EP1734928A2 - Serumstabile amphotere liposomen - Google Patents

Serumstabile amphotere liposomen

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Publication number
EP1734928A2
EP1734928A2 EP05740433A EP05740433A EP1734928A2 EP 1734928 A2 EP1734928 A2 EP 1734928A2 EP 05740433 A EP05740433 A EP 05740433A EP 05740433 A EP05740433 A EP 05740433A EP 1734928 A2 EP1734928 A2 EP 1734928A2
Authority
EP
European Patent Office
Prior art keywords
liposomal
formulation according
chol
molar composition
membrane
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Withdrawn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
EP05740433A
Other languages
German (de)
English (en)
French (fr)
Inventor
Gerold Endert
Yvonne Kerwitz
Monika Fellermeier
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.)
Marina Biotech Inc
Original Assignee
Novosom AG
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 DE200410016020 external-priority patent/DE102004016020A1/de
Application filed by Novosom AG filed Critical Novosom AG
Publication of EP1734928A2 publication Critical patent/EP1734928A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/902—Specified use of nanostructure
    • Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/906—Drug delivery
    • Y10S977/907—Liposome
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2984—Microcapsule with fluid core [includes liposome]

Definitions

  • the invention relates to amphoteric liposomal formulations which show particular serum stability and are suitable for the intracellular delivery of oligonucleotides.
  • RNA short interfering ribonucleic acids
  • Another approach is to use a carrier that protects the siRNA from attack by enzymes and can transport it to the site of action.
  • Liposomes have been used as pharmaceutical carriers for drugs for some time.
  • Vdrformed liposomes and nucleic acids are also produced for the above-mentioned applications.
  • the complex formation or the mostly not stable in serum Liposomal formulations mean that the stability of the oligonucleotides is not guaranteed over a long period of time.
  • Amphoteric liposomes are a new class of liposomes that offer advantageous properties over conventional liposomes and purely cationic systems for intracellular delivery. Negatively charged active ingredients, such as the Nucleic acids can be effectively packed inside these liposomes, although the total charge of the liposomes remains negative at physiological pH. By changing the ambient pH, as occurs in the endosomal uptake of liposomes in cells, from neutral to slightly acidic, the charge of the amphoteric liposomes also changes from anionic to slightly cationic and the advantages of a cationic transfection reagent are exploited. These liposomes are described for the first time in WO 02/66012 A2. WO 02/66490 and WO 02/66489 (WO 03/070220 and WO 03/070735) present pH-sensitive lipids which are suitable for the construction of amphoteric liposomes.
  • amphoteric liposomes which enclose small oligonucleotides, such as siRNA and / or antisense molecules, in their interior and do not or only release a small part under serum conditions and are therefore suitable for parenteral administration ,
  • the invention solves the problem comprehensively by providing liposomal formulations with an aqueous interior
  • amphoteric lipids with a proportion of 5-30 mol%, or mixtures of cationic and anionic lipids with a total proportion of at most 50 mol%, the formulation as an active ingredient in the aqueous interior comprising at least one oligonucleotide.
  • the oligonucleotides relevant for this embodiment of the invention are composed of 5-100, preferably 5-40 and particularly preferably 10-25 nucleotides or base pairs.
  • the oligonucleotides can be present as a single strand (e.g. antisense oligonucleotides), as a double strand (e.g. small-interfering RNA, decoy-oligonucleotides) or in complex folds (e.g. aptamers, Spiegelmers, ribozymes).
  • oligonucleotides relevant to this invention are made up of deoxyribonucleotides or ribonucleotides and their chemically modified derivatives, such as phosphorothioate DNA (PS), 2'-O-methyl RNA (OMe), 2'-O-methoxy-ethyl RNA (MOE) , Peptide nucleic acid (PNA), N3'-P5 'Phosphoroamidate (NP), 2'-fluoro-arabino nucleic acid (FANA), Locked nucleic acid (LNA), Morpholino phosphoroamidate (MF), Cyclohexene nucleic acid (CeNA), Tricyclo-DNA (tcDNA)).
  • PS phosphorothioate DNA
  • OMe 2'-O-methyl RNA
  • MOE 2'-O-methoxy-ethyl RNA
  • PNA Peptide nucleic acid
  • NP N3'-P5 'Phosphor
  • aptamers or Spiegelmers are included in the liposomes.
  • Aptamers are DNA or RNA-based oligonucleotides with a complex three-dimensional structure. Due to this structure, aptamers can bind to protein targets in a very specific and highly affine manner and thus have a therapeutic, usually extracellular, effect. Their functionality is almost identical to that of monoclonal antibodies.
  • Spiegelmers are made up of L-ribose and L-2'-deoxyribose units. Just like aptamers, these mirror-image nucleic acids bind specifically to protein targets. Due to the chiral inversion, Spiegelmers, in contrast to conventional D-oligonucleotides, have increased stability against enzymatic degradation.
  • the basic structure of the amphoteric liposomes according to the invention is formed by neutral framework lipids which have a membrane share of 5 to 65 mol%, preferably 10 to 60 mol%.
  • Suitable lipids are phosphatidylcholines, such as DMPC, DPPC, DSPC, DOPC and POPC, which can be of synthetic as well as natural or semi-synthetic origin.
  • the liposomes according to the invention preferably have a content of 30 to 50 mol%, preferably 35 to 45 mol%, of cholesterol in the membrane.
  • lipids that can be used as charge carriers for amphoteric liposomes are derived from the basic chemical structure of cholesterol (for example the compounds disclosed in WO 02/66490 and WO 02/6648). Surprisingly, it was found that these cholesterol derivatives in general, especially MoChol, HisChol, Chems, HistChol, do not have the stabilizing effect of native cholesterol, so it is advantageous if the sum of all sterol-based lipids does not exceed 60 mol%.
  • amphoteric charge behavior can be formed in two ways: by using amphoteric lipids or by suitable mixtures of pH-sensitive cationic and anionic lipids, as are disclosed in WO 02/066012. If amphoteric lipids are used, the membrane part is between 5 and 30 mol%, more preferably between 5 and 20 mol%, in the case of mixtures the total charge carrier fraction is preferably not more than 50 mol%, more preferably between 15 and 45 mol%. In a preferred embodiment of the invention, amphoteric formulations are composed as follows:
  • - Cholesterol with 35 - 45 mo_% - amphoteric lipid, selected from the group HistChol, HistDG, isoHistSuccDG, Acylcamosin, HCChol with 5 - 20 mol%.
  • formulations are composed as follows: base lipid selected from the group DMPC, DPPC, DSPC with 10 to 50 mol%
  • cationic and anionic lipids of which at least one is pH-sensitive, selected from groups a) cationic: DMTAP, DPTAP, DOTAP, DC-Chol, MoChol, HisChol, DPIM, CHIM, DORIE, DDAB, DAC-Chol, TC- Chol, DOTMA, DOGS, C (18) 2Gly + N, N-dioctadecylamidoglycin, CTAP, CpyC, DODAP, and DOEPC with 5 to 40 mol%.
  • the formulation and its composition are selected from Table 3.
  • formulations according to the invention for use of the formulations according to the invention as a pharmaceutical carrier, it may be expedient to add substances which increase the shelf life and which serve to regulate the osmotic pressure.
  • the liposomes according to the invention can be produced by methods according to the prior art, such as, for example, extrusion through membranes of defined pore size, ethanol injection or high-pressure homogenization. Exemplary designs are given in the examples.
  • Active substance not included is separated off. Suitable separation processes are used for this, so that at least 90% of the active substance is enclosed in the liposome and less than 10% of the active substance is outside the liposome. Chromatographic methods, centrifugation, dialysis or ultrafiltration can be used for this.
  • the liposomal formulations according to the invention can be used for the therapeutic treatment of a mammal. They can also be used for the therapeutic treatment of people.
  • the liposomal formulations according to the invention are particularly suitable for parenteral administration, preferably for intravenous administration.
  • the invention accordingly also relates to a kit which optionally comprises the liposomal formulation according to the invention together with a suitable carrier, and to the use of the kit in diagnosis and therapy.
  • DOTMA 1-dioIeyloxypropyl) -N, N, N-trimethylammonium chloride
  • DOTAP (1,2-dioleoyloxypropyl) -N, N, N-trimethylammonium salt
  • HistChol N-histidinyl cholesterol hemisuccinate HistChol N-histidinyl cholesterol hemisuccinate.
  • IsoHistSuccDG 1, 2 Dipalmitoylglycerol-O ⁇ -histidinyl-N ⁇ -hemisuccinate, & Distearoyl-, Dimyristoyl, Dioleoyl or palmitoyl-oleoyl derivatives
  • Figure 2 Assignment of a 96-well microtiter plate for the serum stability test, the release of the fluorescent marker CF is measured
  • Figure 3 Fluorescence microscope image next to a phase contrast image for cell localization.
