[go: up one dir, main page]

WO1999033463A9 - LACTONES SESQUITERPENE INHIBANT SPECIFIQUEMENT L'ACTIVATION DU NF-λB EN EMPECHANT LA DEGRADATION DES PROTEINES IλB-α et IλB-$g(b) - Google Patents

LACTONES SESQUITERPENE INHIBANT SPECIFIQUEMENT L'ACTIVATION DU NF-λB EN EMPECHANT LA DEGRADATION DES PROTEINES IλB-α et IλB-$g(b)

Info

Publication number
WO1999033463A9
WO1999033463A9 PCT/IB1998/002108 IB9802108W WO9933463A9 WO 1999033463 A9 WO1999033463 A9 WO 1999033463A9 IB 9802108 W IB9802108 W IB 9802108W WO 9933463 A9 WO9933463 A9 WO 9933463A9
Authority
WO
WIPO (PCT)
Prior art keywords
sls
cells
iκb
cell
tnf
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/IB1998/002108
Other languages
English (en)
Other versions
WO1999033463A1 (fr
Inventor
Michael Heinrich
M Lienhard Schmitz
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Priority to AU15734/99A priority Critical patent/AU1573499A/en
Publication of WO1999033463A1 publication Critical patent/WO1999033463A1/fr
Publication of WO1999033463A9 publication Critical patent/WO1999033463A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones

Definitions

  • Sesquiterpene lactones specifically inhibit activation of NF- ⁇ B by preventing the
  • the present invention is concerned with the use of extracts from certain Mexican indian medicinal plants applied for the treatment of inflammations. These extracts contain
  • the transcription factor NF- ⁇ B is one of the key regulators of genes involved in the
  • F- ⁇ B is
  • ROIs reactive oxygen intermediates
  • I ⁇ B- ⁇ and I ⁇ B- ⁇ are major forms of I ⁇ B proteins.
  • NF- ⁇ B transcription is mainly regulated by NF- ⁇ B include many cytokines, cell adhesion molecules,
  • intercellular adhesion molecule 1 ICM-1
  • VCAM-1 vascular cell adhesion molecule 1
  • acute-phase proteins and immunoreceptors for review see 15
  • NF- ⁇ B has been shown recently to counteract the induction of apoptosis by the
  • Glutamate was found to induce NF- ⁇ B in neuronal cells and acetylsalicylic acid (Aspirin ® )
  • NF-KB inhibitors exerts its inhibitory effects by scavenging ROIs. These inhibitors include N-
  • Another group of inhibitors interferes with the induced degradation of I ⁇ B-
  • inhibitors of NF- ⁇ B exert their effects only in the cell nucleus by impairing the transcriptional
  • glucocorticoids the activated glucocorticoid
  • WO 96/25666 (PCT US96/02122) describes a polypeptide I ⁇ B- ⁇ which binds to and
  • PCT/US9204073 describes the purification, cloning, expression and characterization of I ⁇ B.
  • SL-containing plant extracts are frequently used in the traditional Mexican indian medicine for the treatment of infections of the skin and other organs (for review see 38).
  • the SL parthenolide is also contained in drugs such as Feverfew ® (Tanacetum parthenium) used against migraine, an illness that has been implicated with neurogenic inflammatory processes (39).
  • Feverfew ® Teacetum parthenium
  • the anti-inflammatory activity of the SL-containing plant extracts was confirmed in the hen egg tests where they showed a delay in cell culture experiments (40) and the onset of capillary reactions of the allantois membrane (1).
  • the present invention shows that SLs prevent a common step in NF- ⁇ B activation.
  • ICAM-1 Structural studies identified the exomethylene group in conjugation with the lactone group as the decisive structural feature for the inhibitory activity.
  • SLs Numerous biological activities have been reported for SLs, including antimicrobial (48), antiviral (49) and antitumor activities (50). Furthermore SLs or SL-containing plant extracts were found to have anti-inflammatory properties (51, 52). Anti-phlogistic activities were also seen in hen eggs assays, in the reduced production of the inflammatory cytokine IL- 6 and in cell culture experiments (1, 40). It was previously reported that the anti-inflammatory effects of SLs can be assigned -at least to a certain extent- to the inhibition of transcription
  • DNA-binding subunits of NF- ⁇ B DNA-binding subunits of NF- ⁇ B. Also the I ⁇ B subunits seems not to be a direct target for the
  • I ⁇ B proteins an unlikely candidate for the SLs, since I ⁇ B- ⁇ is completely resynthesized within
  • transducing events leading to NF- ⁇ B include the small GTP-binding proteins Racl (54) and Cdc 42 (55) which then lead to the activation of mitogen-activated protein kinase/ERK kinase kinase- 1 (MEKK-1).
  • MEKK-1 mitogen-activated protein kinase/ERK kinase kinase- 1
  • the SLs tested in this study display a high degree of specificity for their inhibitory activity, since they did not influence the activity of other transcription factors such as AP-1,
  • SLs do not interfere in a non-specific manner with transcription factors or signaling molecules.
  • a potential target-specificity of SLs may well be explained by considering the fact that the combination of the reactive Michael- acceptor system together with the oxygen-substituted isoprenoide rings forms a pattern of potential non-covalent binding sites (e.g. hydrogen bonds). These binding sites would allow the SLs to interact with complementary sites on the surface of the target molecule(s).
  • optimal blocking may presumably be reduced in such a blend, thereby reducing the respective side effects.
  • the inhibition of NF- ⁇ B may be of therapeutic use for the treatment of chronic
  • Alzheimer ' s disease involve the activation of NF- ⁇ B.
  • the amyloid ⁇ peptide which is a major amyloid ⁇ peptide
  • SLs inhibit NF- ⁇ B activation by different stimuli.
  • Fig. 2. SLs do not interfere with DNA-binding of activated NF- ⁇ B.
  • Fig. 3 Effects of SLs on DNA-binding of Oct-1 and the fragmentation of I ⁇ B- ⁇ .
  • SLs inhibit the degradation of I ⁇ B- ⁇ and I ⁇ B- ⁇ induced by various stimuli.
  • Fig. 6 Effect of parthenolide on the cell killing by TNF- ⁇ .
  • Fig. 9 Distinct positions of NF- ⁇ B inhibitors in the activation cascade.
  • Fig. 1 A shows the inhibition of NF- ⁇ B activated by H 2 O 2 .
  • Jurkat JR cells were preincubated with the indicated amounts of parthenolide for one h and stimulated with various concentrations of H 2 O 2 for 90 min. Subsequently total cell extracts were prepared and tested for DNA-binding
  • the filled arrowhead indicates the location of the
  • DNA-NF- ⁇ B complex the circle indicates the position of a constitutively DNA-binding protein
  • Fig. 2 HeLa cells were stimulated for 20 min with PMA and total cell extracts of the stimulated cells were pooled. These extracts were incubated for 1 h with various concentrations of parthenolide as indicated. Subsequently these extracts were tested together with a protein extract from unstimulated HeLa cells as a control for DNA-binding activity of NF- ⁇ B by EMS A. Bound
  • C DNA-binding activity of Oct-1.
  • the indicated cell extracts were tested for the activity of the constitutively DNA-binding protein Oct-1 by EMSA.
  • the open arrowhead points to the position of the unbound oligonucleotide, the filled arrowhead indicates the position of the DNA-protein complex.
  • Fig. 4 Cells were preincubated with 10 ⁇ M of parthenolide 1 h prior to stimulation with 2000 U
  • TNF- ⁇ A
  • 50 ng/ml PMA B
  • ligation of the CD3/CD28 receptors C
  • HeLa cells were preincubated with 5 ⁇ M of parthenolide for 1 h. Subsequently the medium was replaced by parthenolide-free medium and the cells were grown for the indicated
  • parthenolide-free medium was set as 100%. A typical experiment is shown.
  • Fig. 7 A, effect of TNF- ⁇ on the expression of ICAM-1. Jurkat JR cells were stimulated for 10 h
  • Fig. 8. effects of TNF- ⁇ on the concentration of intracellular ROIs.
  • Jurkat JR cells were either
  • T-cells were incubated with 5 ⁇ M parthenolide and further treated and analyzed as described in
  • C comparison of ROI amounts.
  • the mean fluorescence of unstimulated cells was set as 1 and directly compared to the fluorescence of the TNF-stimulated cells in the presence or absence of parthenolide as indicated.
  • B7-1 B-cell activation antigen
  • PKC protein kinase C
  • TCR T-cell receptor
  • TNF- ⁇ and poly (dl-dC) were obtained
  • Antibodies directed against I ⁇ B- ⁇ and NF- B were from Santa Cruz Inc. (Santa Cruz,
  • ⁇ -CD28 antibodies were obtained from Pharmingen Inc. (San Diego, CA) and ⁇ -CD3
  • Electrophoretic mobility shift assay (EMSA)
  • HeLa or L929 cells (5 x 10 5 ) were grown overnight on 10 cm dishes, Jurkat cells (approximately
  • myristate 13 -acetate (PMA) or hydrogen peroxide cells were preincubated with the indicated amounts of the tested substances for 60 minutes at 37°C.
  • the pellet was resuspended in TOTEX buffer (20 mM Hepes/KOH pH 7.9, 0.35 M NaCl, 20% (v/v) glycerol, 1% (v/v) NP-40, 1 mM MgCl 2 , 0.5 mM EDTA, 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF)) and incubated on ice for 30 min. The samples were carefully vortexed every 10 min. The cell debris was pelleted upon centrifligation with 14000 rpm at 4°C for 10 min. Equal amounts of supernatant were tested for DNA binding activity as described in detail elsewhere (1). Briefly, the
  • oligonucleotide on ice in 5 x binding buffer (20% (w/v) Ficoll 400, 100 mM Hepes/KOH pH 7.9, 1 mM DTT and 300 mM KC1) in a final volume of 20 ⁇ l. Subsequently the free and the oligonucleotide-bound proteins were separated by electrophoresis on a native 4% polyacrylamide gel. The gel was dried after electrophoresis and exposed to an X-ray film (Amersham Hyperfilm). The following oligonucleotides (binding site underlined) were used:
  • NF- ⁇ B 5'-AGTTGAGGGGACrrTCCCAGGC-3'
  • TBST buffer 25 mM Tris/HCl pH 7.4, 137 mM NaCl, 5 mM KC1, 0.7 mM CaCl 2 , 0.1 mM MgCl 2 , 0.05 % (v/v) Tween-20).
  • the Src family protein tyrosine kinases p60 src and p59 fyn were expressed in baculovirus-infected Sf9 cells and purified by affinity chromatography as described (41). The effect of SLs on protein kinase activity was determined using enolase as a substrate. Briefly, serial concentrations of
  • parthenoUde and isohelenin were preincubated at 30°C with p ⁇ O 8 " or p59 fyn protein in 50 ⁇ l
  • reaction was ended by the addition of Laemmli buffer.
  • samples were analyzed by SDS- PAGE and subjected to autoradiography.
  • TNF cytotoxic activity of TNF was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide assay (MTT assay) essentially as described by Mosmann (42).
  • L929 cells were seeded at a density of lxl 0 4 cells per well in 96-well microtiter plates (flat bottomed) and incubated for 16 h in 0.2 ml culture medium. The supernatant was then removed and replaced by fresh medium containing TNF (2000 U/ml) or/and parthenolide (5 ⁇ M). At the indicated times 20 ⁇ l of a MTT solution (5 mg/ml PBS) was added to all wells. After another 3 h incubation supernatants were removed followed by addition of 100 ⁇ l of a 24:1 (v/v) isopropanol HCl solution. After 15 min at room temperature the absorbance of each well was determined with an automated plate reader (Digiscan, Asys Hitech, Austria) at 550 nm.
  • DFCH dichlorofluorescein-diacetate
  • PBS phosphate-buffered saline
  • ICAM-1 expression on Jurkat cells was determined using a FITC- conjugated anti-CD 54 (ICAM-1 )-antibody (Dianova, Hamburg, Germany). In both cases measurements were performed in duplicates using a FACScan (Becton Dickinson, Heidelberg, Germany) flow cytometer. Dead cells were excluded by forward/side scatter gating and staining with propidium iodide.
  • Example 1 SLs inhibit a common step in NF- B activation
  • Example 2 SLs do not affect activity of Src family protein tyrosine kinases
  • Tyrosine kinases of the Src family have been implicated in NF- ⁇ B activation in response to various stimuli including UV radiation, T-cell receptor ligation and stimulation with prooxidants.
  • binding complex was confirmed to be a p50/p65 NF- ⁇ B dimer by competition assays with
  • CD3/CD28 ligation is reflected by a degradation of the I ⁇ B- ⁇ and I ⁇ B- ⁇ proteins, which again
  • L929 cells were incubated either with 5 ⁇ M parthenolide or 2000 U/ml TNF- ⁇ alone or by a
  • inhibitor parthenolide is also enhancing the TNF- ⁇ -induced cell killing of mouse L929 cells.
  • the inducible transcription of the ICAM-1 gene in response to TNF- ⁇ , EL- IB and PMA is
  • ICAM-1 surface expression a good read-out to test the effect of SLs on the expression of
  • SLs transcription of NF- ⁇ B target genes is specifically inhibited by SLs.
  • SLs Two structural hallmarks of SLs are an isoprenoide ring system and a
  • lactone ring contains a conjugated exomethylene group. Both groups together form a reactive Michael system which is a target for nucleophilic substrates, e.g. for cysteine residues in proteins.
  • Example 8 SLs inhibit NF- ⁇ B activation without having anti-oxidative properties
  • parthenolide revealed the structure of the parthenolide binding site on I ⁇ B- ⁇ and I ⁇ B- ⁇ .
  • the structural data as resulting from mass spectroscopic studies (results are not shown) are relevant for rational drug design.
  • CHUK conserved helix-loop-helix ubiquitous kinase
  • DFCH dichlorofluorescein-diacetate
  • DMEM Dulbecco's modified Eagle medium
  • DMSO dimethyl sulfoxide
  • EMSA EMSA
  • ICAM-1 intercellular adhesion molecule- 1
  • DCK I ⁇ B
  • kinase kinase
  • JNK c-Jun N-terminal kinase
  • MEKK-1 mitogen-activated protein kinase/ERK kinase kinase- 1
  • MKK4 mitogen-activated protein kinase kinase 4
  • MTT 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide
  • PBS phosphate-buffered saline
  • PMA phorbol- 12- myristate 13 -acetate
  • PMSF phenylmethylsulfonyl fluoride
  • PVDF polyvinylidene difluoride
  • ROIs reactive oxygen intermediates
  • SLs sequite ⁇ ene lactones
  • TNF tumor necrosis factor
  • VCAM-1 vascular cell adhesion molecule- 1.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Des extraits de plantes médicinales des Indiens du Mexique utilisées dans la médecine indigène traditionnelle dans le traitement des inflammations contiennent des lactones sesquiterpène (SL), qui inhibent spécifiquement le facteur de transcription NF-λB (1). Nous démontrons ici que les SL empêchent l'activation du NF-λB par différents stimuli, tels que les phorbolesters, le facteur de nécrose tumorale (TNF)-α, la ligation du récepteur des lymphocytes T et le peroxyde d'hydrogène dans divers types de cellules. Le traitement de cellules avec des SL empêche la dégradation induite des protéines IλB-α et IλB-β par tous ces stimuli, ce qui suggère que ces lactones interfèrent avec une phase plutôt commune de l'activation du NF-λB. Les lactones SL n'interfèrent ni avec l'activité de fixation de l'ADN du NF-λB activé ni avec l'activité des protéines tyrosine-kinases p59?fyn et p60src¿. Des quantités micromolaires de SL empêchent l'expression induite du gène ICAM-1 cible du NF-λB. L'inhibition du NF-λB par les SL entraîne une destruction accrue des cellules des fibroblastes murins par le TNF-α. Les SL à qui il manque un groupe exométhylène en conjugaison avec la fonction lactone ne déploient aucune activité inhibitrice sur le NF-λB. L'analyse de l'état de réduction-oxydation cellulaire par FACS montre que les lactones SL n'ont aucune propriété anti-oxydante directe ou indirecte.
PCT/IB1998/002108 1997-12-23 1998-12-23 LACTONES SESQUITERPENE INHIBANT SPECIFIQUEMENT L'ACTIVATION DU NF-λB EN EMPECHANT LA DEGRADATION DES PROTEINES IλB-α et IλB-$g(b) Ceased WO1999033463A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU15734/99A AU1573499A (en) 1997-12-23 1998-12-23 Sesquiterpene lactones specifically inhibit activation of nf-k B by preventing the degradation of IkB-alpha and IkB-beta

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6856597P 1997-12-23 1997-12-23
US60/068,565 1997-12-23

Publications (2)

Publication Number Publication Date
WO1999033463A1 WO1999033463A1 (fr) 1999-07-08
WO1999033463A9 true WO1999033463A9 (fr) 1999-09-23

Family

ID=22083358

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB1998/002108 Ceased WO1999033463A1 (fr) 1997-12-23 1998-12-23 LACTONES SESQUITERPENE INHIBANT SPECIFIQUEMENT L'ACTIVATION DU NF-λB EN EMPECHANT LA DEGRADATION DES PROTEINES IλB-α et IλB-$g(b)

Country Status (2)

Country Link
AU (1) AU1573499A (fr)
WO (1) WO1999033463A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6890946B2 (en) 1999-12-23 2005-05-10 Indiana University Research And Technology Corporation Use of parthenolide to inhibit cancer
WO2001045699A1 (fr) * 1999-12-23 2001-06-28 Advanced Research And Technology Institute, Inc. Utilisation du parthenolide pour inhiber le cancer
DE10048596A1 (de) * 2000-09-30 2002-04-25 Henkel Kgaa Entzündungshemmende Wirkstoffe
US20060148732A1 (en) * 2000-11-17 2006-07-06 Gutterman Jordan U Inhibition of NF-kappaB by triterpene compositions

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1270843A (fr) * 1982-05-19 1990-06-26 Edward Stewart Johnson Lactones sesquiterpenes
GB9100581D0 (en) * 1991-01-11 1991-02-27 Rhodes Technology Method for the extraction of sesquiterpene lactones
DE4202657C2 (de) * 1992-01-31 1995-10-12 Schaper & Bruemmer Gmbh Pharmazeutisch wirksame Zusammensetzung aus Tanacetum parthenium sowie Verfahren zu deren Extraktion und mit der pharmazeutisch wirksamen Zusammensetzung hergestelltes Arzneimittel
FR2695931B1 (fr) * 1992-09-24 1994-10-28 Madeca Procédé pour l'obtention de lactone sesquiterpénique de parthénolide, et préparation thérapeutique contenant un tel produit pour le traitement de la migraine.

Also Published As

Publication number Publication date
AU1573499A (en) 1999-07-19
WO1999033463A1 (fr) 1999-07-08

Similar Documents

Publication Publication Date Title
Hehner et al. Sesquiterpene lactones specifically inhibit activation of NF-κB by preventing the degradation of IκB-α and IκB-β
Endale et al. Quercetin disrupts tyrosine-phosphorylated phosphatidylinositol 3-kinase and myeloid differentiation factor-88 association, and inhibits MAPK/AP-1 and IKK/NF-κB-induced inflammatory mediators production in RAW 264.7 cells
Wang et al. Plumbagin inhibits LPS-induced inflammation through the inactivation of the nuclear factor-kappa B and mitogen activated protein kinase signaling pathways in RAW 264.7 cells
Hai et al. Characterization of histone deacetylase mechanisms in cancer development
Li et al. Combination of quercetin and cisplatin enhances apoptosis in OSCC cells by downregulating xIAP through the NF-κB pathway
Damia et al. Cisplatinum and taxol induce different patterns of p53 phosphorylation
Park et al. Britanin suppresses LPS-induced nitric oxide, PGE2 and cytokine production via NF-κB and MAPK inactivation in RAW 264.7 cells
Zhou et al. The isomerase PIN1 controls numerous cancer-driving pathways and is a unique drug target
Cho et al. Zedoarondiol isolated from the rhizoma of Curcuma heyneana is involved in the inhibition of iNOS, COX-2 and pro-inflammatory cytokines via the downregulation of NF-κB pathway in LPS-stimulated murine macrophages
Zhai et al. Protective effects of paeonol on inflammatory response in IL-1β-induced human fibroblast-like synoviocytes and rheumatoid arthritis progression via modulating NF-κB pathway
Lin et al. Targeting histone deacetylase in cancer therapy
Philip et al. Osteopontin induces nuclear factor κB-mediated promatrix metalloproteinase-2 activation through IκBα/IKK signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways
Wang et al. Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases
Chang et al. Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade
Gong et al. Triptolide inhibits COX‐2 expression and PGE2 release by suppressing the activity of NF‐κB and JNK in LPS‐treated microglia
Liu et al. A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway
Chang et al. Regulatory role of mitochondria in oxidative stress and atherosclerosis
Lin et al. Ellagic acid protects ovariectomy‐induced bone loss in mice by inhibiting osteoclast differentiation and bone resorption
Shang et al. Antitumor activity of isosteroidal alkaloids from the plants in the genus Veratrum and Fritillaria
Kim et al. Suppression by a sesquiterpene lactone from Carpesium divaricatum of inducible nitric oxide synthase by inhibiting nuclear factor-κB activation
Shi et al. Kanglaite inhibits EMT caused by TNF-α via NF-κΒ inhibition in colorectal cancer cells
Zhang et al. Vomifoliol isolated from mangrove plant Ceriops tagal inhibits the NFAT signaling pathway with CN as the target enzyme in vitro
Martínez-Martínez et al. Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells
Zhang et al. Polo-like kinase 2: from principle to practice
Wang et al. Apigenin alleviates myocardial reperfusion injury in rats by downregulating miR-15b

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AT AU AZ BA BB BG BR BY CA CH CN CU CZ CZ DE DE DK DK EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: C2

Designated state(s): AL AM AT AT AU AZ BA BB BG BR BY CA CH CN CU CZ CZ DE DE DK DK EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/10-10/10, DRAWINGS, REPLACED BY NEW PAGES 1/10-10/10; AFTER RECTIFICATION OF OBVIOUS ERRORSAS AUTHORIZED BY THE INTERNATIONAL SEARCHING AUTHORITY

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase