WO2004084902A1 - Utilisation d'une trioxopyrimidine dans le traitement de lesions chroniques - Google Patents

Utilisation d'une trioxopyrimidine dans le traitement de lesions chroniques Download PDF

Info

Publication number
WO2004084902A1
WO2004084902A1 PCT/EP2004/003203 EP2004003203W WO2004084902A1 WO 2004084902 A1 WO2004084902 A1 WO 2004084902A1 EP 2004003203 W EP2004003203 W EP 2004003203W WO 2004084902 A1 WO2004084902 A1 WO 2004084902A1
Authority
WO
WIPO (PCT)
Prior art keywords
mmp
trione
piperazin
pyrimidine
wound
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/EP2004/003203
Other languages
English (en)
Inventor
Nuray Dagdelen
Hans-Willi Krell
Frank Roesken
Okan Safak
Christian Sommerhoff
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.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche 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
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Publication of WO2004084902A1 publication Critical patent/WO2004084902A1/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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A61K31/515—Barbituric acids; Derivatives thereof, e.g. sodium pentobarbital
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Definitions

  • the present invention relates to the use of a trioxopyrimidine compound for the treatment and prevention of chronic wounds.
  • MMPs Matrix Metalloproteinases
  • TMPs endogenous inhibitors
  • ECM extracellular matrix
  • MMPs are produced by several different types of cells in skin including fibroblasts, keratinocytes, macrophages, endothelial cells, mast cells, and eosinophils.
  • MMPs are not constitutively expressed in skin but are induced temporarily in response to exogenous signals such as cytokines, growth factors, cell-matrix interactions and altered cell-to-cell contacts.
  • MMPs also play important roles ⁇ n various normal physiologic situations, including morphogenesis of tissues during embryological development, angiogenesis, parturition, ovulation and remodelling of scar tissue by proteolytically restructuring the extra-cellular matrix.
  • MMPs can also contribute to pathological conditions that are characterized by the excessive breakdown of ECM components such as in rheumatoid arthritis, tumor cell invasion and metastasis.
  • MMPs seem to be involved in various processes.
  • MMPs In the first phase of wound repair, MMPs participate in the removal of de-vitalized tissue. During the repair phase, MMP activities are necessary for angiogenesis, for contraction of wound matrix, for migration of fibroblasts, and for keratinocyte migration and epithelialisation.
  • TIMPs are relatively small proteins (21 000 to 29 000 M r ) yet they have several biological functions, including inhibition of active MMPs, stimulation of cell division, binding to ECM, inhibition of angiogenesis and induction of apoptosis.
  • Matrix metalloproteases are a family of zinc- and calcium-dependent proteases that are capable of degrading the extracellular matrix (ECM) and basement membrane (Egeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161- 174; Overall, CM., and Lopez-Otin, C, Nat. Rev. Cancer 2 (2002) 657-672). They are believed to have pivotal roles in embryonic development and growth (Holmbeck, K., et al., Cell 99 (1999) 81-92; Vu, T.H., et al, Cell 93 (1998) 411-422) as well as in tissue remodeling and repair (Shapiro, S.D., Curr. Opin. Cell Biol. 10
  • MMPs may therefore contribute to the pathogenesis of many tissue-destructive processes, including tumor progression (Egeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161-174; Overall, CM., and Lopez-Otin, C, Nat. Rev. Cancer 2 (2002) 657-672) and aneurysm formation (Carmeliet, P., et al.,
  • MMP effects are far from being restricted to ECM degradation (Chang, C, and Werb, D., Trends Cell Biol. 11 (2001) S37-43).
  • Peptide growth factors that are sequestered by ECM proteins become available once degraded by MMP-9 (Manes, S., et al, J. Biol. Chem. 274 (1999) 6935-6945).
  • MMPs can increase the bioavailability of VEGF (Bergers, C, et al., Nat. Cell Biol. 2
  • angiogenesis inhibitors such as angiostatin by cleavage of plasminogen (Dong, Z., et al., Cell 88 (1997) 801-810).
  • MMPs Tissue Inhibitors of Metalloproteases
  • Tissue Inhibitors of Metalloproteases Tissue Inhibitors of Metalloproteases
  • Low molecular weight inhibitors of MMPs are derived from the hydroxamic acid compound class and inhibit MMPs in a broad manner, being not selective for MMP-2 and MMP-9, the key MMPs in tumor invasion, metastatic spread, and angiogenesis.
  • MMP inhibiting molecules from another structural class, the trioxopyrimidines have been described, e.g. in WO 97/23465 and WO 01/25217. This class of compounds is extremely potent, and highly selective, with an almost exclusive specificity for MMP-2, MMP-9, while sparing most other members of the MMP family of proteases.