  • a mixture of the lipids named in Table 1 is dissolved in the molar ratios given in chloroform and then completely dried in a rotary evaporator in vacuo.
  • the lipid film is mixed with 10 mM Hepes, 150 mM NaCl, pH 7.5 that a 100 mM
  • the liposomes are repeatedly passed through a membrane with a
  • Pore width of 400 nm extruded is 400 nm extruded.
  • Lipid film uses a solution of the dye, after: 500 mg CF in 130 ul 5
  • the CF which is not included, is separated off by gel filtration.
  • a lipid mixture of the following composition DMPC / MoChol / DG-Succ / Chol 40: 10: 10: 40 (mol%) is dissolved in chloroform at 50 ° C. and then completely dried in a rotary evaporator in vacuo.
  • the liposomes are extruded several times through a membrane with a pore size of 200 nm or 400 nm (Avestin LiposoFast, polycarbonate membrane with a pore size of 200 or 400 nm).
  • a pH of 7.5 is adjusted by adding a 1 mol / L HEPES stock solution.
  • the fraction of the enclosed Cy5.5-anti-CD40-ODN (antisense oligonucleotide) is determined after separation of the freely available active ingredient by sedimentation three times in the ultracentrifuge at 60,000 x g for 45 min using fluorescence spectroscopy.
  • the inclusion efficiency of the oligonucleotide is seen by determining the lipid content and the fluorimetric Cy5.5 determination in relation to the material use of lipid and ODN and is 53% for the formulation.
  • Carboxyflourescein (CF) is used as a model drug, which like oligonucleotides is negatively charged at physiological pH.
  • the serum stability of the CF-filled liposomes is observed over a period of 24 h at 37 ° C.
  • the release of the CF from the liposomes is observed over time by fluorescence measurement.
  • Liposome formulations can be tested per 96-well plate. To determine the percentage of CF released, the sample incubated in buffer is measured both directly (base value) and after opening the liposomes by adding Triton (Triton value or 100% value).
  • Sterile-filtered serum and 1 mM liposomes are combined in a total volume of 500 ⁇ l.
  • the same approach is prepared as a control with buffer instead of serum.
  • a 96-well plate is prepared as follows before the respective sampling: In the wells of a row, columns 1, 3, 5, 7, 9 and 11, 5 ⁇ l buffer (10 mM Hepes, 150 mM NaCl, pH 7.5 ) submitted. In columns 2, 4, 6, 8, 10 and 12 each 5 ⁇ l 10% Triton X-100.
  • the 96-well plate is loaded as shown in Figure 2.
  • Columns 1-6 each contain 5 ⁇ l of the liposomes incubated in buffer, columns 7-12 each in serum incubated.
  • 290 ⁇ l buffer are added to the 5 ⁇ l sample plus 5 ⁇ l buffer or Triton.
  • a base and a triton value are used again.
  • the times are as follows: zero, zero, 15 minutes, 30 minutes, 1 hour, 3 hours, 5 hours, 24 hours.
  • a plate reader with 485/530 nm filters is used to measure the fluorescence.
  • the relative release values are calculated from the measured fluorescence data, in that the triton value corresponds to 100% release.
  • Table 1 shows a number of formulations tested. It can be seen that only compositions according to the invention have a high serum stability.
  • the antisense oligonucleotide (ODN) used is an 18-th phosphorothioate with a FITC label at the 5 'end (fluorescein isothiocyanate).
  • ODN antisense oligonucleotide
  • Liposomes with FITC-ODN were prepared: 0.5 ml of a 1 mM lipid solution with 9 ⁇ g ODN.
  • the liposomes were floated in a discontinuous sucrose gradient with 0, 0.8 and 1, 2 M sucrose in Hep 10 , NaCI 150 (10mM Hepes, 150mM NaCI).
  • siRNA anti GFP
  • amphoteric liposomes 5: 1 to 10: 1 is chosen as the charge ratio of cationic lipid to anionic siRNA.
  • the siRNA in 10mM Hepes, 10mM NaCl pH 7.2
  • the hydration buffer (10mM NaAcetate, 10mM NaCl, 280mM sucrose, pH 4.5) and applied to the lipid film so that a 5-10 mM lipid suspension is formed.
  • the lipids are detached from the piston wall by ultrasound treatment (max. 10 min). It is hydrated for 15 min at room temperature (PO PC as carrier lipid) or at 50 ° C (with DMPC, DPPC as carrier lipid).
  • the liposome fractions are mixed with the same volume of 2.4 M sucrose in H 2 O (ie a 1.2 M solution is formed).
  • the gradient is layered with buffer (10 mM Hepes, 150 mM NaCl, pH 7.5), including 0.8 M sucrose in buffer and below the liposomes in 1.2 M sucrose in H 2 O.
  • the volume of the gradient is maximal 4.5 ml.
  • the flotation is carried out at room temperature for 45 min at 50,000 rpm in an ultracentrifuge.
  • the liposomes, which are located between the 0.8 M sucrose and the buffer layer, are removed.
  • the assay is carried out in a final volume of 200 ⁇ l.
  • a calibration series of the siRNA is made between 1 ng and 10 ng (10-100 ⁇ l 100 ng / ml siRNA).
  • the Ribo Green is diluted 1: 2000 in TE buffer. 100 ⁇ l of Ribo Green are added to each batch, then incubated for 5 min at room temperature and the fluorescence is measured at 485/520 nm.
  • the batches are mixed with 4 ⁇ l of 10% Triton (final concentration 0.2 mM). This calibration curve will later be used to determine the amount of siRNA enclosed in liposomes. After about 15 minutes, the fluorescence is measured again.
  • the results are entered graphically in a diagram (one curve each with and without Triton) and calibration lines with the corresponding equations are generated from the values.
  • the liposomes are diluted in two different concentrations (e.g. 1:50 and 1: 100) and 2-3 different volumes of the dilutions are measured (e.g. 5, 10 and 15 ⁇ l of the dilutions to 100 ⁇ l TE plus 100 ⁇ l Ribo Green reagent ) without and with Triton.
  • concentration of the siRNA of a formulation must roughly match in the different measurements. If this is not the case, the siRNA amount was outside the calibration curves and these values should not be included in the calculation of the concentration.
  • the efficiency with which the siRNA could be included in various formulations is shown in Table 2.
  • Unmodified siRNA is broken down very quickly in the serum.
  • the concentration of the included siRNA is determined before the serum test. Liposomes with 4 ⁇ g siRNA per time are used for each time to be tested. Tested times: zero, 1 h, 2 h and 4 h.
  • the liposomes with 4 ⁇ g siRNA inside are diluted to a volume of 60 ⁇ l with the buffer in which the liposomes are located (usually 10 mM Hepes, 150 mM NaCl, pH 7.5). 60 ⁇ l of serum are added to the batches and up to the incubated at 37 ° C at appropriate times. The zero time is determined separately.
  • a phenol / chloroform extraction is carried out with PLG-Eppis (phase lock gel Eppendor tubes) for a good separation of the serum components and the lipids from the siRNA.
  • the PLG-Eppis are centrifuged for 1 min at 13,000 rpm and placed on ice (in the following, work is carried out at max. 4 ° C). 280 ⁇ l of buffer (as above) and 45 ⁇ l of 5 M NaCI are added to the PLG-Eppis.
  • the liposomes in the serum (120 ⁇ l) are added to this solution. Immediately afterwards, 300 ⁇ l phenol / chloroform mixture is added. For the zero value, 60 ⁇ l serum is also added to the buffer with NaCI in the PLG-Eppi. Everything is mixed immediately. Centrifugation for 10 min at 13,000 rpm and 4 ° C follows. the aqueous phase contains the free siRNA. A further 300 ⁇ l of chloroform are added to the gradient and mixed briefly. After a further centrifugation as above, the aqueous phase is clear and can be removed as completely as possible. The siRNA is now precipitated from the aqueous phase.
  • the quality of the siRNA is checked in the denaturing and possibly also in the native acrylamide gel.
  • siRNA The quality of siRNA is determined over the full length of the single strands and the
  • Double-strandedness defined. Using a native acrylamide gel, you can
  • the gel must be pre-run with TBE buffer for 30 min (80 V, 100 mA) to achieve the required
  • a maximum of 2 ⁇ g siRNA per slot should be applied, otherwise the gel will be overloaded.
  • the samples are taken up in a total volume of 9 ⁇ l. Then add 1 ⁇ l of Blue Juice loading buffer. The samples are applied to the previous gel and separated for 1 h at 100 mA.
  • Staining the gel The gel is removed from the apparatus after electrophoresis. Approx. 200 ml Stains All working solution (20 ml stock solution, 180 ml formamide, 200 ml water) are added to the gel. The gel is stained in the dark for 30-60 min in the Stains All solution. The solution is then removed from the gel and the gel is decolorized under the influence of light in the water (approx. 30 min-2 h depending on the light intensity) . The siRNA remains colored, whereas the background is completely decolored again.
  • HepG2 cells are sown in 96-well plates 2 days before the transfection, so that they show a cell density of 60-80% on the day of the transfection (0.02-0.2 x 10 5 in 100 ⁇ l, usually 1 x 10 4 ) , Two plates (centrifugation and control incubation) are prepared, which are otherwise treated identically.
  • liposome dilutions with and without cargo
  • transfection mix of commercial control transfection reagents (e.g. Lipofectamine 2000, oligofecate mine, siPort amine, siPort lipid) on a 96-well plate with serum-free medium (approx. 160 ng antisense / well).
  • the liposome dilution will be added to the cells in a volume of 25-50 ⁇ l.
  • the cells are washed with serum-free medium (1x) and mixed with serum-containing or serum-free medium, e.g. 75 ⁇ l per well if the liposome dilutions were prepared in such a way that the corresponding amount of antisense / well is contained in 25 ⁇ l.
  • a multiwell plate is centrifuged at 1,500 rpm (342 g), 1 h, 37 ° C., the control plate is incubated for 1 h (37 ° C., 5% CO 2 ). Then both plates are incubated for 3 h (37 ° C, 5% CO 2 ). Remove medium from all wells completely, wash 1 x with PBS, 2 x with serum-containing medium; Add serum-containing medium to all wells, microscopic control for vitality, then incubate both plates overnight (37 ° C, 5% CO 2 ).
  • Figure 3 shows a fluorescence image next to a phase contrast image for cell localization.
  • Example 7 Determination of the serum stability of amphoteric liposomes with and without cholesterol and / or matrix lipid
  • E1 DC-Chol / DOPA (66/34) E2 DC-Chol / DOPA / Chol (40/20/40) E3 DMPC / DC-Chol / DOPA (60/27/13) E4 DMPC / DC-Chol / DOPA / Chol (20/27/13/40) E5 DMPC / DC-Chol / DOPA / Chol (30/20/10/40) E6 DMPC / DC-Chol / DOPA / Chol (40/13/7/40)
  • the liposomes were floated after extrusion to separate the outside FITC dextran.
  • the following data were measured on the Zetasizer at pH 3.9 (10 mM Hepes 150 NaCI HAc) or pH 9.0 (10 mM Tris 150 mM NaCI):
  • ⁇ 1 mM liposomes were incubated for 3 hours at 37 ° C. in human serum and then floated.
  • the fluorescence was determined both in the non-floated sample and in the lower layer (serum layer) of the floated sample and the cargo release was calculated therefrom.
  • the proportion outside FITC-dextran at the start of the test was determined in the same way by appropriate dilution in buffer pH 3.9 and subsequent flotation.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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EP05740433A 2004-03-28 2005-03-29 Serumstabile amphotere liposomen Withdrawn EP1734928A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE200410016020 DE102004016020A1 (de) 2004-03-28 2004-03-28 Serumstabile amphotere Liposomen
DE102004054730A DE102004054730A1 (de) 2004-03-28 2004-11-05 Serumstabile amphotere Liposomen
PCT/DE2005/000589 WO2005094783A2 (de) 2004-03-28 2005-03-29 Serumstabile amphotere liposomen

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EP1734928A2 true EP1734928A2 (de) 2006-12-27

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US (1) US8236770B2 (enExample)
EP (1) EP1734928A2 (enExample)
JP (1) JP2007530462A (enExample)
AU (1) AU2005229485A1 (enExample)
CA (1) CA2561247C (enExample)
DE (1) DE102004054730A1 (enExample)
WO (1) WO2005094783A2 (enExample)

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WO2005094783A3 (de) 2006-03-02
US8236770B2 (en) 2012-08-07
AU2005229485A2 (en) 2005-10-13
JP2007530462A (ja) 2007-11-01
AU2005229485A1 (en) 2005-10-13
CA2561247A1 (en) 2005-10-13
WO2005094783A2 (de) 2005-10-13
DE102004054730A1 (de) 2006-05-11
US20080311181A1 (en) 2008-12-18
CA2561247C (en) 2014-12-09

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