[go: up one dir, main page]

WO1999047130A1 - Nanoparticules, leur procede de production et leur utilisation - Google Patents

Nanoparticules, leur procede de production et leur utilisation Download PDF

Info

Publication number
WO1999047130A1
WO1999047130A1 PCT/EP1999/001452 EP9901452W WO9947130A1 WO 1999047130 A1 WO1999047130 A1 WO 1999047130A1 EP 9901452 W EP9901452 W EP 9901452W WO 9947130 A1 WO9947130 A1 WO 9947130A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanoparticles
oxy
particles
oxo
hexafluorophosphate
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/EP1999/001452
Other languages
German (de)
English (en)
Inventor
Javier Manero
Jennifer Filbey
Peter Boderke
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.)
Aventis Research and Technologies GmbH and Co KG
Original Assignee
Aventis Research and Technologies GmbH and Co KG
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 Aventis Research and Technologies GmbH and Co KG filed Critical Aventis Research and Technologies GmbH and Co KG
Priority to EP99911744A priority Critical patent/EP1061904A1/fr
Priority to JP2000536370A priority patent/JP2002506814A/ja
Priority to AU30318/99A priority patent/AU3031899A/en
Publication of WO1999047130A1 publication Critical patent/WO1999047130A1/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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes

Definitions

  • Nanoparticles processes for their production and their use
  • the invention relates to nanoparticles, methods for producing nanoparticles and the use of nanoparticles.
  • formulations and combinations of active substances are becoming increasingly interesting, the form of use of which can not only be applied in a gentle manner, but also have a targeted influence on the distribution, bioavailability or absorption of the drug.
  • particulate systems so-called micro- or nanoparticles, which have a particle size in the range of less than 100 ⁇ m, have proven to be promising forms of application for delivering a wide variety of pharmaceuticals to the body.
  • Particles can be modified in a variety of ways, for example to increase the retention time. It is also mentioned, for example, that the particles can be provided with antigens in order to release the pharmaceuticals which are in the particles in a very targeted manner at their intended site of action. However, it is problematic that the depot effect of the particles is relatively limited, since the release takes place very quickly. Furthermore, the stability of the particles described is relatively low.
  • Particles on the mucous membranes increased so that they are increasingly absorbed into the body.
  • the problem here is that the particles are produced in emulsions. This makes it possible for residues of the solvent to remain on or in the particles. This is caused in particular by adsorption of the hydrophobic solvent onto hydrophobic polymers or bioactive substances, such as proteins. These solvent residues are of particular concern for pharmaceutical applications.
  • the positive charge of the particles which is measured by a positive zeta potential, can furthermore be disadvantageous for the surface modifications described in WO 96/20698.
  • microparticles described are generally too large (1-100 ⁇ m) to ensure efficient absorption through biological membranes. Larger particles (> 5 ⁇ m) can also have a toxic effect because they can get stuck in the fine blood capillaries of the lungs.
  • Patent specification EP 0 454 044 B1 describes the production of pharmaceutical preparations in microparticulate form, which are made up of a polyelectrolyte complex
  • biocompatible and biodegradable polyelectrolyte complex of polycations and polyanions is treated with a crosslinking agent during or after its formation, active ingredients that are present in the nanoparticles are released in a controlled manner
  • Sensitive active substances can be superbly protected against degradation by the nanoparticles according to the invention, since they are well embedded in the nanoparticles
  • the nanoparticles are much more stable than conventional ones without losing their biodegradability
  • the particles according to the invention are particularly easily absorbed
  • the nanoparticles are essentially free of residues of organic solvents, since they can be formed in an aqueous solution
  • the charge of the polyelectrolyte complex can be varied by the crosslinking in order to change the hydrophilicity of the particles. This causes a modification of the binding of plasma proteins to the nanoparticles. Furthermore, the retention time of the particles on the mucous membranes and the residence time of the particles in the body can also be influenced
  • the swelling capacity or the water absorption of the particle can be influenced by this measure.
  • nanoparticles refer to particles with an average size of 10 to 1000 nm, preferably 10 to 500 and particularly preferably 50 to 250 nm, which have a biocompatible and biodegradable polyelectrolyte complex 10.
  • the shape of the particles can be regular or irregular.
  • biocompatible means that the compounds which are preferably used for the preparation of the polyelectrolyte complex are compatible with application, that is to say, for example, are not or only to a reasonable extent toxic and / or have only a very slight allergenic effect. So that there is no accumulation of the polymers in the body, they should be biodegradable or excreted. Depending on their use, the polymers are preferably also biocompatible.
  • the polyelectrolyte complex can result from the bringing together of polyanions and polycations, as is described in the patent specification EP 0 454 044 B1.
  • the surface of the 25 nanoparticles formed can be positively or negatively charged (a positive or negative zeta
  • the polyacids can in particular have 30 charged phosphonate, phosphate, sulfonate, sulfate and / or carboxy groups. Preferred, without being restricted thereby, 6
  • These charged polymers can also be partially substituted or in the form of a salt.
  • the polyanions can also be used in copolymer form.
  • the weight average molecular weight of the polyanions is preferably 1,000 to 2,000,000 daltons, particularly preferably 40,000 to 600,000 daltons. They are generally commercially available. However, the polyanions can also be prepared in any manner known to those skilled in the art
  • the polyanions are preferably used in a concentration of 0.1 to 40 g / l, particularly preferably 1 to 20 g / l
  • Suitable biodegradable or excretable polycations in the context of the invention include, without limitation, collagen and collagen derivatives, gelatin, poly-N-alkylvinylpy ⁇ dine, polyethyleneimine, polyvinylamine, polyallylamine and polyacrylate, poly-L-Lysi ⁇ , poly - ⁇ , ß- (d ⁇ methylam ⁇ noethyl) -D, L-aspartam ⁇ d (PDAA), copolymers of PDAA and hydrophobically esterified poly- ⁇ , ß- (2-hydroxyethyl) -D, L-aspartam ⁇ d (PHEA), chitosan and derivatives, lysinoctadecyl ester , aminated dextrans, aminated cyclodextins, 7
  • aminated cellulose ethers aminated pectins and their partly substituted derivatives and salts.
  • the polycations can also be used in copolymer form. These include copolymers of various monomers that can be used to produce the above-mentioned polycations, as well as copolymers of these monomers with other biodegradable monomers. How they can be used to produce the biodegradable polymers listed below, to understand. Mixtures of these polymers / copolymers are likewise suitable in the context of the present inventions. Chitosan is preferred here, since its particularly high compatibility is recognized
  • the weight average molecular weight of the polycations is preferably 1,000 to 2,000,000 daltons, particularly preferably 40,000 to 600,000 daltons.
  • Their preparation is known to the person skilled in the art. They are generally commercially available
  • the polycations are preferably used in a concentration of 0.1 to 40 g / l, particularly preferably 1 to 20 g / l
  • the nanoparticles can be further biocompatible and / or biodegradable
  • polysaccharides such as dextran and its derivatives
  • polyalkyl cyanoacrylates such as polyalcohols
  • polymethylidene malonates such as polymethylidene malonates
  • polyesters such as PLGA (polylactic-polyglycolic acid copolymer) and polycaprolactone , Polyether, like
  • Polyethylene glycol, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyphosphazenes and biodegradable polyamides and polyurethanes are particularly preferred. These functional groups are not intended to restrict the hydroxyl, amino, the thiol, the
  • biodegradable polymers can also be used in copolymer form. These include copolymers of various monomers, which can be used to produce the biodegradable polymers listed above. Mixtures of these polymers / copolymers are also suitable in the context of the present invention
  • the polymers can serve the surface-modifying agents mentioned below, to bind the bioactive substances to the nanoparticles and / or to stabilize the substances
  • the weight average molecular weight of the polymers is more than 1,000, preferably 30,000 to 2,000,000, particularly preferably 50,000 to 300,000 daltons
  • the biodegradable polymers are preferably used in a concentration of 0 to 100 g / l, particularly preferably 0 to 40 g / l
  • Bioactive substances are substances that influence the properties or the behavior of living systems
  • prophylactic substances include, for example, contrast agents such as oxygen or noble gases, etc. be understood.
  • bioactive substances are, for example, active peptides and proteins, such as insulin, interferons, enzymes, somatropin, erythropoietin, G-CSF, human growth hormone, calcionin, LHRH, factor VIII, tPA, enkephaline, glucagon, TRH, thymopoietin, thymopentin, thymocartin as well as analogs and fragments;
  • active peptides and proteins such as insulin, interferons, enzymes, somatropin, erythropoietin, G-CSF, human growth hormone, calcionin, LHRH, factor VIII, tPA, enkephaline, glucagon, TRH, thymopoietin, thymopentin, thymocartin as well as analogs and fragments;
  • Enzyme inhibitors such as HIV protease inhibitors
  • Antigens and immunogens for example influenza viruses or subunits of antigens;
  • Antibiotics such as ß-lactam antibiotics (penicillins, cephalosporins, monobactams, carbapenems, etc.), aminoglycosides (e.g. streptomycin), tetracyclines,
  • Chloramphenicol macrolide antibiotics (e.g. erythromycin), lincomycine, fosfomycin, fusidic acid, polymyxine, vancomycine and the like. Teicoplanin;
  • Analgesics such as hypnoanalgesics, in particular opium alkaloids, 4-phenylpiperidine derivatives (pethidine), 3,3-diphenylpropylamine derivatives (methadone), fentanyl derivatives, tramadol and nefopam and non-opioid analgesics, antipyretics and
  • Anti-inflammatory drugs especially derivatives of salicylic acid (e.g. acetylsalicylic acid), 10
  • the aniline e.g. paracetamol
  • the anthranilic acid mefenammic acid
  • the pyrazole metalamizole, phenazone, propyphenazone
  • aryless ⁇ g- u -propionsaure ⁇ indomethacm, diclofenac, ibuprofen, phenonobutene
  • sterucocoblene steruco -De ⁇ vate
  • Anti-rheumatic drugs such as oxyphenbutazone, arylacetic acid and propionic acid derivatives, in particular indomethacm, diclofenac, ibuprofen, ketoprofen, oxicams such as piroxicam, gold (l) preparations, D-penicillamine, chloroqum and immunosuppressants,
  • Hormones and antagonists such as peptide hormones, in particular
  • Cytostatics such as alkylating agents, especially mechloroethamm,
  • Cyclophosphamide Ifosfamide, Mephalan, Chlorambucil, Hexamethylmelamm, Thitepa, Busulfan, Carmustm, lomustm, Semustm, Steptozocm and dacarbazin,
  • Antimetabohten especially methotrexate, fluorouracil, Floxu ⁇ dm, Cytarabm, Mercaptopurm, Thioguanm, Pentostatin,
  • RNA DNA
  • DNA such as nucleotides, oligonucleotides, polynucleotides, genes or gene segments, plasmids and / or vectors and their derivatives, which are particularly active in HIV, rheumatoid arthritis, cancer, hormone deficiency diseases, hypertension, atherosclerosis, vascular diseases, viral infections and a lack of endogenous synthesis Peptides and proteins are used 1 1
  • Toxme or Vaccme such as bacterial vaccines, such as tetanus and cholera toxin, such as viral vaccines, such as AIDS antigens or viral hepatitis components,
  • Carbohydrates such as mono- or polysacchand, dextran, agar, agarose-denvate,
  • Protooglycans such as Heparm, Heparan, dermatan sulfates
  • Lipids such as phosphopides, choleste ⁇ n, Tngylce ⁇ de and Lipoproteme u
  • the nanoparticles according to the invention are particularly suitable dosage forms, particularly for unstable preparations, since the particles are particularly stable and thus protect the active ingredients, for example proteins, against decomposition by, for example, gastric acid.
  • the active ingredient can therefore be released in a particularly targeted manner, so that, for example, after oral administration of the Active ingredient is not released in the stomach or intestine where it was broken down, but only when it has been taken ms blood
  • the active ingredient can be in at least four different ways
  • the bioactive agent / the bioactive agent mixture is preferably used in a concentration of 0.1 to 40 g / l, particularly preferably 1 to 20 g / i
  • the polyelectrolyte complex after its formation is additionally treated with at least one crosslinking agent. These compounds link the polymers of the nanoparticles so that they become more stable and pharmaceutical
  • crosslinking agents include, but are not intended to restrict them.
  • halogenated triazine pvates such as 2,4,6-trichloro-1, 3,5-tricazione, 2,4-dichloro-6-methoxy-1, 3,5-tricacade,
  • Phosphomum salts such as
  • Tris-di-methyl-amino-azido-phosphonium hexafluorophosphate tris-di-methyl-amino-trichloromethyl-phosphonium hexafluorophosphate, tris-di-methylamino-trifluoromethyl-phosphonium hexafluorophosphate, tris-di-methyl-amino-phenoxy- phosphonium hexafluorophosphate, tris-dimethylamino-p-nitrophenoxyphosphonium hexafluorophosphate,
  • Tris-pyrrolidino-chloro-phosphonium-hexafluorophosphate tris-pyrrolidino-bromo-phosphonium-hexafluorophosphate, tris-pyrrolidino-cyano-phosphonium-hexafluorophosphate, tris-pyrrolidino-isothiocyanoto-phosphonium-hexafluorophosphoronophosphate, tris
  • 1,2-dihydro-2-oxo-1-pyridyl-oxy-biscyclohexylidenuronium chloride 1, 2-dihydro-2-oxo-1-pyridyl-oxy-biscyclohexylidenuronium hexafluorophosphate, 1,2-dihydro-2-oxo-1-pyridyl oxy-biscyclohexylidenuronium perchlorate, 1, 2-dihydro-2-oxo-1-pyridyl-oxy-biscyclohexylidenuronium tetrafluoroborate,
  • N-maleinimidyl-oxy-biscyclohexylidenuronium chloride N-malemimidyl-oxy-biscyclohexylidenuronium hexafluorophosphate, N-maleinimidyl-oxy-biscyclohexyhdenuronium perchlorate, N-maleinimidyl-oxy-biscyclohexy-denoronidylonatylonuronium xurobluronium xyluronium oxychloride
  • N-Maleinimidyl-oxy-biscyclopenty denuronium hexafluorophosphate N-Maleinimidyl-oxy-biscyclopentylidenuronium perchlorate, N-Maleinimidyl-oxy-biscyclopentyhdenur ⁇ iumtetrafluoroborat, N-Male ⁇ n ⁇ m ⁇ dyl-oxy-N-N, N, Male N, N ', N'-tetramethyluronum hexafluorophosphate,
  • N-maleimidyl-oxy-NNN'.N'-tetramethyluronium perchlorate N-maleimidyl-oxy-N, N, N ', N'-tetramethyluronium tetrafluoroborate
  • N-succinimidyl-oxy-biscyclohexylidenuronium chloride N-succinimidyl-oxy-biscyclohexyiidenuronium hexafluorophosphate
  • N-succinimidyl-oxy-biscyclopentylidenuronium chloride N-succinimidyl-oxy-biscyclopentylidenuronium hexafluorophosphate
  • N-succinimidyl-oxy-biscyclopentylidenuronium perchlorate N-succinimidyl-oxy-biscyclopentyluronium-ninoxin-n-oxy-n-oxy-n-oxy-n-oxy-n-oxy-n-oxy-n-cyclo
  • N-succinimidyl-oxy-N, N, N ', N'-tetramethyluronium hexafluorophosphate N-succinimidyl-oxy-N, N, N', N'-tetramethyluronium perchlorate
  • N-succinimidyl-oxy-N, N, N ', N 'tetramethyluronium tetrafluoroborate N-phthalimidyl-oxy-biscyclohexylidenuronium chloride, N-phthalimidyl-oxy-biscyclohexylidenuronium hexafluorophosphate,
  • N-phthalimidyl-oxy-biscyclohexylidenuronium perchlorate N-phthalimidyl-oxy-biscyclohexylid ⁇ uronium tetrafluoroborate
  • N-phthalimidyl-oxy-biscyclopentylidenuronium chloride N-phthalimidyl-oxy-biscyclopentylorylidononylylurylidylonylurylidyluronium
  • N-phthalimidyl-oxy-biscyclopentylidenuronium tetrafluoroborate N-phthalimidyl-oxy-N, N, N ', N'-tetramethyluronium chloride, N-phthalimidyl-oxy-N, N, N', N'-tetramethyluronium hexafluorophimidate, N-phthalate N, N, N ', N'-tetramethyluronium perchlorate, N-phthalimidyl-oxy-N, N, N', N'-tetramethyluronium tetrafluoroborate,
  • N-perhydrophthalimidyl-oxy-biscyclohexylidenuronium chloride N-perhydrophthalimidyl-oxy-biscyclohexylidenuronium hexafluorophosphate
  • N-perhydrophthalimidyl-oxy-biscyclohexylidenuronium perchlorate N-perhydrophthalimidyl-oxy-biscyclo-oxychloride-oxychloride
  • N-perhydrophthalimidyl-oxy-biscyclopentylidenuronium perchlorate N-perhydrophthalimidyl-oxy-biscyclopentylidenuronium tetrafluoroborate, N-perhydrophthalimidyl-oxy-N, N, N ', N'-tetramethyluronium chloride, N-perhydrophthalimide, tetramethyluronium hexafluorophosphate, N-perhydrophthalimidyl-oxy-N, N, N ', N'-tetramethyluronium perchlorate,
  • reactive carbonic acid derivatives such as carbodiimides, in particular N- (3-dimethylaminopropyl) -N-ethylcarbodiimide.
  • ester or amide groups are preferred because the resulting ester or amide groups can be biodegraded particularly well.
  • These include all halogenated triazine derivatives, all phosphonium salts, all uronium salts and reactive carbonic acid derivatives.
  • N- (3-Dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDAP) and O- (N-succinimidyl) -N, N, N ' , N ' - tetramethyluronium tetrafluoroborate (TSTU) are particularly preferred.
  • the degree of crosslinking can be influenced by the concentration of the polyelectrolyte complex and the crosslinking agent and the reaction time.
  • the reaction time depends, among other things, on the type, reactivity and concentration of the selected crosslinking agent and the polyelectrolyte complex, as well as the reaction temperature and the pH of the solution. Under certain circumstances, it can be influenced by catalysts. At room temperature, it is preferably 1 minute to 24 hours, particularly preferably 5 to 120 minutes.
  • the surface of the nanoparticles can be modified. This modification is described in the above-mentioned patent applications WO 96/20698, US 5,449,720 and WO 92/17167, which are intended to be included in the disclosure.
  • the properties of the nanoparticles can be influenced in a targeted manner by the modification. For example, antithrombocytic properties can be generated, the absorption of the particles via the intestine can be improved or substances can be transferred to the modification.
  • Particles are bound so that the particles in very defined areas in the 21
  • Body will be enriched.
  • Antigens against cancer cells which can be linked to the particles, are mentioned as an example, so that the drugs are released directly from the drug saturated drug depots in the cancer cells.
  • This modification can be achieved in that at least one of the charged polymers of the polyelectrolyte complex is additionally treated with an agent which modifies the surface before, during or after the formation of the complex.
  • agents include, without being limited by this, various synthetic polymers, biopolymers, low molecular weight
  • Oligomers natural substances and surface-active substances.
  • the synthetic polymers with which the surface of the nanoparticles can be modified include carboxymethyl cellulose, cellulose, cellulose acetate, cellulose phthalate, polyethylene glycol (carbowax), polyvinyl alcohol
  • PVA hydroxypropyl methyl cellulose phthalate, hydroxypropyl cellulose, sodium or potassium salts of carboxymethyl cellulose, polyvinyl pyrolidone, polystyrene and silicates such as bentonite.
  • the biopolymers with which the surface of the nanoparticles can be modified include, in particular, proteins and peptides, such as gelatin, casein, albumins (ovalbumin), myoglobin, hemoglobin, monoclonal and polyclonal antibodies, cytokines, such as growth factors, interferons, lymphokines, monokines, Interleukins and chemokines; as well as polysaccharides and pectins.
  • proteins and peptides such as gelatin, casein, albumins (ovalbumin), myoglobin, hemoglobin, monoclonal and polyclonal antibodies
  • cytokines such as growth factors, interferons, lymphokines, monokines, Interleukins and chemokines
  • pectins such as polysaccharides and pectins.
  • the natural substances with which the surface of the nanoparticles can be modified include, in particular, cofactors, such as coenzymes, such as vitamins, in particular vitamin B12, and prosthetic groups, such as the heme group; Lipids, especially phospholipids such as lecithin and cholesterol; and prostaglandins. 22
  • the surface-active substances with which the surface of the nanoparticles can be modified include non-mastic surfactants, in particular sorbitan fatty acid esters, in particular polyoxyethylene sorbitan fatty acid esters, fatty alcohols, such as cetylaic alcohols or stearyl alcohols, and polyether sulfonates, anionic surfactants, in particular sodium, for example, fatty dodecyl sulfates Palmitic acid, stearic acid and oleic acid), Gylce ⁇ nester of fatty acids (for example Glyce ⁇ nmonostearat) and sodium and potassium salts of fatty acids (Natnumoleat, sodium palmitate, Nat ⁇ umstearat, among others), Polyoxylstearat, Polyoxylethylenlaurylether, Sorbitansesanolamine, Methyldimbromidom, Ethylammonyl, Methylammonium Ethyl, Methylammonium Ethyl,
  • agents which can be used to modify the surface of the nanoparticles, preferably include agents which enable active transport (for example absorption) of the particles. These agents are known as car ⁇ ers
  • carners include bile acids, adhesins, invasme,
  • Toxins such as plant or bacterial toxins, cobalamins, viral Hamaglutimne, Lectme, Transfer ⁇ n, Riboflavm and peptides that are transported intestinally (which use Car ⁇ ersysteme for mtestinal peptide transport) derivatives of these substances, which also use the respective Camer systems , can also be used
  • Cobolamines which are suitable as carriers, include, for example, substances such as vitamin B12 or analogues that bind to the int ⁇ nsic factor (IF), a glycoprotem of gastric juice. Through this binding, the nanoparticles are actively absorbed by the mucous membranes from the digestive tract. To the analogues belong, for example, without being restricted thereby, 23
  • these analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, nitrocobalamin
  • Benzimidazolecyanocobalamm derivatives such as 5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole, trimethylbenzimidazole, as well as adenosylcyanocobalamm [(Ade) CN-Cbl], cobalt amalgamation, cobalt ammonium lactam and the anilide, ethyl or deoxy amide and dicarboxylate, monocarboxylate, monocarboxyl Corresponding analogues Further analogues of vitamin B12 result from the substitution of the cobalt atom by zinc or nickel
  • these surface-modifying agents can also be used as mixtures in order to combine the various properties of the surface-modifying agents or to achieve synergistic effects
  • the surface-modifying agents are preferably used in a concentration of 0 to 200 g / l, particularly preferably 0 to 20 g / l
  • These surface-modifying agents can be added directly to the aqueous solution. These agents can preferably be bound covalently or ionically to at least one of the hydrophilic polymers, to the crosslinking agent or to the other above-mentioned biocompatible and biodegradable polymers and to the bioactive agent, in order to do so to connect as firmly as possible with the polyelectrolyte complex
  • the surface-modifying agents can be activated with other substances.
  • the hydrophilic polymers, the crosslinking agents or to the other above-mentioned biocompatible and biodegradable 24 are also possible.
  • activators include, for example, without limitation, disuccinimidyl suberate, B ⁇ s (sulfoaucc ⁇ n ⁇ m ⁇ dyl) suberate, ethylene glycol b ⁇ s (succ ⁇ n ⁇ mdylsucccolnate), ethylene glycol (sulfosucc ⁇ n ⁇ mdylsucc ⁇ nat), p-Aminophenylessigsaure, D ⁇ th ⁇ o-b ⁇ s (succ ⁇ n ⁇ m ⁇ dylprop ⁇ onat), 3 3'D ⁇ th ⁇ o- b ⁇ s (sulfosucc ⁇ n ⁇ m ⁇ dylprop ⁇ onat) Disuccmimidyltartrat, Disulfosuccmimidyltartrat, -ethylene b ⁇ s [2- (Succ ⁇ n ⁇ m ⁇ dooxycarbonyloxy) -ethylene] sulfone, b ⁇ s [
  • epoxides can be used as activators. These epoxides include, for example, ethylene oxide, 1, 2-propylene oxide, glycidyl ethers, such as diglycidylbutane diol ether, diglycidylethane diol ether, and erytholic acid anhydride
  • activators which have a thiol group and are particularly readily biodegradable.
  • These activators include, for example, N-succinomidyl-3- (2-pyridyld ⁇ th ⁇ o) propionate, iminothiolane, sulfosuccinidimidyl-6- [3- (2-pyridyld ⁇ th ⁇ o ) prop ⁇ onam ⁇ do] hexanoate, succinomidyl-6- [3- (2- py ⁇ dyld ⁇ th ⁇ o) prop ⁇ onam ⁇ do] hexanoate, sulfosucc ⁇ n ⁇ m ⁇ dyl-6- [-methyl- - (2-pyr ⁇ dyl- d ⁇ th ⁇ o) toluam ⁇ do] hexanoate, 1'-4-d ⁇ (2'py ⁇ dyld ⁇ th ⁇ o) prop ⁇ onam ⁇ do] butane, 4- -Succ ⁇ n ⁇ m ⁇ d
  • the activators are preferably used in a concentration of 0 to 40 g / l, particularly preferably 0 to 2 g / l
  • the nanoparticles can be produced, for example, by polyelectrolyte complexation, emulsion techniques, spray drying, solvent evaporation, solvent extraction, coacervation, extrusion, precipitation and filtration or other processes known to the person skilled in the art.
  • the nanoparticles are preferably produced by polyelectrolyte complexation.
  • the nanoparticles can be brought together by bringing together an aqueous solution of polycations, an aqueous solution of polyanions and at least one bioactive agent and, if appropriate, further substances (further polymers, auxiliaries, etc.) which may be bound to one of the two ionic polymers or which are present in free form can be obtained, and subsequent treatment with a crosslinking agent.
  • the at least two aqueous solutions of the hydrophilic polymers are brought together in such a way that nanoparticles of the desired size and
  • Form size distribution This can be done, for example, by controlled dropping of one of the two solutions into the other of the two solutions.
  • the complex that forms during mixing precipitates as a result of neutralization. It may be necessary to adjust the pH in order to dissolve the substances, such as the polymers, bioactive substances, etc. These pH values depend, among other things, on the particular polyelectrolyte and are known to the person skilled in the art. In preferred embodiments, the person skilled in the art can orientate himself, for example, at the isoelectric point.
  • the particle size can be controlled by the manner in which they are brought together, for example when adding drops, the dilution of the at least two solutions, the speed of the stirrer, the pH and the diameter of the nozzles used for the drops and the dropping speed.
  • the particle size can also be influenced by ultrasound.
  • auxiliaries may be necessary. Excipients may also be indispensable in the treatment with the crosslinking agent
  • emulsion processes can be used depending on the bioactive active substance and the polymer used. This may be necessary, for example, if particularly hydrophobic active substances or additionally hydrophobic polymers are to be incorporated into the nanoparticles. These emulsion processes are described in WO 96/05810
  • one of the hydrophilic polymers is dissolved in water. This solution is added to a non-polar solvent with vigorous stirring by dissolving the hydrophobic active ingredient. Then, for example, the second of the hydrophilic polymers can be added to the resulting emulsion, so that the polyelectrolyte complex is formed.
  • This complex can be crosslinked in situ by adding one of the crosslinking agents mentioned above. It is preferred that this emulsion is stabilized by suitable means, for example dioctylsulphosuccmate
  • a non-polar polymer can also be dissolved in a hydrophobic solvent in order to introduce it into the polyelectrolyte complex. If both a hydrophobic bioactive agent and a non-polar polymer are to be introduced into the polyelectrolyte complex, it is possible to vary the process explained above slightly so that a multiple emulsion (oil-in-water-oil-oil emulsion) is formed
  • the particles can also be formed by spray drying.
  • a suitable solution of at least one polyanion, at least one polycation and at least one bioactive active ingredient and optionally further substances is sprayed through a suitable nozzle, so that particles of the desired size are formed. These particles are then dried 27
  • the resulting particles can be crosslinked in situ by adding crosslinking agents in order to obtain the nanoparticles according to the invention.
  • crosslinking agents for example, one of the above-mentioned crosslinking agents can be added and preferably stirred at room temperature for a further 10 minutes to 24 hours, depending on the desired degree of crosslinking and crosslinking agent. The exact one
  • the regulation for the implementation of the particles depends on the crosslinking agent and can be optimized by a person skilled in the art with a few routine tests.
  • Crosslinking can be determined using methods known from the literature, such as NMR, NIR or exclusion chromatography.
  • the resulting particles which contain a polyelectrolyte complex and at least one bioactive agent, can also be crosslinked later.
  • these particles can be in a suitable solvent, for example water or a dipolar aprotic solvent, such as DMF (dimethylformamide) or
  • DMSO Dimethylsoulfoxid
  • the particles can then be isolated. This separation can take place, for example, by filtration or centrifugation.
  • the particles are then preferably washed with water and dried, for example by lyophilization.
  • nanoparticles thus obtained can be sterilized by radiation, as is well known in the art. However, the nanoparticles can also be produced under sterile conditions. 28
  • the particles can be administered in any manner known to the person skilled in the art. This includes, in particular, without being restricted by this, the oral form of administration. However, they can also be administered parenterally, for example by injection intravenously, intraarterially, intramuscularly, subcutaneously, intrathecally or intralumbally.
  • the nanoparticles can also be administered nasally, occularly, rectally, vaginally, buccally, orally, transdermally and by inhalation.
  • the suspension was then ultrafiltered over a 100 kD membrane (PLHK membrane from Millipore) (Amicon 8050 ultrafiltration cell, nitrogen pressure 0.2 bar, purity> 99.9%), the residue being washed with 5 ml of water.
  • the retentate was then transferred to a 100 ml round bottom flask, frozen (with a mixture of isopropanol / dry ice) and freeze-dried overnight (model LDC-1, Christ).
  • the release of the insulin was tested by suspending 5 mg of the dried particles in 10 ml of pH 7.4 phosphate buffer (produced with Sigma phosphate buffer tablets) and heating them at 37 ° C. in a drying cabinet. After 30 minutes, a sample is taken, ultrafiltered (Filter Millipore PLHK) and examined by means of fluorescence spectroscopy for FITC insulin content according to the method known from the literature (excitation wavelength: 494 nm, emission wavelength: 518 nm).
  • Example 1 was repeated. However, the resulting particles were not cross-linked. This means that the resulting suspension was not mixed with glyoxal, but washed, ultrafiltered and dried directly as described above.
  • the release of the FITC insulin was tested as in Example 1. It was found that 59.4% of the FITC insulin was released into the solution.
  • BSA bovine serum albumin
  • BSA-FITC bovine serum albumin
  • the suspension was stirred for 15 mm at room temperature
  • the suspension was cleaned with the aid of an ultrafiltration (Amicon 8050 ultrafiltration cell, nitrogen pressure 1 bar) over a 300 kD membrane (PLMK from Millipore).
  • the residue was washed 3 times with 30 ml of water, 100 ml of permeate being formed
  • the retentate was then transferred to a 250 ml round bottom flask, frozen and freeze-dried overnight
  • the release of the BSA-FITC was tested by suspending 5 mg of the dried particles in 10 ml of phosphate buffer pH 7.4 and incubating at 37 ° C in the drying cabinet. After 4 hours a sample was taken, ultrafiltered (filter
  • Example 3a Inclusion of tetracycline
  • Example 2a 20 mg of tetracycline (Sigma) were used this time and processed according to Example 2a.
  • the release of the tetracycline was measured according to the method known from the literature using UV spectroscopy at 356 nm. Without crosslinking, the release of the tetracycline was 70% after 4 hours. If, as mentioned in Example 2a, the particles were crosslinked with 200 ⁇ l of a 40% glyoxal solution, the release of tetracycline was only 10%

Landscapes

  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des nanoparticules comportant un complexe polyélectrolytique biocompatible et biodégradable, constitué de polycations et de polyanions, et au moins un principe bioactif. Ces nanoparticules sont obtenues par le fait que le complexe polyélectrolytique est en outre, après sa formation, traité avec au moins un agent réticulant. De plus, l'invention concerne un procédé de production desdites nanoparticules, selon lequel on met en contact un principe actif sous forme liée ou non liée, une solution aqueuse d'une substance polymère acide et d'une substance polymère basique, le polyélectrolyte se constituant ensuite sous forme nanoparticulaire ou étant éventuellement mis sous forme nanoparticulaire. Ce procédé se caractérise en ce que le complexe polyélectrolytique nanoparticulaire est ensuite traité avec un agent de réticulation. Les avantages présentés par ces particules sont, en particulier, leur stabilité élevée, leur libération contrôlée des principes actifs et le fait qu'elles évitent d'avoir un effet de décharge. En outre, les nanoparticules selon l'invention ne présentent, grâce à leur procédé de fabrication, que des résidus extrêmement faibles de solvants organiques.
PCT/EP1999/001452 1998-03-13 1999-03-06 Nanoparticules, leur procede de production et leur utilisation Ceased WO1999047130A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99911744A EP1061904A1 (fr) 1998-03-13 1999-03-06 Nanoparticules, leur procede de production et leur utilisation
JP2000536370A JP2002506814A (ja) 1998-03-13 1999-03-06 ナノ粒子、ナノ粒子の製造法およびその使用
AU30318/99A AU3031899A (en) 1998-03-13 1999-03-06 Nanoparticles, method for producing nanoparticles and use of the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19810965.2 1998-03-13
DE1998110965 DE19810965A1 (de) 1998-03-13 1998-03-13 Nanopartikel, Verfahren zu ihrer Herstellung und ihre Verwendung

Publications (1)

Publication Number Publication Date
WO1999047130A1 true WO1999047130A1 (fr) 1999-09-23

Family

ID=7860805

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1999/001452 Ceased WO1999047130A1 (fr) 1998-03-13 1999-03-06 Nanoparticules, leur procede de production et leur utilisation

Country Status (6)

Country Link
EP (1) EP1061904A1 (fr)
JP (1) JP2002506814A (fr)
CN (1) CN1292687A (fr)
AU (1) AU3031899A (fr)
DE (1) DE19810965A1 (fr)
WO (1) WO1999047130A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003004004A1 (fr) * 2001-07-05 2003-01-16 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Preparation pharmacologique obtenue a partir d'un complexe nanoparticulaire mesomorphe polyelectrolyte-lipide et d'au moins une substance active
EP2266546A1 (fr) 2009-06-08 2010-12-29 Advancell Advanced in Vitro Cell Technologies,S.A. Procédé pour la préparation de systèmes colloïdaux pour la livraison de composés actifs
US8628801B2 (en) 2004-04-29 2014-01-14 Universidad De Navarra Pegylated nanoparticles
US8895067B2 (en) 2004-04-29 2014-11-25 Universidad De Navarra Immune response stimulating composition comprising nanoparticles based on a methyl vinyl ether-maleic acid copolymer
US8920842B2 (en) 1999-11-15 2014-12-30 Piramal Healthcare (Canada) Ltd. Temperature controlled and pH dependent self gelling biopolymeric aqueous solution
US20240189246A1 (en) * 2017-07-14 2024-06-13 Drexel University Multifunctional Nanoparticles For Prevention And Treatment Of Atherosclerosis

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19952073A1 (de) * 1999-10-29 2001-07-12 Cognis Deutschland Gmbh Verfahren zur Herstellung von nanopartikulären Chitosanen oder Chitosan-Derivaten
DE10118852A1 (de) * 2001-04-17 2002-10-31 Fricker Gert Bdellosomen
DE10127526A1 (de) * 2001-05-31 2002-12-12 Novosom Ag Verfahren zur Herstellung und Auflösung von Nano- und Mikrokapseln
DE60334224D1 (de) * 2002-07-16 2010-10-28 Biosyntech Canada Inc Zusammensetzung für die herstellung zellkompatibler, injizierbarer, selbstgelierender chitosan lösungen zum einkapseln und verabreichen von lebenden zellen oderbiologisch aktiven faktoren
EP1548439A4 (fr) * 2002-09-13 2008-04-23 Hitachi Chemical Co Ltd Porteur de fixation et phase solide
WO2008093195A2 (fr) * 2007-01-29 2008-08-07 Medipol Sa Particules colloïdales à base de chitosane pour la délivrance d'arn
ITRM20070327A1 (it) * 2007-06-11 2008-12-12 Univ Palermo Vettori colloidali a struttura poliamminoacidica per il rilascio orale di peptidi e proteine e relativo metodo di produzione.
WO2009035438A1 (fr) * 2007-09-13 2009-03-19 Janos Borbely Nanoparticules polymériques obtenues par interactions ion-ion
US9095568B2 (en) * 2007-09-26 2015-08-04 Mark Berninger Therapeutic and vaccine polyelectrolyte nanoparticle compositions
CN101396351B (zh) * 2007-09-28 2011-04-27 上海交通大学医学院附属瑞金医院 可响应磷酸酶浓度的载药聚电解质胶囊及其制备方法
JP5530934B2 (ja) 2008-01-24 2014-06-25 ユニバーシティ オブ ユタ リサーチ ファンデーション 接着複合コアセルベートならびにその作製および使用方法
US8283384B2 (en) 2008-01-24 2012-10-09 University Of Utah Research Foundation Adhesive complex coacervates and methods of making and using thereof
CN103025360A (zh) 2010-05-24 2013-04-03 犹他大学研究基金会 增强的粘合剂复合物凝聚层和其制备和使用方法
CA2812599A1 (fr) 2010-11-12 2012-05-18 University Of Utah Research Foundation Coacervats adhesifs simples et procedes de fabrication et d'utilisation de ceux-ci
CN102813937A (zh) * 2012-06-12 2012-12-12 天津大学 含有疏水性药物的聚电解质复合物及其制备方法和应用
US10077324B2 (en) 2013-02-06 2018-09-18 Kci Licensing, Inc. Polymers, preparation and use thereof
US9913927B2 (en) 2014-07-14 2018-03-13 University Of Utah Research Foundation In situ solidifying complex coacervates and methods of making and using thereof
JP7410876B2 (ja) 2018-01-26 2024-01-10 フルイデックス メディカル テクノロジー，リミティッド ライアビリティ カンパニー 血管閉塞のためにその場凝固複合コアセルベートを使用する装置及び方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0427190A2 (fr) * 1989-11-09 1991-05-15 Hoechst Aktiengesellschaft Complexes de polyélectrolytes pour le traitement et la prophylaxie de maladies virales et leur procédé de préparation
EP0454044A2 (fr) * 1990-04-25 1991-10-30 Hoechst Aktiengesellschaft Produit pharmacologique contenant des composés de polyélectrolytes sous forme de microparticules et au moins un agent actif
WO1992017167A1 (fr) * 1991-04-02 1992-10-15 Biotech Australia Pty. Ltd. Systemes de liberation orale de microparticules
EP0671169A1 (fr) * 1994-03-09 1995-09-13 Hoechst Aktiengesellschaft Nanoparticules contenant un ingrédient actif et un polymère de polycétals-amides d'acide tartrique, leur procédé et leur utilisation
WO1999018934A1 (fr) * 1997-10-09 1999-04-22 Vanderbilt University Dispositif d'administration de microparticules ou de nanoparticules de polymeres

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0427190A2 (fr) * 1989-11-09 1991-05-15 Hoechst Aktiengesellschaft Complexes de polyélectrolytes pour le traitement et la prophylaxie de maladies virales et leur procédé de préparation
EP0454044A2 (fr) * 1990-04-25 1991-10-30 Hoechst Aktiengesellschaft Produit pharmacologique contenant des composés de polyélectrolytes sous forme de microparticules et au moins un agent actif
WO1992017167A1 (fr) * 1991-04-02 1992-10-15 Biotech Australia Pty. Ltd. Systemes de liberation orale de microparticules
EP0671169A1 (fr) * 1994-03-09 1995-09-13 Hoechst Aktiengesellschaft Nanoparticules contenant un ingrédient actif et un polymère de polycétals-amides d'acide tartrique, leur procédé et leur utilisation
WO1999018934A1 (fr) * 1997-10-09 1999-04-22 Vanderbilt University Dispositif d'administration de microparticules ou de nanoparticules de polymeres

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8920842B2 (en) 1999-11-15 2014-12-30 Piramal Healthcare (Canada) Ltd. Temperature controlled and pH dependent self gelling biopolymeric aqueous solution
WO2003004004A1 (fr) * 2001-07-05 2003-01-16 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Preparation pharmacologique obtenue a partir d'un complexe nanoparticulaire mesomorphe polyelectrolyte-lipide et d'au moins une substance active
US8628801B2 (en) 2004-04-29 2014-01-14 Universidad De Navarra Pegylated nanoparticles
US8895067B2 (en) 2004-04-29 2014-11-25 Universidad De Navarra Immune response stimulating composition comprising nanoparticles based on a methyl vinyl ether-maleic acid copolymer
EP2266546A1 (fr) 2009-06-08 2010-12-29 Advancell Advanced in Vitro Cell Technologies,S.A. Procédé pour la préparation de systèmes colloïdaux pour la livraison de composés actifs
US20240189246A1 (en) * 2017-07-14 2024-06-13 Drexel University Multifunctional Nanoparticles For Prevention And Treatment Of Atherosclerosis

Also Published As

Publication number Publication date
CN1292687A (zh) 2001-04-25
DE19810965A1 (de) 1999-09-16
EP1061904A1 (fr) 2000-12-27
AU3031899A (en) 1999-10-11
JP2002506814A (ja) 2002-03-05

Similar Documents

Publication Publication Date Title
EP1061904A1 (fr) Nanoparticules, leur procede de production et leur utilisation
DE69812887T2 (de) Mikropartikel aus chitosan-gelatin a
DE69230254T2 (de) Herstellung von Mikropartikeln
DE69526787T2 (de) Biokompatibele und biodegradierbare Nanokapseln zur Absorption und Verabreichung von proteinischen Arzneimitteln
DE69221568T2 (de) Systeme zur oralen freisetzung von mikropartikeln
Sinha et al. Chitosan microspheres as a potential carrier for drugs
KR101181563B1 (ko) 고분자 미립구의 제조방법 및 그 방법에 의해 제조된 고분자 미립구
DE69131677T2 (de) Arzneimittel freisetzendes System mit Wechselwirkung zwischen Calcitonin und einem hydrophoben, biologisch abbaubaren Polymeren
EP2819659B1 (fr) Procédé de fabrication de nanoparticules chargées d'une substance active
EP1289642B2 (fr) Nanocapsules a enveloppe de polyelectrolyte
DE112006002617T5 (de) Verfahren zur Herstellung von Core-Shell-Kompositen bzw. Kern-Hüllen-Kompositen mit vernetzten Hüllen und daraus entstehende Core-Shell-Komposite
HUE026134T2 (en) Microparticles containing diketopiperazine salts for drug delivery
WO2000040259A1 (fr) Microsphere a liberation prolongee utilisee pour encapsuler des analogues de la luteoliberine, et son procede de production
DE112006002618T5 (de) Verfahren und Zusammensetzungen zum selektiven Entfernen von Kaliumionen aus dem Gastrointestinaltrakt eines Säugers
EP1397200A1 (fr) Microcapsules et nanocapsules solubles, procede de production associe et utilisation
EP0615442B1 (fr) Matrice thermocollo dale reglee par des sols a base de gelatine utile dans des formes d'administration perorale a action prolongee
EP0315875A1 (fr) Procédé de préparation de microcapsules biodégradables contenant des peptides et des protéines solubles dans l'eau et les microcapsules obtenues par ledit procédé
DE69100535T2 (de) Verfahren zur herstellung von azo- und/oder disulfid enthaltenden polymeren.
DE69706969T2 (de) Metall valproat-tabletten mit verzoegerter freisetzung
WO2004030649A2 (fr) Procede pour preparer et stabiliser des microsuspensions et des nanosuspensions au moyen d'amphiphiles et de polyelectrolytes
US20180250231A1 (en) Polymeric microspheres with spontaneous pore-closing functionality and methods for preparign the same
DE102006011507A1 (de) Wirkstoffbeladene Nanopartikel auf Basis hydrophiler Proteine
JP2006241321A (ja) キトサンナノ粒子の製造方法、キトサン粒子、コーティング用組成物、徐放性製剤、注射剤
Di et al. Enhanced Anti‐inflammatory Effects of Diclofenac Delivered Orally via Polyvinylpyrrolidone K30/Silk Fibroin Nanoparticles in a Murine Model of Carrageenan‐Induced Paw Edema
EP1333806A2 (fr) Bdellosomes

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 99803733.8

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA CN IL JP NZ US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1999911744

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 09623899

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1999911744

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1999911744

Country of ref document: EP