EP1949110A2 - Verfahren zur diagnostizierung von lebererkrankungen und zum screening von molekülen zur behandlung dieser erkrankungen - Google Patents

Verfahren zur diagnostizierung von lebererkrankungen und zum screening von molekülen zur behandlung dieser erkrankungen

Info

Publication number
EP1949110A2
EP1949110A2 EP06831103A EP06831103A EP1949110A2 EP 1949110 A2 EP1949110 A2 EP 1949110A2 EP 06831103 A EP06831103 A EP 06831103A EP 06831103 A EP06831103 A EP 06831103A EP 1949110 A2 EP1949110 A2 EP 1949110A2
Authority
EP
European Patent Office
Prior art keywords
apo
blood
liver
tissues
cells
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Withdrawn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
EP06831103A
Other languages
English (en)
French (fr)
Inventor
Pierre Casellas
Hubert Vidal
Isabelle Buisson
Jean Marchand
Dominique Simon
Sylvaine Galiegue
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.)
Sanofi SA
Original Assignee
Sanofi Aventis France
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 Sanofi Aventis France filed Critical Sanofi Aventis France
Publication of EP1949110A2 publication Critical patent/EP1949110A2/de
Withdrawn legal-status Critical Current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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides

Definitions

  • the present application relates to a method for diagnosing liver diseases. It also relates to a method for screening molecules for the treatment of these diseases.
  • Apolipoprotein A4 (Apo A4) is an abundant circulating apolipoprotein.
  • the Apo A4 has multiple functions. It regulates lipid transport and metabolism through various mechanisms such as activation of cholesterol fluxes from liver tissue and stimulation of lipoprotein iipase activity (Stan et al., Biochim Biophys acta, 1631). 2003, 177-187).
  • the Apo A4 is also known to be a factor of satiety.
  • Hepatic diseases are a major public health problem. It is therefore essential to diagnose all liver diseases as early as possible, using specific and sensitive assays.
  • the Applicant has surprisingly demonstrated that Apo A4 is expressed in the liver in humans and that the level of hepatic expression of the gene encoding Apo A4 is significantly increased in liver diseases.
  • the present invention relates to a method for detecting, diagnosing or prognosing liver disease comprising measuring the expression of Apo A4 in liver cells or tissues or extracts from such cells and tissues, or in blood and its derivatives.
  • the object of the present invention is to measure the expression of Apo A4 not only in liver cells or tissues but also in the blood, in which is found Apo A4 of hepatic origin.
  • Such a method may include the steps of:
  • liver diseases any disease affecting the tissues and liver cells, whether chronic or etiological (alcoholic, viral, toxic, food, environmental ).
  • the subject of the present invention is a method for the detection, diagnosis or prognosis of steatohepatitis, carcinogenesis of the liver, cirrhosis, viral hepatitis, hepatocellular insufficiency, cholestasis, portal hypertension, steatosis and steatohepatitis, vein and hepatic artery disease, fibrosis, obesity, and metabolic syndromes.
  • blood derivative is understood to mean any cellular or acellular fraction derived from the blood by physical (for example by centrifugation), biological (for example by coagulation) or chemical treatment or by any other treatment making it possible to obtain such derivatives.
  • such derivatives are plasma and serum.
  • said method comprises measuring the amount of messenger RNA (mRNA) transcribed from the gene or genes encoding Apo A4 in the cells or liver tissues of an individual in which one wishes to diagnose or prognose liver disease.
  • the liver cells or tissues or in the blood and its derivatives of said mammal are removed and treated by methods known to those skilled in the art, in particular to avoid the degradation of messenger RNAs and proteins and the amount of transcribed messenger RNA. by the gene (s) encoding Apo A4 is measured.
  • the amount of transcribed messenger RNA is measured by amplification according to the so-called quantitative Polymerase Chain Reaction (PCR) or QPCR method.
  • PCR quantitative Polymerase Chain Reaction
  • the total RNAs are extracted and purified and the messenger RNAs contained in the extract are converted firstly into DNAs. (cDNA), using reverse transcriptase.
  • the polymerase preferably used in the present invention is Taq polymerase, but it can be any other enzyme having a polymerase activity and usable under the operating conditions of the PCR. Due to its properties, this enzyme allows a doubling of the amount of initial DNA at each synthesis cycle.
  • cDNAs derived from the mRNAs contained in the biological sample is performed by quantitative PCR in real time.
  • the cDNAs are amplified using primers and probes specific for the sequence of the Apo A4, according to a method which essentially comprises a repetition of the cycle comprising the following steps:
  • the sense and antisense primers comprise at least 15 nucleotides and have an identity of at least 80%, preferably 90%, and still more preferably 95% with the sequence of the human Apo A4, corresponding to the sequence SEQ ID N ° 13 (Gene bank No. NM 000482), or with its complementary sequence.
  • the product of the reaction, or amplimer is detected using a probe, consisting of an oligonucleotide comprising at least 15 nucleotides and having an identity of at least 80%, preferentially 90%, and even more preferably 95% with the sequence of the human Apo A4, corresponding to the sequence SEQ ID No. 13 (Gene bank No. NM 000482), or with its complementary sequence.
  • a probe consisting of an oligonucleotide comprising at least 15 nucleotides and having an identity of at least 80%, preferentially 90%, and even more preferably 95% with the sequence of the human Apo A4, corresponding to the sequence SEQ ID No. 13 (Gene bank No. NM 000482), or with its complementary sequence.
  • the two primers are respectively selected on the sense and antisense strands, and so as to allow the amplification of a DNA fragment.
  • the probe is in turn targeted so as to hybridize with the DNA fragment resulting from the amplification delimited by the position of the two primers.
  • the probe has a theoretical melting temperature Tm greater than about 10 0 C + 0.5 to the theoretical Tm of the primers.
  • said oligonucleotides (primers and probe) comprise between 15 and 25 nucleotides.
  • the primers used for the amplification of the gene encoding human Apo A4 preferentially present the sequences
  • SEQ ID No. 1 gcagctggctccctatgct and SEQ ID No. 2: ggaaggtcaggccctcaag.
  • the probe preferably has the sequence SEQ ID No. 3: cacgcaggagaag ctcaaccacca.
  • the probe comprises a molecule or a system of developer molecules.
  • the developer system is preferably a reporter dye and a fluorescent quencher dye, respectively attached to the 5 'and 3' ends of the probe.
  • the developer system is constituted by the "reporter / quencher” pair represented by 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) respectively fixed at 5 'and 3' of the probe. .
  • the quantification of the number of targets initially set in the reaction is very reliable and reproducible. Detection of the PCR product is done during PCR cycles using a fluorescent probe. This is necessary to detect the PCR product and takes place in full exponential phase of PCR and not in end point; this detection principle is therefore more sensitive and more specific.
  • Another advantage of the QPCR lies in the fact that nonspecific amplifications are avoided, thanks to the principle of "hot start", real-time PCR being performed in the presence of a thermostable DNA polymerase activating at the first denaturation.
  • the method according to the present invention comprises measuring the amount of protein or fragments of the Apo A4 protein expressed in the liver cells or tissues or in the blood and its derivatives. Particularly advantageously the amount of protein or fragments of the protein Apo A4 is measured using at least one antibody specific for this protein.
  • These antibodies can be obtained by a method of injecting animals, such as rodents, with an emulsion of the purified human Apo A4 protein or a peptide sequence of Apo A4 of about 20 amino acids, optionally in the presence of an adjuvant such as Freund's adjuvant. Injections are repeated and the antiserum is harvested.
  • animals such as rodents
  • an adjuvant such as Freund's adjuvant
  • the measurement of the binding between these antibodies and the Apo A4 expressed in the liver cells or tissues or in the blood and its derivatives of the individuals in whom it is desired to quantify the hepatic Apo A4 can be carried out by any suitable means.
  • it can be done by the ELISA technique (abbreviation for Enzyme-Linked Immunosorbent Assay) and even more preferably by a so-called "Sandwich” method.
  • the so-called "Sandwich” method uses two antibodies: a first capture antibody is bound to a solid phase such as a microplate, a second detection antibody makes it possible to quantify the protein.
  • the detection antibodies are coupled with a peroxidase.
  • the antibodies bound to the Apo A4 protein are isolated from the unbound antibodies and placed in the presence of a peroxidase substrate, preferentially the O-phenylenediamine dihydrochloride.
  • a peroxidase substrate preferentially the O-phenylenediamine dihydrochloride.
  • the colorimetric reaction makes it possible to quantify the number of bound antibodies and therefore the amount of bound protein. This colorimetric reaction is measured using a suitable apparatus, for example by measuring the optical density.
  • An amplification of the reaction can be carried out using at least two antibodies, on the one hand anti-Apo A4 antibodies not coupled to peroxidase and on the other hand peroxidase-coupled antibodies recognizing the first antibodies.
  • another anti-Apo A4 antibody can be used to fix the antibody-Apo A4 complexes on a support thus facilitating the isolation of the complexes and the separation of the non-fixed antibodies.
  • the binding between these antibodies and Apo A4 can also be measured using antibodies labeled with a radioactive isotope.
  • the antibodies bound to the Apo A4 protein are isolated and the radioactivity of the antibody-Apo A4 complexes is measured using a measuring device adapted to the isotope used.
  • the present invention further relates to a method for screening compounds for the prevention or treatment of liver diseases comprising the steps of:
  • the mammal in cells or blood from which the measurement of Apo A4 expression can be made is any mammal in which Apo A4 is expressed in the liver. It can be advantageously a rodent and preferably a mouse.
  • animal lines with a natural tendency to obesity are used.
  • C57BI / 6J is preferably used.
  • the measurement of the expression of Apo A4 can be carried out by measuring the amount of Apo A4 expressed in the liver cells or tissues or in the blood and its derivatives of said mammal or by measuring the amount of RNA messenger transcribed by the gene or genes encoding Apo A4 expressed in cells or tissues hepatic of said mammal, as indicated above.
  • a piece of liver (75-130 mg) was collected in 2 ml tubes containing 1.5 ml of RNA Later (Ambiom).
  • liver and the plasma were stored at -20 ° C. for the assay of the mRNAs and the Apo A4 protein.
  • cDNA analysis performed by quantitative PCR in real time. The relative expressions of the gene are given in% relative to an invariant internal reference gene, such as beta2-microglobulin or 18S ribosomal RNA.
  • tissue lysates 80 mg of mouse liver are excised and immediately immersed in 2 ml of RNAlater in order to preserve the integrity of 1 RNA before any manipulation.
  • the piece of liver is transferred dry in an Eppendorf tube into which 2 ml of lysis buffer (RNAtotal extraction kit, RNeasy Midi Kit marketed by QIAGEN) and 2 steel balls (diameter, 3 mm) are added.
  • Tissue lysis is performed with Tissuelyser (marketed by QIAGEN) by stirring for 5 min at 30HZ. At this stage, the lysates can be stored at -20 ° C.
  • the lysates are centrifuged for 3 min at 14000xg and filtered on QIAshredder
  • RNA concentration is obtained by measuring the optical density (OD) at 260 nm.
  • the cDNA is synthesized using the Superscipt III reverse transcription kit (marketed by INVITROGEN). The cDNA obtained is recovered in 20 .mu.l of final volume.
  • the sequences of the primers and probes used for the amplification of the gene encoding human and mouse Apo A4 and beta2-m are shown in Table VII (SEQ ID NO: 1 to SEQ ID NO: 12).
  • the gene amplification reaction is carried out with temperature cycles (denaturation 95 ° C / 15s then hybridization and synthesis 60 ° C / 1 min) in microplates of 96 or 384 wells. The duration of the analysis is 90 min.
  • the amplification kinetics (amplification signal function of the number of cycles) are analyzed in a linear representation of the exponential phase (software: SDS Enterprise Database).
  • Ct 1 cycle number measured constant amplification signal the Ct is inversely proportional to the amount of messenger RNA present in the original sample.
  • the capture antibody is goat anti-Apo A4 antibody marketed by
  • Anti serum against mouse Apo A4 is obtained from rabbits immunized with a peptide corresponding to the C-terminal sequence (amino acid
  • a peptide emulsion coupled to a carrier protein (KLH) in complete Freund's adjuvant is injected subcutaneously to the animals. Subsequent injections are made with a peptide emulsion coupled in incomplete Freund's adjuvant.
  • KLH carrier protein
  • the antiserum is harvested 10 days after the third booster and the antibody is purified by affinity chromatography on the peptide coupled to a chromatography gel.
  • Table IA shows the tissue distribution of Apo A4 in mice.
  • Apo A4 is expressed in smooth muscle, small intestine, liver, colon, placenta, stomach, ovaries, rectum, adipose tissue ... There is a strong expression in the intestine hail.
  • Table IB confirms strong expression in small bowel and localization of Apo A4 in different regions (duodenum, jejunum, ileum).
  • Table II shows the expression of Apo A4 in liver and plasma and shows that this expression is increased in liver and plasma when comparing normal and obese mice.
  • the cDNAs are prepared from human liver and the measurements performed as described in the previous example for the preparation from mouse liver.
  • Table III shows the tissue distribution of Apo A4 in humans: presence in fundus uterus, esophagus, retina, placenta, epididymis, and adipose tissue.
  • Apo A4 as a potential marker for these conditions is demonstrated by the comparative analysis of Apo A4 from other apolipoprotein classes: Apo A1, Apo A2 and Apo A5 (Tables IV and V-B).
  • Example 3 Measurement of the amount of Apo A4 protein in plasma in humans by sandwich ELISA method
  • the capture antibody is goat anti-Apo A4 antibody marketed by (Santa Cruz Biotechnology, Inc. 21414 Delaware Avenue Santa Cruz, California 95060 U.S.A.).
  • Purified human Apo A4 is obtained from human serum as described by Weinberg RB, Hopkins RA, Jones JB. (Methods Enzymol 1996, 263: 282-96, Purification, isoform characterization, and quantitation of human apolipoprotein A4).
  • Anti serum against human Apo A4 is obtained from rabbits.
  • An h-Apo A4 emulsion purified in complete Freund's adjuvant is injected subcutaneously to the animals. Subsequent injections are made with a purified Apo A4 emulsion in incomplete Freund's adjuvant. The antiserum is harvested 10 days after the third booster.
  • the wells of a 96-well plate are incubated with 100 ⁇ l of goat anti-Apo A4 antibody at 4 ⁇ g / ml in PBS for 4 hours at 20 ° C. After washing with PBS containing 0.1% Tween 20 non-specific binding sites are blocked with PBS buffer containing 0.5% bovine serum albumin (BSA) for 60 min at room temperature. 100 ⁇ l of plasma samples (dilution in PBS containing 0.1% BSA from 1/3000 to 1/20000) are added and incubated overnight at 4 ° C. After washing, 100 ⁇ l of rabbit anti-human Apo A4 polyclonal serum antibodies (dilution 1/5000 in PBS BSA) are added and incubated for 1 h at room temperature.
  • BSA bovine serum albumin
  • a study focused on obesity was performed by analyzing the expression of apoA4 in the liver of overweight patients. This pathology is correlated with the body mass index (IMC ⁇ Poids in kg / (height in meter) 2 ). Normal weight load corresponds to a BMI ⁇ 25.
  • Experimentally apolipoprotein expression was analyzed by the previously described QPCR technique from a collection of human liver RNA provided by Asterand, Inc. (TechOne Bldg, Suite 501, 440 Burroughs, Str. Ml 48202, US) . Body Mass Index (BMI) is given by Asterand.
  • Table 1B Expression of Apo A4 in Small Intestine Regions (% / Beta2-m, Mean +/- Standard Deviation (4 animals))
  • Table II Expression of 1 mRNA of Apo A4 in the liver and measurement of Apo A4 in plasma of normal (C57 B16) or obese (C57 B16 / obob) mice.
  • Table VB Expression of Apo A1, 2 & 5 Human in Liver Samples.
  • Table VII Sequences of primers and probes for the quantitative PCR assay of beta2-microqlobulin and Apo A4 Human and Mouse.
  • Table VIII Expression of human hepatic apoA4 in overweight patients

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  • Urology & Nephrology (AREA)
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EP06831103A 2005-11-10 2006-11-10 Verfahren zur diagnostizierung von lebererkrankungen und zum screening von molekülen zur behandlung dieser erkrankungen Withdrawn EP1949110A2 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR0511432A FR2893136A1 (fr) 2005-11-10 2005-11-10 Procedes de diagnostic de maladies hepatiques et de criblage de molecules pour le traitement de ces maladies
PCT/FR2006/002503 WO2007057548A2 (fr) 2005-11-10 2006-11-10 Procedes de diagnostic de maladies hepatiques et de criblage de molecules pour le traitement de ces maladies

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EP1949110A2 true EP1949110A2 (de) 2008-07-30

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EP06831103A Withdrawn EP1949110A2 (de) 2005-11-10 2006-11-10 Verfahren zur diagnostizierung von lebererkrankungen und zum screening von molekülen zur behandlung dieser erkrankungen

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US (1) US20090220958A1 (de)
EP (1) EP1949110A2 (de)
JP (1) JP2009515514A (de)
KR (1) KR20080074121A (de)
CN (1) CN101310187A (de)
AR (1) AR058510A1 (de)
AU (1) AU2006314369A1 (de)
BR (1) BRPI0618490A2 (de)
CA (1) CA2627082A1 (de)
FR (1) FR2893136A1 (de)
RU (1) RU2008123385A (de)
TW (1) TW200804811A (de)
WO (1) WO2007057548A2 (de)

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CA2719001A1 (en) * 2008-03-21 2009-09-24 Podiceps B.V. Diagnostic of pre-symptomatic metabolic syndrome
EP2381259B1 (de) * 2008-12-24 2016-09-21 Keio University Verfahren zur messung von leberkrankheitsmarker sowie testverfahren für die pharmazeutische herstellung
CA2886748A1 (en) * 2012-10-17 2014-04-24 Enterome Gene signatures of inflammatory disorders that relate to the liver
WO2019167874A1 (ja) 2018-02-27 2019-09-06 エーザイ・アール・アンド・ディー・マネジメント株式会社 Ａｐｏａ４に対するモノクローナル抗体、免疫学的測定方法及び測定用キット
CN112891561A (zh) * 2021-03-09 2021-06-04 百码科技(深圳)有限公司 一种基因治疗脂肪肝的生物药剂及其制备方法
CN113018459A (zh) * 2021-03-09 2021-06-25 百码科技(深圳)有限公司 一种基因治疗减肥的生物药剂及其制备方法

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FR2686605B1 (fr) * 1992-01-27 1994-03-11 Rhone Poulenc Rorer Sa Nouveaux polypeptides, leur preparation et leur utilisation.
US5925333A (en) * 1995-11-15 1999-07-20 Massachusetts Institute Of Technology Methods for modulation of lipid uptake
WO2001077124A2 (en) * 2000-04-05 2001-10-18 Genaissance Pharmaceuticals, Inc. Haplotypes of the apoa4 gene
US6727066B2 (en) * 2000-07-28 2004-04-27 Incyte Corporation Genes expressed in treated human C3A liver cell cultures
US20060009410A1 (en) * 2002-11-13 2006-01-12 Crooke Rosanne M Effects of apolipoprotein B inhibition on gene expression profiles in animals
ATE462001T1 (de) * 2002-12-24 2010-04-15 Nitto Boseki Co Ltd Markerproteine zur diagnose einer lebererkrankung und verfahren zur diagnose einer lebererkrankung damit

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Title
See references of WO2007057548A2 *

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CA2627082A1 (fr) 2007-05-24
FR2893136A1 (fr) 2007-05-11
AU2006314369A1 (en) 2007-05-24
TW200804811A (en) 2008-01-16
WO2007057548A3 (fr) 2007-07-26
US20090220958A1 (en) 2009-09-03
RU2008123385A (ru) 2009-12-20
JP2009515514A (ja) 2009-04-16
CN101310187A (zh) 2008-11-19
AR058510A1 (es) 2008-02-06
WO2007057548A2 (fr) 2007-05-24
KR20080074121A (ko) 2008-08-12
BRPI0618490A2 (pt) 2011-08-30

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