adenoviruses in humans are currently being developed in anticancer indications.
  • adenoviral vectors as therapeutic agents can only be envisaged if methods are available which allow the preparations to be preserved and stored for sufficient long periods without significant loss of infectious power of the viruses in question. This is why the present invention is particularly interesting.
  • a viral suspension is prepared as follows: the 293 cells are cultured in a CellCube (Costar) in DMEM medium supplemented with 10% fetal calf serum. When they reach confluence, the cells are infected at a multiplicity of infection of 2 with an aliquot of the working library of the adenovirus expressing the p53 gene. Five days after infection, the supernatant of production is collected by drainage and clarified by passage through a train of filters of decreasing porosity 10/1 / 0.8-0.2 ⁇ m. This supernatant is then concentrated 20 times in volume by tangential ultrafiltration on a Millipore membrane having a cutoff threshold of 300 kDa.
  • the virus is then purified by chromatography on a balanced Source 15Q column and eluted with a sodium chloride gradient in 20 mM Tris / HCl buffer, pH 8.0.
  • the virus peak is collected, and the virus is concentrated by tangential ultrafiltration on a Millipore membrane having a cutoff threshold of 300 kDa.
  • the viral preparations thus obtained are of very high purity and contain ⁇ 50 ng of serum bovine albumin and ⁇ 10 ng of DNA of the host cell for 1E12 viral particles.
  • the virus is then formulated in the various buffers selected.
  • the buffer change is carried out by chromatography on a PD-10 column (Amersham-Pharmacia Biotech) filled with Sephadex G-25 balanced and eluted with the selected buffer according to the supplier's instructions. After dosing by chromatography, the concentration of the virus is adjusted if necessary to the target value by dilution in the selected formulation buffer.
  • the viral stability in the various formulations is then studied by placing, for each of the selected formulations, 2 ml of viral suspension in a polypropylene tube at the temperature studied (-20 ° C, + 4 ° C, or + 20 ° C). The samples are then stored at this temperature for a period determined for each of the experiments in question (see examples below).
  • the analyzes by electron microscopy are carried out by depositing the solution to be analyzed on a carbon grid which is then treated in negative staining with 1.5% uranyl acetate.
  • the apparatus used for these analyzes is a Jeol 1010 electron microscope operating at a voltage of 50 kV to 100 kV.
  • Viral particle size distribution analyzes are performed by photon correlation spectroscopy (PCS) using a Coulter N4 + device (Coultronics).
  • HPLC analyzes high performance liquid chromatography
  • This column mounted on an HPLC system equipped with a UV / visible detection system operating in the absorbance range 200- 300 nm, is used for the separation and the quantification of the viral particles.
  • the column is equilibrated at 30 ° C in a 20 mM Tris / HCl buffer, pH 7.5 at a flow rate of 1.5 ml / min.
  • the sample to be analyzed containing the viral particles is injected onto the column.
  • the column is washed with 5 volumes of the same buffer, and the fixed species are eluted with a linear gradient of 0 to 1 M of sodium chloride in the 20 mM Tris / HCl buffer, pH 7.5 over 30 column volumes. At the end of the gradient, the column is washed with 2 volumes of 0.5 N sodium hydroxide column before re-equilibration for the next analysis.
  • a standard curve at 260 nm is constructed with a preparation of adenovirus particles purified by chromatography. This standard preparation was previously titrated into particles by its absorbance at 260 nm in a 0.1% SDS solution using the conversion factor of 1 x 10 particles per unit of absorbance at 260 nm). The samples are filtered through a filter (0.22 ⁇ m) before analysis by chromatography.
  • the adenovirus titration technique is described by FL Graham et al., Molecular Biotechnology, 3, 207 (1995).
  • the technique for measuring the expression of the penton protein by immunotitration followed by quantification by flow cytometry is described in Boyle et al., "Determination of adenoviral vector activity using an immunotitration method", poster presented in "5 , h annual meeting of viral vector and vaccines, (1998) ”.
  • compositions according to the invention Storage at + 4 ° C of compositions according to the invention; physical stability of viral particles:
  • the infectious power of viral particles is measured by the capacity of the particles to express the transgene which they contain and to lead to the expression of the corresponding protein.
  • the protein here is the penton protein (protein III) which is detected by immunotitration using a labeling method with an antibody (anti-penton) followed by an analysis by flow cytometry. The result obtained is expressed in infection units per ml of solution (IU / ml).
  • the calculation of the pv / IU ratio represents a second way of expressing the evolution of the infectious titer over time. Under the experimental conditions used, this ratio has a value of approximately 20 ⁇ 10 for a viral preparation freshly obtained before storage.
  • the table below shows the infectious power of the particles formulated as glycerol or sucrose after 6 or 12 months of storage at + 4 ° C or after 1 month of storage at + 4 ° C then 3.5 months of storage at +20 ° C.
  • the low concentration formulation (0.36 x 10 12 pv / ml) sees its title decrease after a storage of one month at + 4 ° C followed by a storage of 3.5 months at + 20 ° C.
  • Formulations containing sucrose are totally unsuitable when the concentration is increased.
  • the particles appear native, full, whole, and symmetrical. Virtually no free subunit is detectable in the medium.
  • the particles are for the most part aggregated either into masses containing approximately 50 particles, or into filamentous structures. Some particles have lost part of their capsomers and have a more rounded or ovoid structure. The capsomers and the fibers released into the medium remain completely dispersed and are very easily observable. This is observed both at + 4 ° C and + 20 ° C. After 12 months at 4 ° C, the particles stored in 20% glycerol appear unchanged (solid, whole and symmetrical).
  • the infectivity of the particles is determined by the titration in pfu / ml.
  • pfu plaque forming unit
  • the term pfu corresponds to the determination of the infectious power of an adenovirus solution. This determination is made by infection of an appropriate cell culture, and measures, generally after 15 days of incubation, the number of plaques of infected cells. These dosages are based on biological methods and the values obtained are to a certain extent a function of the operating conditions used [J. Nirol., 70, 7498 (1996)].
  • the table below shows the infectious titer, measured in a pfu test, of a viral preparation stored for 2 months at -20 ° C.
  • the value of the measurement of the infectious titre, and more particularly the pv / pfu ratio, indicates that the viral particles were not altered by the freeze-thaw stage, nor by the storage for 2 months at -20 ° C. (This ratio has a value of approximately 20 to 30 for the initial viral preparation).
  • compositions according to the invention Storage at + 4 ° C of compositions according to the invention; physical stability of viral particles:

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
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  • Virology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
EP00917147A 1999-04-09 2000-04-07 Composition destinee a la conservation d'adenovirus recombinants infectieux Withdrawn EP1194529A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
FR9904443 1999-04-09
FR9904443A FR2791999B1 (fr) 1999-04-09 1999-04-09 Composition destinee a la conservation d'adenovirus recombinants infectieux
US13212099P 1999-04-30 1999-04-30
US132120P 1999-04-30
PCT/FR2000/000879 WO2000061726A1 (fr) 1999-04-09 2000-04-07 Composition destinee a la conservation d'adenovirus recombinants infectieux

Publications (1)

Publication Number Publication Date
EP1194529A1 true EP1194529A1 (fr) 2002-04-10

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Application Number Title Priority Date Filing Date
EP00917147A Withdrawn EP1194529A1 (fr) 1999-04-09 2000-04-07 Composition destinee a la conservation d'adenovirus recombinants infectieux

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US (2) US6734008B2 (cs)
EP (1) EP1194529A1 (cs)
JP (1) JP2002541792A (cs)
KR (1) KR20020013521A (cs)
CN (1) CN1347450A (cs)
AU (1) AU778894B2 (cs)
BR (1) BR0009595A (cs)
CA (1) CA2364562A1 (cs)
CZ (1) CZ20013560A3 (cs)
HK (1) HK1045716A1 (cs)
HU (1) HUP0200821A3 (cs)
IL (2) IL145447A0 (cs)
MX (1) MXPA01010124A (cs)
NO (1) NO20014765D0 (cs)
NZ (1) NZ514731A (cs)
PL (1) PL351553A1 (cs)
WO (1) WO2000061726A1 (cs)

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CN1347450A (zh) 2002-05-01
WO2000061726A1 (fr) 2000-10-19
CZ20013560A3 (cs) 2002-01-16
PL351553A1 (en) 2003-05-05
KR20020013521A (ko) 2002-02-20
NO20014765L (no) 2001-10-01
JP2002541792A (ja) 2002-12-10
IL145447A0 (en) 2002-06-30
HUP0200821A3 (en) 2004-11-29
US20040191909A1 (en) 2004-09-30
AU778894B2 (en) 2004-12-23
HK1045716A1 (zh) 2002-12-06
IL145447A (en) 2006-10-05
CA2364562A1 (fr) 2000-10-19
NZ514731A (en) 2004-03-26
HUP0200821A2 (hu) 2002-07-29
AU3825500A (en) 2000-11-14
NO20014765D0 (no) 2001-10-01
US20020061592A1 (en) 2002-05-23
BR0009595A (pt) 2001-12-26
MXPA01010124A (es) 2002-06-26
US6734008B2 (en) 2004-05-11

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