[go: up one dir, main page]

WO2008038022A1 - Test concernant une infection à h. pylori - Google Patents

Test concernant une infection à h. pylori Download PDF

Info

Publication number
WO2008038022A1
WO2008038022A1 PCT/GB2007/003703 GB2007003703W WO2008038022A1 WO 2008038022 A1 WO2008038022 A1 WO 2008038022A1 GB 2007003703 W GB2007003703 W GB 2007003703W WO 2008038022 A1 WO2008038022 A1 WO 2008038022A1
Authority
WO
WIPO (PCT)
Prior art keywords
nitro
hpa
hydroxyphenylacetic acid
pylori infection
helicobacter pylori
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.)
Ceased
Application number
PCT/GB2007/003703
Other languages
English (en)
Inventor
Kevin Peter Moore
Alireza Mani
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.)
UCL Business Ltd
Original Assignee
UCL Business Ltd
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 UCL Business Ltd filed Critical UCL Business Ltd
Priority to EP07804410A priority Critical patent/EP2078200A1/fr
Publication of WO2008038022A1 publication Critical patent/WO2008038022A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to a novel biomarker for Helicobacter pylori infection in human subjects. More specifically, the invention relates to the identification of urinary 3-nitro-hydroxyphenylacetic acid as a marker for H. pylori infection and to a test for H. pylori infection based on detection of 3-nitro-hydroxyphenylacetic acid in urine.
  • H. pylori infection is one of the most common chronic bacterial infections of human populations worldwide. H. pylori infection occurs in approximately 30% of the UK population and is recognised as the major cause of gastritis and peptic ulcer disease. Infection also increases the risk of gastric carcinoma in chronic carriers by 4-5 fold.
  • 3-nitro-p- hydroxyphenylacetic acid 3-nitro-p- hydroxyphenylacetic acid
  • 3-nitro-HPA can also be formed by a second pathway involving metabolism of nitrotyrosine, which is itself formed by direct nitration of tyrosine.
  • urinary 3-nitro-HPA would serve as a general time-integrated biomarker of the formation of reactive nitrogen species in vivo, based on the erroneous assumption that the majority of urinary 3-nitro- HPA is derived from nitrotyrosine metabolism.
  • the inventors now know that nitrotyrosine metabolism only accounts for a minor fraction of 3-nitro-HPA excreted in the urine, and that the vast majority is formed by nitration of para- hydroxyphenylacetic acid in the stomach.
  • the present inventors have unexpectedly found that the nitration of salivary or gastric p-hydroxyphenylacetic acid is markedly up-regulated in the stomach of patients with Helicobacter pylori infection and this is reflected in elevated levels of 3-nitro-HPA in the urine of infected patients, as compared to "normal" individuals not infected with H. pylori.
  • Urinary 3-nitro-HPA thus provides a useful specific biomarker for the presence of H. pylori infection. Measurement of urinary 3-nitro-HPA can form the basis of a simple and sensitive assay for H. pylori infection which avoids many of the drawbacks of the currently available tests.
  • a method of testing for Helicobacter pylori infection in a human subject which comprises testing a sample comprising urine from the subject for the presence of 3-nitro-hydroxyphenylacetic acid, wherein the presence of an elevated level of 3-nitro-hydroxyphenylacetic acid, compared to control subjects who do not have Helicobacter pylori infection, is taken as an indication that the subject is infected with Helicobacter pylori.
  • the method of the invention may be based on detection of urinary 3-nitro-hydroxyphenylacetic acid produced by nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid to form 3-nitro- hydroxyphenylacetic acid which is then excreted in the urine.
  • the inventors have observed that the nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid is markedly up-regulated in individuals infected with H. pylori.
  • the inventors postulate that nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid is caused by reactive nitrogen species formed during H. pylori infection.
  • an elevated level of urinary 3-nitro- hydroxyphenylacetic acid provides a biomarker for H. pylori infection.
  • the invention further provides kits and devices for detecting urinary 3-nitro-hydroxyphenylacetic acid which can be used in the method of the invention.
  • FIG 1 illustrates the biochemical pathways for the formation of 3-nitro-HPA in human subjects.
  • Tyrosine can be nitrated by reactive nitrogen species (RNS) such as peroxynitrite to form nitrotyrosine.
  • RNS reactive nitrogen species