  • MMP inhibitors predominantly of the hydroxamic acid substance class with broad substrate specificity were, and in part still are, in clinical testing for anti- tumor treatment. All of the published clinical results with these inhibitors were disappointing, showing little or no clinical efficacy (Fletcher, L., Nat. Biotechnol. 18 (2000) 1138-1139). The reason for this lack of efficacy in the clinic most likely is the fact that patients could not be given high enough doses for anti-tumor or anti- metastatic activity because of the side effects associated with these broadly acting inhibitors. These dose-limiting side effects were predominantly arthralgias and myalgias (Drummond, A.H., et al., Ann. N.Y. Acad. Sci.
  • inhibitors of metalloproteinases for wound healing in general is known in the state of the art.
  • Such inhibitors are for example hydroxamic acids (US 2003/0,050,310; US 6,465,508). Description of the Invention
  • trioxopyrimidine-based MMP inhibitors which are highly selective for MMP-2, MMP-9 and MMP-14 are useful for the promoting of wound healing, preferably of skin wounds, especially of chronic skin wounds.
  • the invention therefore provides the use of a trioxopyrimidine compound having an inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 defined as
  • IC 50 values are measured by an in vitro assay for MMP enzymatic activity.
  • an assay is described by Stack, M.S., and Gray, R.D., J. Biol. Chem. 264 (1989) 4277- 4281. This assay is based on the determination of MMP enzymatic activity on a dinitrophenol substrate and fluorescence measurement of the substrate after cleaving by MMPs.
  • the invention further provides the use of such trioxopyrimidine compounds for the manufacturing of a medicament for the promoting of wound healing.
  • wound denotes a bodily injury with disruption of the normal integrity of tissue structures in the skin.
  • the term is also intended to encompass the terms “sore”, “lesion”, “necrosis” and “ulcer”.
  • the term “sore” is a popular term for almost any lesion of the skin and the term “ulcer” is a local defect, or excavation, of the surface of an organ or tissue, which is produced by the sloughing of necrotic tissue.
  • Lesion generally relates to any tissue defect. Necrosis is related to dead tissue resulting from infection, injury, inflammation or infarctions.
  • wound denotes any wound and at any particular stage in the healing process including the stage before any healing has initiated, and especially a chronic wound.
  • wounds which can be prevented and/or treated in accordance with the present invention are, e.g., aseptic wounds, contused wounds, incised wounds, lacerated wounds, non-penetrating wounds (i.e. wounds in which there is no disruption of the skin but there is injury to underlying structures), open wounds, penetrating wound, perforating wounds, puncture wounds, septic wounds, subcutaneous wounds, etc.
  • sores are bed sores, cancer sores, chrome sores, cold sores, pressure sores etc.
  • ulcers are, e.g., peptic ulcer, duodenal ulcer, gastric ulcer, gouty ulcer, diabetic ulcer, hypertensive ischemic ulcer, stasis ulcer, ulcus cruris (venous ulcer), sublingual ulcer, submocous clear, symptomatic ulcer, trophic ulcer, tropical ulcer, veneral ulcer, e.g. caused by gonorrhoea (including urethritis, endocervicitis and proctitis).
  • wounds or sores Conditions related to wounds or sores which may be successfully treated according to the invention are burns, anthrax, tetanus, gas gangrene, scalatina, erysipelas, sycosis barbae, folliculitis, impetigo contagiosa, or impetigo bullosa, etc. Therefore as mentioned above, in the present context the term “wound” encompasses the term "ulcer”,
  • skin is used in a very broad sense embracing the epidermal layer of the skin and— in those cases where the skin surface is more or less injured— also the dermal layer of the skin. Apart from the stratum corneum, the epidermal layer of the skin is the outer (epithelial) layer and the deeper connective tissue layer of the skin is called the dermis.
  • the body provides mechanisms for healing injured skin or mucosa in order to restore the integrity of the skin barrier or the mucosa.
  • the repair process for even minor ruptures or wounds may take a period of time extending from hours and days to weeks. However, in ulceration, the healing can be very slow and the wound may persist for an extended period of time, i.e. months or even years.
  • Matrix metalloproteinases are well-known in the state of the art and are defined, e.g., by their EC numbers (MMP-1 EC 3.4.24.7; MMP-2 EC 3.4.24.24; MMP-3 EC 3.4.24.17, MMP-9 EC 3.4.24.35, MMP-14 EC 3.4.24).
  • Trioxopyrimidines useful for the invention are compounds from a well-known structural class. Such compounds are described in, for example, US Patent Nos.
  • the exact dosage of the MMP inhibitors will vary, but can be easily determined. In general, the daily dosage of the inhibitors will range between 1 nmol/kg and day to 1 mmol/kg and day.
  • the pharmaceutical compositions are aqueous compositions having physiological compatibility.
  • the compositions include, in addition, auxiliary substances, buffers, preservatives, solvents and/or viscosity modulating agents.
  • Appropriate buffer systems are based on sodium phosphate, sodium acetate or sodium borate.
  • Preservatives are required to prevent microbial contamination of the pharmaceutical composition during use. Suitable preservatives are, for example, benzalkonium chloride, chlorobutanol, methylparabene, propylparabene, phenylethyl alcohol, sorbic acid. Such preservatives are used typically in an amount of 0.01 to 1% weight/volume.
  • a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • a pharmaceutically acceptable substances include saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • compositions of the invention for parenteral administration contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphtalenes, and the like.
  • polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphtalenes, and the like.
  • biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxethylene-polyoxypropylene copolymers are examples of excipients for delayed release of a compound of the invention in vivo.
  • Other suitable parenteral delivery systems include ethylene-vinyl acetate copolymer particles and liposomes.
  • Formulations for inhalation administration may contain excipients such as lactose, if desired.
  • composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol.
  • phospholipids particularly high-phase-transition-temperature phospholipids
  • steroids especially cholesterol.
  • Other phospholipids or other lipids may also be used.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • the therapeutic agents useful in the method of the invention can be administered parenterally by injection or by gradual perfusion over time. Administration may be intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride
  • lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents and inert gases and the like.
  • Figure 1 shows the growth of granulation tissue after local application of
  • Gelatinase-selective MMP-Inhibitors were investigated using a rat wound model.
  • Isofluran gas (1.5%/3l O 2 )
  • a 3 cm median section in the dorsum was performed and a subcutaneous pocket was formed on both sides of the section.
  • a preliminary cut was made with a punch (diameter 1.1 cm) above each pocket and skin as well as subcutaneous tissue was excised by using microsurgical forceps and scissors to create a circular wound area on the top of the latissimus dorsi muscle.
  • a polyethylene ring was implanted on the fascia of latissimus dorsi muscle and fixed by four sutures (Prolene 4.0, Ethicon).
  • the ring consist of eight perforations arranged circularly at the side of the ring (Diameter 0.4 cm) to facilitate the growth of granulating tissue and epithelium string from the margin of the wound.
  • the area of the open wound was documented by digital image analysis in a standardized format. The pictures of the wound have been analyzed off-line by computer-assisted planimetry. Parallel documentation of body weight, blood sugar and healthy status. Blood sugar was measured using venous blood from the tail vain.
  • Gelatinase inhibitor could not detected in the serum after local application within the wound area.
  • serum levels around 150 ⁇ g/ml could be detected in orally treated animals.
  • systemic side effects can be eliminated by the local treatment.
  • Inhibitors were tested in a modified fluorescence-assay as described by Stack, M.S., and Gray, R.D., J. Biol. Chem. 264 (1989) 4277-4281.
  • Human MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 are commercially available (e.g. Calbiochem).
  • the pro-enzymes were activated with 1 mM APMA (incubation for 30 min at 37°C) immediately before testing.
  • Activated enzyme is diluted to 100 ng/ml in incubation buffer (50 mM Tris, 100 mM NaCl, lOmM CaCl2, pH 7.6).
  • the compounds were dissolved in 100% DMSO. For IC 50 determination a minimum of 8 dilution steps between 0.5 - 1000 nM have been prepared.
  • DNP-substrate (Bachem M1855,
  • the test tube contains 970 ⁇ l incubation buffer, lO ⁇ l inhibitor solution and lO ⁇ l enzyme solution. The reaction was started by adding the lO ⁇ l substrate solution.
  • ICso's are defined as the concentration of inhibitor that gives a signal that is 50% of the positive enzyme control.
  • IC 5 Q values (nM) are shown in Table 1.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dermatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne l'utilisation d'un composé de trioxopyrimidine ayant une activité inhibitrice contre MMP-1, MMP-2, MMP-3, MMP-9 et MMP-14 défini comme suit a) une valeur IC50 inférieure à 5 µM pour MMP-2, MMP-9 et MMP-14 chacun; b) un rapport ne dépassant pas 100 pour les valeurs IC5O de MMP-1:MMP-2, MMP-1: MMP-9, MMP-l:MMP-14; et c) un rapport ne dépassant pas 10 pour les valeurs IC50 de MMP- 3:MMP-2, MMP-3: MMP-9, MMP-3:MMP-14, pour la production d'une médicament destiné à favoriser la cicatrisation de lésions.
PCT/EP2004/003203 2003-03-28 2004-03-26 Utilisation d'une trioxopyrimidine dans le traitement de lesions chroniques Ceased WO2004084902A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP03007121 2003-03-28
EP03007121.1 2003-03-28

Publications (1)

Publication Number Publication Date
WO2004084902A1 true WO2004084902A1 (fr) 2004-10-07

Family

ID=33040926

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2004/003203 Ceased WO2004084902A1 (fr) 2003-03-28 2004-03-26 Utilisation d'une trioxopyrimidine dans le traitement de lesions chroniques

Country Status (1)

Country Link
WO (1) WO2004084902A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023465A1 (fr) * 1995-12-23 1997-07-03 Boehringer Mannheim Gmbh Nouveaux derives d'acide barbiturique, leurs procedes de production et substances pharmaceutiques contenant lesdits derives
WO2001012611A1 (fr) * 1999-08-12 2001-02-22 Pfizer Products Inc. Inhibiteurs de pyrimidine-2,4,6-trione metalloproteinase
WO2001025217A1 (fr) * 1999-10-01 2001-04-12 F. Hoffmann-La Roche Ag Nouveaux derives de pyrimidine-2,4,6-trione, leurs procedes de production et les agents pharmaceutiques contenant ces composes
WO2002034753A2 (fr) * 2000-10-26 2002-05-02 Pfizer Products Inc. Inhibiteurs de metalloproteinase spiro-pyrimidine-2,4,6-trione
WO2002034726A2 (fr) * 2000-10-26 2002-05-02 Pfizer Products Inc. Inhibiteurs de la pyrimidine-2,4,6-trione metalloproteinase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023465A1 (fr) * 1995-12-23 1997-07-03 Boehringer Mannheim Gmbh Nouveaux derives d'acide barbiturique, leurs procedes de production et substances pharmaceutiques contenant lesdits derives
WO2001012611A1 (fr) * 1999-08-12 2001-02-22 Pfizer Products Inc. Inhibiteurs de pyrimidine-2,4,6-trione metalloproteinase
WO2001025217A1 (fr) * 1999-10-01 2001-04-12 F. Hoffmann-La Roche Ag Nouveaux derives de pyrimidine-2,4,6-trione, leurs procedes de production et les agents pharmaceutiques contenant ces composes
WO2002034753A2 (fr) * 2000-10-26 2002-05-02 Pfizer Products Inc. Inhibiteurs de metalloproteinase spiro-pyrimidine-2,4,6-trione
WO2002034726A2 (fr) * 2000-10-26 2002-05-02 Pfizer Products Inc. Inhibiteurs de la pyrimidine-2,4,6-trione metalloproteinase

Similar Documents

Publication Publication Date Title
US7638511B2 (en) Methods and compositions for treating primary and secondary tumors of the central nervous system (CNS)
EP0996448B1 (fr) Traitement et prevention des adherences
US20080279820A1 (en) Compositions and Methods for Treating Burns
US20100266709A1 (en) Compositions and Methods for Treating Burns
EP3300729A1 (fr) Compositions thérapeutiques contenant du macitentan
CN101506151A (zh) 改善药物中具有脒官能团的活性物质的生物利用度
KR20010062061A (ko) 발기 기능장애 또는 발기부전을 치료하기 위한프로스타글란딘 작용제의 용도
WO2017220810A1 (fr) Cilastatine pour une utilisation dans le traitement de la sepsie
US9044427B2 (en) Compositions and methods for prevention and treatment of wounds
JP4630661B2 (ja) 線維症および瘢痕形成の治療におけるコンバターゼインヒビターの使用
US20060293228A1 (en) Therapeutic compositions and methods using transforming growth factor-beta mimics
JP7809731B2 (ja) 全身性硬化症の予防または治療用薬学的組成物
KR20190064686A (ko) 혈액 응고를 저해하는 화합물들의 살서제로서의 사용
DE69732784T2 (de) Inhibierung von invasiver remodulierung
TW201500042A (zh) 與血液透析有關的低血壓的治療
US20050142129A1 (en) Methods of preventing morbidity and mortality by perioperative administration of a blood clotting inhibitor
WO2002032881A1 (fr) Nouveaux agents therapeutiques ou prophylactiques contre l'angiostenose
RU2834641C2 (ru) Фармацевтическая композиция для предупреждения или лечения системного склероза
Kinami et al. Clinical effects of anticancer drugs to pancreatic diseases as protein synthesis inhibitors
JP2006089455A (ja) 動脈瘤予防および／または治療剤
JP4674339B2 (ja) 脳損傷の予防・治療剤
TW202317115A (zh) 組成物用於治療高血壓的用途
WO2006132245A1 (fr) Inhibiteur de croissance tumorale
US20160106747A1 (en) Treating postoperative mechanical stress with an ectoine
HK1157221A (en) Therapeutic compositions containing macitentan

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES 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 MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)