  • Nitrotyrosine is metabolised to several products.
  • the major urinary metabolite of nitrotyrosine is 3-nitro-HPA (designated NHPA in the Figure) .
  • the majority of urinary 3-nitro-HPA is formed by direct nitration of salivary/gastric p- hydroxyphenylacetic acid (PHPA) , which is itself formed as a major product of tyrosine metabolism.
  • PHPA salivary/gastric p- hydroxyphenylacetic acid
  • Day 1 was on a low nitrate diet
  • day 2 on a high nitrate diet
  • day 3 a high nitrate diet with gastric acid suppressed by omeprazole. It can be clearly seen that there is a marked elevation of the urinary excretion of 3-nitro-HPA (NHPA) in patients with H. pylori infection compared to healthy controls.
  • NHPA 3-nitro-HPA
  • the invention provides a method of testing for the presence of Helicobacter pylori infection in human subjects which involve testing for 3-nitro-hydroxyphenylacetic acid (3-nitro-HPA) in a sample comprising urine from the subject under test, wherein urinary 3-nitrohydroxyphenylacetic acid is a biomarker for infection with Helicobacter pylori.
  • 3- nitro-HPA is generated by nitration of endogenous salivary or gastric p-hydroxyphenylacetic acid in the presence of reactive nitrogen species which form during H. pylori infection.
  • this method requires only the collection of a suitable urine sample which can be tested for the presence of 3-nitro-HPA as a biomarker for H. pylori infection.
  • test sample a sample which must comprise urine from the subject under test.
  • additional components may be added to the subject's urine to form the test sample.
  • additional components may include (but are not limited to) buffers, diluents, preservatives, stabilisers or other components required for long term storage.
  • the test sample may consist only of urine from the test subject. The latter would typically be preferred in the case of simple point-of-care testing.
  • a urine sample may be tested immediately after it is given (or tested midstream) or, if required, the test sample comprising or consisting of urine from the test subject may be stored for a period of time and/or concentrated prior to testing, or re-testing.
  • the urine may be frozen and thawed or freeze-dried and reconstituted prior to testing.
  • Testing for the presence of 3-nitro-HPA in the test sample may be carried out using any suitable technique, the invention is not intended to be limited with respect to the methodology used to detect/measure 3-nitro-HPA in the test sample.
  • Suitable techniques for detecting/measuring 3-nitro-HPA include, but are not limited to, analytical techniques such - S -
  • 3-nitro-HPA may be based on a specific binding assay.
  • 3-nitro-HPA may be detected using an immunological technique, for example an immunoassay using an antibody specific for 3-nitro-HPA, or a fragment thereof which retains specific binding capability.
  • an immunological technique for example an immunoassay using an antibody specific for 3-nitro-HPA, or a fragment thereof which retains specific binding capability.
  • specific refers to immunological specificity.
  • Immunoassays for example ELISA, radioimmunoassays, immunochromatographic assays and the like, are well known to those skilled in the art (see Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996) .
  • Immunoassays for the detection of small molecules such as 3-nitro-HPA generally require the use of at least one antibody (or antibody fragment) that exhibits specific immunological reactivity with the molecule of interest.
  • the antibody may be immobilised on a solid support .
  • a sample to be tested for the presence of the molecule of interest is brought into contact with the antibody and if the molecule of interest is present in the sample it will immunologically react with the antibody to form complexes which may then be detected or quantitatively measured by any suitable means, e.g. based on colorimetric, fluorescent or radioactive labelling of the complex.
  • Binding agents other than antibodies and fragments thereof can also be used if they display binding specificity for 3- nitro-HPA.
  • Antibodies specific for 3-nitro-HPA may be raised using standard techniques available in the art.
  • One approach which may be used in order to raise antibodies to a small immunogen, such as 3-nitro-HPA is based on coupling of the small immunogen (e.g.
  • 3-nitro-HPA to a carrier protein which facilitates generation of antibodies specific for the small immunogen.
  • Suitable carrier proteins include lysozyme, keyhole limpet haemocyanin, purified protein derivative or albumin etc.
  • 3-nitro-4 hydroxyphenylacetic acid can be coupled to a carrier protein using carbodiimide and hydroxysuccinimide. The resulting conjugate may then be used as a challenging antigen in a suitable host animal in order to raise antibodies immunologically specific for 3- nitro-HPA.
  • Immunoassays specific for 3-nitro-HPA may also be based on the use of antibody fragments which retain immunological specificity for 3-nitro-HPA.
  • Suitable antibody fragments which may be used include, inter alia, F(ab') 2 fragments, scAbs, Fv and scFv fragments etc.
  • antibodies or antibody fragments specific for 3-nitro-HPA should exhibit little or no cross-reactivity with p-hydroxyphenylacetic acid (PHPA) .
  • antibodies or antibodies specific for 3-nitro- HPA may be labelled to facilitate detection.
  • Suitable detectable label include, for example, radiolabels, enzymatic labels, including enzymatic labels which permit colorimetric detection (e.g. horseradish peroxidase, etc), redox labels or particulate labels (e.g. colloidal gold) .
  • the format of the binding assay is not limited and could include, for example, assays performed in standard microtiter plates, test strips, lateral flow devices, dipsticks and the like, the latter being generally preferred for point-of-care testing or home testing kits. Binding assays, including immunoassays, may be performed in a competitive format, or sandwich format or can be based on direct detection of a labelled binding agent (e.g. labelled antibody or antibody fragment) bound to 3-nitro-HPA.
  • a labelled binding agent e.g. labelled antibody or antibody fragment
  • testing for urinary 3-nitro-HPA may be based on a specific chemical reactions with 3-nitro-HPA.
  • an assay could take the form of a test strip or dipstick test suitable for point-of-care or home testing, or may be carried out remotely using specific reagent kits .
  • a positive result i.e. a determination that H. pylori infection is present, is indicated by an elevated level of urinary 3-nitro-HPA as compared to control subjects who are known to be free of H. pylori infection (e.g. subjects negative for H. pylori on the basis of accepted clinical criteria, such as CLO test and/or C 13 -urea breath test) .
  • the method may involve a quantitative determination of the amount of 3-nitro-HPA present in the test sample from the test subject, the result of which can be compared to a predetermined cut-off value. Other tests may be based on a simple yes/no determination.
  • Test strips, dipstick tests or the like may include an internal standard which would enable the user to immediately determine (e.g. by visual inspection) whether the test result is positive or negative without the need to calculate the absolute amount of 3- nitro-HPA present.
  • the method of the invention may include suitable controls or calibrators to take into account background nonspecific levels of urinary 3-nitro HPA, for example 3-nitro HPA derived from metabolism of nitrotyrosine . All human subjects produce background low levels of urinary 3-nitro HPA by metabolism of nitrotyrosine, irrespective of H. pylori infection status.
  • the pathway by which 3- nitro HPA is formed by metabolism of nitrotyrosine is separate from the pathway by which 3-nitro HPA is produced by direct nitration of salivary or gastric p- hydroxyphenylacetic acid (PHPA) .
  • PHPA gastric p- hydroxyphenylacetic acid
  • the present inventors have observed that (i) the majority (typically greater than 90%) of urinary 3-nitro HPA is in fact formed by the direct nitration of salivary or gastric PHPA, and (ii) this pathway is markedly up-regulated in H. pylori infection.
  • the alternative pathway by which 3-nitro HPA is formed by metabolism of nitrotyrosine typically accounts for less than 10% of urinary 3-nitro HPA.
  • the invention also relates to assay kits or devices for detection of urinary 3-nitro-hydroxyphenylacetic acid which can be used in the method of the invention.
  • the invention provides assay kits comprising at least one antibody or antibody fragment immunologically specific for 3-nitro-hydroxyphenylacetic acid.
  • Such kits may include further reagents needed to perform an immunoassay for 3- nitro-hydroxyphenylacetic acid, such as for example secondary antibodies .
  • the invention also provides a dipstick device, test strip or lateral flow device for detection of urinary 3- nitro-hydroxyphenylacetic acid.
  • dipstick devices, test strips and lateral flow devices may be based on a specific binding assay for detection of 3-nitro-HPA, in which case the dipsticks, test strips or lateral flow device will comprise at least one specific binding reagent specific for 3-nitro-HPA.
  • Suitable specific binding reagents include antibodies or antibody fragments immunologically specific for 3-nitro-HPA.
  • the general features of dipstick tests, test strips and lateral flow devices embodying binding assays specific for a particular analyte are known in the art.
  • Such devices may be based on a competitive binding assay format, or a sandwich binding assay format or even direct detection of a labelled specific binding reagent which binds the analyte of interest, i.e. 3-nitro-HPA.
  • any of the methods described herein can be used diagnostically, for example to confirm whether H. pylori infection is present in a patient who presents with symptoms consistent with peptic ulcer disease, including but not limited to gastric irritation, dyspepsia or indigestion, or if H. pylori infection is suspected for any other reason.
  • the test may be used as a tool for point-of-care testing, for example in a GP surgery, outpatients clinic or pharmacy, allowing a diagnosis to be made and appropriate treatment to be prescribed in a single consultation.
  • the methodology used to test for the presence of 3-nitro-HPA provide an immediate indication of a positive result.
  • the invention also contemplates home-testing kits for detection of urinary 3-nitro-HPA.
  • the method described herein may be used as a screening tool, for example to screen a whole population of test subjects, who may be asymptomatic for H. pylori infection or any associated condition, in order to identify those subjects within the population who do in fact carry an H. pylori infection, or to identify those subjects requiring further investigation with more invasive test procedures in order to confirm the presence of H. pylori. Infected subjects may then be selected for appropriate treatment to eradicate the infection.
  • the method according to the invention may be used as a tool to monitor the course of an infection with H. pylori, and in particular to monitor response to treatment.
  • a major advantage of the method of the invention as compared to prior art tests for H. pylori infection is that it is non-invasive, requiring only that the test subject provide a sample of urine each time the method is to be carried out. Accordingly, the test may be repeated as often as necessary in order to monitor response to treatment and as a follow-up to check that the infection has not recurred. This can be done with minimal inconvenience and discomfort to the patient.
  • the simple nature of the method is particularly suited to point-of-care and home testing.
  • One important aspect of the invention therefore relates to provision of home testing kits and point-of-care testing kits for detection of urinary 3-nitro- HPA.
  • Example 1 measurement of urinary 3-nitro-HPA as a biomarker for Helicobacter pylori infection
  • 3-nitro-HPA was measured in urine using a gas chromatography mass spectrometry (GCMS) based assay, as described in Mani et al. Biochem. J. (2003) 374, 521-527, and in Dynamic assessment of nitration reactions in vivo.
  • GCMS gas chromatography mass spectrometry
  • urinary 3-nitro-HPA was markedly increased in patients with Helicobacter pylori infection and this was unaffected by alterations in dietary nitrate or gastric pH.
  • l-ethyl-3- (3-di ⁇ nethylaminopropyl) carbodiimide was pre-reacted with N-hydroxysuccinimide in buffer at pH 4.6, followed by the addition of a 4-fold excess of 3-nitro-p- hydroxyphenylacetic acid. Excess unreacted reagents were reduced with mercaptoethanol , and the resulting solution added to lysozyme in water, and left for 2-3 hours at room temperature . The final product was dialysed against water for 2 days, and then analysed by mass spectrometry. Two molecules of 3-nitro-p-hydroxyphenylacetic acid were coupled to each molecule of lysozyme . This method can be used to generate a conjugate with a protein which can then be used as an immunogen to generate antibodies specific for 3-nitro- p-hydroxyphenylacetic acid.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne la détection de l'acide 3-nitro-hydroxyphénylacétique urinaire servant de biomarqueur dans l'infection à H. pylori. L'invention concerne également des trousses de test et des dispositifs utilisés dans la détection de l'acide 3-nitro-hydroxyphénylacétique urinaire.
PCT/GB2007/003703 2006-09-28 2007-09-28 Test concernant une infection à h. pylori Ceased WO2008038022A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07804410A EP2078200A1 (fr) 2006-09-28 2007-09-28 Test concernant une infection à h. pylori

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0619166A GB0619166D0 (en) 2006-09-28 2006-09-28 Test for H.pylori infection
GB0619166.2 2006-09-28

Publications (1)

Publication Number Publication Date
WO2008038022A1 true WO2008038022A1 (fr) 2008-04-03

Family

ID=37434858

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/003703 Ceased WO2008038022A1 (fr) 2006-09-28 2007-09-28 Test concernant une infection à h. pylori

Country Status (3)

Country Link
EP (1) EP2078200A1 (fr)
GB (1) GB0619166D0 (fr)
WO (1) WO2008038022A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001368A1 (fr) * 1989-07-24 1991-02-07 Dynal A.S. Liaison d'affinite d'haptene/antihaptene dans la separation des cellules

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001368A1 (fr) * 1989-07-24 1991-02-07 Dynal A.S. Liaison d'affinite d'haptene/antihaptene dans la separation des cellules

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOSE R ET AL: "Immunochemical properties of some monoclonal IgE antibodies to 4-hydroxy-3-nitrophenylacetyl (NP)", IMMUNOLOGY 1984 UNITED KINGDOM, vol. 53, no. 4, 1984, pages 801 - 809, XP002458722, ISSN: 0019-2805 *
MANI ALI R ET AL: "Nitration of endogenous para-hydroxyphenylacetic acid and the metabolism of nitrotyrosine.", BIOCHEMICAL JOURNAL, vol. 374, no. 2, 1 September 2003 (2003-09-01), pages 521 - 527, XP002458721, ISSN: 0264-6021 *
TARI AKIRA ET AL: "Does serum nitrite concentration reflect gastric carcinogenesis in Japanese Helicobacter pylori-infected patients?", DIGESTIVE DISEASES AND SCIENCES, vol. 47, no. 1, January 2002 (2002-01-01), pages 100 - 106, XP002458720, ISSN: 0163-2116 *

Also Published As

Publication number Publication date
EP2078200A1 (fr) 2009-07-15
GB0619166D0 (en) 2006-11-08

Similar Documents

Publication Publication Date Title
Graham et al. Noninvasive versus histologic detection of gastric atrophy in a Hispanic population in North America
Leja et al. The validity of a biomarker method for indirect detection of gastric mucosal atrophy versus standard histopathology
Ebule et al. Helicobacter pylori infection and atrophic gastritis
EP1348129A1 (fr) Procede servant a diagnostiquer la gastrite atrophique
Lau et al. Qualification and application of an ELISA for the determination of Tamm Horsfall protein (THP) in human urine and its use for screening of kidney stone disease
CN105717308A (zh) 快速定量检测粪便乳铁蛋白的免疫层析试剂盒
KR100237239B1 (ko) 심장 트로포닌 1의 초감도 분석방법
Wunderlich et al. A high-sensitivity enzyme-linked immunosorbent assay for serum thyroglobulin
WO2020005947A9 (fr) Procédés et kits de détection de 11-déshydro-thromboxane b2
US7252955B2 (en) Method of detecting colon cancer
Pelerito et al. Evaluation of rapid test Assure® Helicobacter pylori for diagnosis of H. pylori in pediatric population
EP2078200A1 (fr) Test concernant une infection à h. pylori
EP3816629B1 (fr) Kit de suivi et de diagnostic du degré de l'hépatite chronique progressive et de la fibrose hépatique par la mesure de l'asialo a1 glycoprotéine-acide en tant que marqueur de lésions hépatocellulaires et son utilisation
WO2023281160A1 (fr) Panel gastrique unifié en une microplaque
JP5663494B2 (ja) 尿検体中のサブスタンスｐの測定方法
WO2010142984A2 (fr) Dosage de l'aspirine
Demian et al. Evaluation of the analytical performance of the novel NS-Prime system and examination of temperature stability of fecal transferrin compared with fecal hemoglobin as biomarkers in a colon cancer screening program
US20090011431A1 (en) Diagnosis of Sepsis by the Selective Determination of the Concentration of Cu/Zn Superoxide Dismutase (Cu/Zn Sod) in Patient Samples
WO1997024141A1 (fr) Anticorps monoclonaux et methode de capture immunologique pour la quantification et la differenciation d'especes d'hematozoaires
US20130157291A1 (en) DIAGNOSTIC MARKER FOR LUNG CANCER COMPRISING HPaR AS ACTIVE INGREDIENT
RU2338462C1 (ru) Способ определения прогрессирования течения опухолевого процесса
RU2180117C1 (ru) Способ оценки сенсибилизации к химическим соединениям
Bandara et al. Glycated Haemoglobin A1c in Contrast to Conventional and Novel Biomarkers in the Diagnosis of Diabetes and Monitoring of Glycemic Control: A Review
KR20080048200A (ko) 알파1산 당단백질과 아사이알로 알파1산 당단백질의 비율을 이용한 간질환 진단킷트
Shakerian et al. Different combinations of monoclonal antibodies and polyclonal antibodies in the design of neonatal hypothyroidism diagnostic kit

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07804410

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2009529769

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2007804410

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP