WO1999027897A9 - Site-specific preparation of polyethylene glycol-grf conjugates - Google Patents
Site-specific preparation of polyethylene glycol-grf conjugatesInfo
- Publication number
- WO1999027897A9 WO1999027897A9 PCT/EP1998/007748 EP9807748W WO9927897A9 WO 1999027897 A9 WO1999027897 A9 WO 1999027897A9 EP 9807748 W EP9807748 W EP 9807748W WO 9927897 A9 WO9927897 A9 WO 9927897A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hgrf
- peg
- lys
- conjugates
- peptide
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
Definitions
- the present invention relates to a method for the site-specific preparation of hGRF-PEG conjugates containing one or more than one PEG units (per hGRF) covalently bound to Lys 12 and/or Lys 21 and/or N ⁇ , characterized in that the conjugation reaction between the hGRF peptide and activated PEG is carried out in solution and the desired hGRF-PEG conjugate is purified by chromatographic methods.
- the conjugates prepared by this method are also an object of the present invention.
- GRF also called Somatorelin
- GRF is a peptide secreted by the hypothalamus which acts on its receptor and can promote the release of growth hormone (GH) from the anterior pituitary. It exists as 44-, 40-, or 37-amino acid peptide; the 44-amino acids form may be converted physiologically into shorter forms. All three forms are reported to be active, the activity residing mainly in the first 29 amino acid residues.
- a synthetic peptide corresponding to the 1-29 amino acid sequence of human GRF [hGRF(l-29)], also called Sermorelin, has been prepared by recombinant DNA technology as described in European Patent EP 105 759.
- Sermorelin has been used in the form of acetate for the diagnosis and treatment of growth hormone deficiency.
- GRF has indeed a therapeutic value for the treatment of certain growth-hormone related disorders.
- the use of GRF to stimulate the release of GH is a physiological method in promoting long bone growth or protein anabolism.
- GRF GRF-29
- DPP-IV dipeptidylpeptidase IV
- Polyethylene glycol (PEG) is a hydrophilic, biocompatible and non-toxic polymer of general formula H(OCH 2 CH 2 ) confrontOH, wherein n > 4. Its molecular weight could vary from 200 to 20,000 daltons.
- PEG in its mono- methoxylated form to proteins and/or peptides significantly increases their duration of biological action.
- PEG provides a protective coating and increases the size of the molecule, thus reducing its metabolic degradation and its renal clearance rate.
- PEG conjugation is an already established methodology for peptide and protein delivery pioneered by the fundamental studies of Davis and Abuchowski (Abuchowski et al., 1977a and 1977b). PEG conjugation to peptides or proteins generally resulted in non-specific chemical attachment of PEG to more than one amino acid residue.
- One of the key issues with this technology is therefore finding appropriate chemical methods to covalently conjugate PEG molecule(s) to specific amino acid residues.
- the trichlorotriazine-activated PEG which was found to be toxic and reacted in a non-specific way, was later on replaced by various PEG reagents with chemical linkers that could react specifically to amino groups (Benchamp et al., 1983; Veronese et al., 1985; Zalipsky et al., 1983; Zalipski et al., 1990; and Delgado et al., 1990), to sulphydryl groups (Sartore et al., 1991; and Morpurgo et al., 1996) or to guanidino residues (Pande et al., 1980).
- the active site could be protected in advance, thus allowing enzyme pegylation to occur at non-active site(s) (Caliceti et al , 1993)
- the present invention relates to site-specific pegylation of hGRF in solution phase hGRF was found to have a low solubility in a neutral/alkaline buffer solution, a chemical condition whereby most efficient pegylation reaction occurs.
- a diluted hGRF solution the hydrolysis of the activated PEG (such as the PEG ester) tends to decrease the yield of the pegylation reaction.
- N ⁇ through out the present invention means the amino group at the N-terminal position of the peptide (Tyr).
- the main embodiment of the present invention is a method for the site- specific preparation of different hGRF-PEG conjugates containing one or more than one PEG units (per hGRF) covalently bound to Lys 12 and/or Lys 21 and/or N ⁇ , characterized in that the pegylation reaction is carried out in solution and the desired hGRF-PEG conjugate is purified, for example, by chromatographic methods.
- hGRF-PEG conjugates containing one or more PEG units (per mole of hGRF) covalently bound to Lys 12 and/or Lys 21 and/or N ⁇ are also covered by the present invention.
- Lys 12 or to Lys 21 are the preferred products of the present invention.
- the pegylation reaction will give directly the desired conjugate with specific pegylation sites, which can then be isolated from the reaction mixture, for example, by ultrafiltration or other chromatographic methods.
- the preparation method can further, optionally, comprise a de-protection reaction.
- the de-protection reaction is preferably carried out according to known methods and depending on the chemical protective group to be removed.
- hGRF unless otherwise specified, is intended to cover any human GRF peptides, with particular reference to the 1-44, 1-40, 1-29 peptides and the corresponding amides thereof (containing an amide group at the N-terminus or C-terminus).
- the preferred hGRF peptide is hGRF(l-29)-NH 2 . whose amino acid sequence is reported in SEQ ID NO: 1.
- activated PEG is an acylating agent, it preferably contains either a norleucine or ornithine residue bound to the PEG moiety via an amide linkage. These residues allow a precise determination of the linked PEG units per mole of peptide (see for example Sartore et al., 1991). Therefore, more in particular, the preferred activated PEG is mono- methoxylated PEG 5, ooo linked by means of an amide bond to the alpha amino group of norleucine, that is activated at the carboxy group as succinimidyl ester
- Branched PEGs are also in common use
- the branched PEGs can be represented as R(-PEG-OH) m in which R represents a central core moiety such as pentaerythritol or glycerol, and m represents the number of branching arms
- R represents a central core moiety such as pentaerythritol or glycerol
- m represents the number of branching arms
- the number of branching arms (m) can range from three to a hundred or more
- the hydroxyl groups are subject to chemical modification
- Chromatographic methods means any technique that is used to separate the components of a mixture by their application on a support (stationary phase) through which a solvent (mobile phase) flows The separation principles of the chromatography are based on the different physical nature of stationary and mobile phase
- chromatographic methods include liquid, high pressure liquid, ion exchange, absorption, affinity, partition, hydrophobic, reversed phase, gel filtration, ultrafiltration or thin-layer chromatography
- PEGlation is the reaction by which a PEG-protein/peptide conjugate is obtained starting from the activated PEG and the corresponding protein/peptide
- the molar ratio PEG hGRF can be 1 1, 2 1 or 3 1, depending on which conjugate is sought at high yields
- the solvent of the pegylation reaction is selected from the group consisting of a highly concentrated nicotinamide aqueous solution, a buffered aqueous solution of a defolding agent (such as urea) or a polar organic solvent selected among dimethyl sulfoxide, dimethyl formamide/buffer or acetonitrile/buffer
- a non-limitative list of protective chemical groups for Lys 12 and Lys 21 includes Alloc (allyloxycarbonyl), Dde (l-(4,4-Dimethyl-2,6-dioxocyclohex-l-ylidene)ethyl), Adpoc (l-(l'-Adamantyl)-l-methyl-ethoxycarbonyl) or 2-Cl-Z (2- Chlorobenzyloxycarbonyl) Alloc is the preferred protective group for the lysine group
- Alloc can be removed according to one of the methods described in Greene T W et al , 1991) Dde can be removed with 2% hydrazine in DMF (see W C Chan et al , 1995) Adpoc can be removed similarly to Alloc (see also Dick F et al , 1997) 2-Cl-Z can be requires a stronger acid deprotection (HF, TFMSA, HBr) or hydrogenation (see also Tarn et al , 1 87)
- the protective groups for N ⁇ can be an alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, benzyl or cyclohexyl Isopropyl is the preferred one
- alkyl groups can be introduced by reductive alkylation (see Murphy et al , 1988 or Hocart et al , 1987)
- the present invention provides the use of the conjugates of the invention in the manufacture of a medicament for treatment, prevention or diagnosis of growth hormone-related disorders, such as for example growth hormone deficiency(GHD), in particular pediatric growth hormone deficiency.
- growth hormone-related disorders such as for example growth hormone deficiency(GHD), in particular pediatric growth hormone deficiency.
- the medicament is preferably presented in the form of a pharmaceutical composition comprising the conjugates of the invention together with one or more pharmaceutically acceptable carriers and/or excipients.
- Such pharmaceutical compositions form yet a further aspect of the present invention.
- An embodiment of the invention is the administration of a pharmacologically active amount of the conjugates of the invention to subjects at risk of developing a growth hormone-related disease or to subjects already showing such pathology.
- a further object of this invention is a method of treatment, prevention or diagnosis of growth hormone-related disorders, comprising administering an effective amount of the conjugates of the invention, in the presence of one or more pharmaceutically acceptable excipients.
- an “effective amount” refers to an amount of the active ingredients that is sufficient to affect the course and the severity of the disorders described above, leading to the reduction or remission of such pathology.
- the effective amount will depend on the route of administration and the condition of the patient.
- “Pharmaceutically acceptable” is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which is administered.
- the above active ingredients may be formulated in unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
- compositions of the invention can also comprise minor amounts of additives, such as stabilizers, excipients, buffers and preservatives.
- the preferred is the parenteral administration, such as subcutaneous, intramuscular or intravenous injection.
- the dose of the active ingredient to be administered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient.
- the dosage of the active ingredient for the human therapy can be between 5 and 9
- 6,000 ⁇ g/Kg body weight and the preferable dose is between 10 and 300 ⁇ g/Kg body weight
- Figure 1 shows the amino acid sequence of hGRF(l-29)-NH Arrows indicate the possible site(s) of pegylation
- Figure 2 shows the reversed-phase HPLC chromatography of the mixture obtained after the pegylation reaction in DMSO carried out as described in Example 1
- the first two major peaks are the conjugates containing 1 PEG chain per mole of hGRF
- the following minor peak is the conjugate hGRF 2PEG and the last minor peak the conjugate hGRF 3PEG
- Figure 3 a reports the degradation of hGRF(l-29) and of the PEG conjugates of the present invention by subtilisin
- Figure 4 shows the spectroscopic characterization of [Lys(MPEG 5 ,ooo -CH -CO-Nle-
- Figure 5 shows the biological effect of various hGRF-PEG conjugates (from a 1 st
- Figure 7 shows the biological effect of various hGRF-PEG conjugates (from a nicotinamide preparation) in the CHO-hGRFR-LUC in vitro assay Data represent the average of two independent experiments
- Figure 9 shows the biological effect of various hGRF-PEG conjugates (from a 2nd
- Figure 11A shows the time-response curve of serum GH levels after the i v injection of 400 ⁇ g/rat of hGRF-PEG conjugates (DMSO preparation) in male rats Each point represents the mean value obtained for three rats
- Figure 11B shows the time-response curve of plasma hGRF levels after the i v injection of 400 ⁇ g/rat of hGRF-PEG conjugates (DMSO preparation) in male rats Each point represents the mean value obtained for three rats
- Figure 13 shows the restriction map of plasmid pTF5-53 LUC used in the reporter gene assay for the evaluation of GRF activity
- Acetonitrile ACN
- allyloxycarbonyl Alloc
- Benzyl BZL
- tert-Butyloxycarbonyl Boc
- Dichloromethane DCM
- DIEA Diisopropylethylamine
- DMEA Dimethyl Formamide
- DMF dimethyl sulphoxide
- FMOC 9-Fluorenylmethyloxycarbonyl
- 2-[ lH-Benzotriazole- 1 -yl]- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate HBTU
- 1-hydroxybenzotriazole HBt,
- methyl-t-butyl ether MTBE
- norleucine Nle
- NMP N-methyl pyrrolidone
- NMP 2,2,5,7,8-Pentamethyl-chroman-6-sulfonyl
- Pmc 2,2,5,7,8-Pentamethyl-chroman-6-sulf
- the following 4 hGRF-PEG conjugates (A1-A4) are obtained Al [Lys(MPEG 5, ooo-CH 2 -CO-Nle-CO) 12 -hGRF(l-29)-NH 2 ], A2 [Lys(MPEG 5 .ooo-CH 2 -CO-Nle-CO) 21 -hGRF(l-29)-NH 2 ], A3 [Lys(MPEG 5 .ooo -CH 2 -CO-Nle-CO) 12 " 21 -hGRF(l-29)-NH 2 ] and A4 N ⁇ -(MPEG 5.
- the hGRF-PEG conjugates were isolated by gel filtration chromatography or alternatively by reversed-phase chromatography
- the hGRF-PEG conjugate eluted at 14 4 min was found to be compound A3 (GRF-2PEG)
- the hGRF-PEG conjugate eluted at 15.5 min. was found to be compound A4
- the dry products were obtained by solvent evaporation/lyophilization.
- EXAMPLE 3a Solution-phase pegylation of hGRF, using PEGm nnn
- the solvent was removed and the residue fractionated by gel exclusion chromatography in a preparative column Superose 12 TM. Two peaks, corresponding to two hGRF-PEG conjugates were eluted. The first minor peak corresponded to the conjugate having two PEGio.ooo units bound to hGRF, the second major peak corresponded to the conjugate containing one PEG ⁇ o,ooo unit per hGRF.
- This branched PEG has been obtained by linking to each amino group of lysine a PEG ⁇ o,ooo-
- the solvent was removed by lyophylization and the residue fractionated by gel exclusion chromatography in a preparative column Superose 12 TM A single peak was obtained, corresponding to the conjugate containing one PEG 20 ,ooo unit per hGRF
- the MALDI- mass spectrometry was used to reveal the molecular weight of the conjugates and their polydispersivity resulting from the polydispersivity of the starting PEG
- the spectroscopic characterization of the unconjugated hGRF and hGRF-PEG conjugates was carried out by circular dichroism analysis in the range of 190-300 nm.
- the samples 50 ⁇ g/ml
- the samples were dissolved in 10 mM acetic acid or methanol/10 mM acetic acid in 30:70 and 60:40 molar ratios.
- the unconjugated hGRF and the hGRF-PEG conjugates presented a superimposable behavior, as shown in Figure 4 for compound A3.
- acetic acid solution the peptides were in random conformation, whereas by increasing the methanol content the peptide assumed an ⁇ -helix structure.
- proteolytic stability of hGRF and of the hGRF-PEG conjugates was investigated using proteolytic enzymes, such as subtilisin and chymotrypsin.
- subtilisin was performed by incubation at 4°C of a 0.297 mM peptide solution in 0.1 M Tris HCl 0.05 M CaCl 2 pH 8.0 with a peptide/protease molar ratio 1 :50,000.
- peptide was dissolved in 0.08 M Tris HCl, 0.1 M CaCl 2 pH 7.8 and a peptide/protease molar ratio of 1:15,000 was used.
- EXAMPLE 6 Pegylation with Alkylating PEG hGRF was conjugated with mono-methoxylated PEG activated with different acylating groups as well as alkylating groups.
- MEM alpha medium with ribonucleosides and deoxyribonucleosides (Gibco) supplemented with 10% fetal bovine serum (Gibco) plus 600 ⁇ g/ml geneticin G418 sulfate (Gibco);
- EBSS Earle's Balanced Salts
- Rat GH radioimmunoassay kit supplied by Amersham
- CHO-hGRFR-LUC (clone 1-1 1-20) is a cloned cell line that had been obtained by cotransfection of the pcDNA3-hGRF-R and pTF5-53 LUC vectors into CHO-DUKX cell line
- the plasmid pcDNA3-hGRF-R was constructed by inserting the human growth hormone releasing factor receptor (hGRF-R) cDNA into pcDNA3 expression vector
- the Bluescript plasmid containing hGRF-R cDNA was kindly provided by Dr B Gaylinn (University of Virginia) the pcDNA3 mammalian expression vector was obtained from Invitrogen
- the hGRF-R coding sequence was driven by the human cytomegalovirus (CMV) promoter Its restriction map is reported in Figure 12
- the plasmid pTF5-53LUC was constructed by inserting the c-fos cAMP response element along with its endogenous promoter upstream of the luciferase coding sequence in plasmid poLuc
- the cAMP response element and the c-fos promoter were obtained from the plasmid pTF5-53 (described in Fish et al, 1989)
- the promoterless reporter gene vector (poLuc) with multiple cloning sites upstream of the luciferase coding sequence was obtained from Dr Brasier (University of Texas, Galveston) Its restriction map is reported in Figure 13
- These CHO-DUKX cells obtained by the above co-transfection were routinely grown in MEM alpha medium containing ribonucleosides and deoxyribonucleosides and supplemented with 10% fetal calf serum plus 600 ⁇ g/ml geneticin G418 sulfate
- the cells were seeded (40,000 cells/well) in white 96-well plates (Dynatech) and incubated for 16-18 hrs in 200 ⁇ l growth medium before the assay
- CHO-hGRFR-LUC cells were washed twice with 200 ⁇ l of PBS (Sigma) and then lysed by adding 50 ⁇ l of a cell culture lyses reagent (Promega) to each well After a further 15-minute incubation at room temperature, the plates were read in a luminometer (Dynatech) after introducing 150 ⁇ l of a luciferase assay reagent (Promega)
- CHO-hGRFR-LUC cells seeded at 50,000cells/well, at the end of incubation with different hGRF-PEG conjugates , were washed with PBS, as above discussed
- PBS containing calcium and magnesium ions
- plates were read in luminometer (Lumicount - Packard)
- the animals SPF male Sprague-Dawley rats 200 g, b w ) were sacrificed by CO 2 inhalation and the pituitaries removed The tissue was finely minced and put into a bottle with the enzyme solution for tissue digestion The bottle was placed in an incubator at 37°C for 1 hour
- the digested tissue was recovered and the cells washed twice, counted and adjusted to a concentration of 5xl0 5 /ml
- the cells were plated out in a 48-well plate (200 ⁇ l/well) and the plate placed in an incubator for 72 hrs
- the animal was injected i v with hGRF(l-29) (400 ⁇ g/rat) A few minutes before blood collection, the animal was anaesthetized (ketamine-xylazine) Two ml of blood were withdrawn from the inferior vena cava from each rat The sample was divided into two aliquots 1 ml was collected as such and serum was obtained after an incubation period of about 3 hours at 37°C and subsequent centrifugation, the remaining 1 ml was collected into a vial containing 50 ⁇ l of a 4 mg/ml heparin solution, immediately stored on ice and plasma was obtained after centrifugation at 4°C
- Blood sampler were collected at different time points from the injection of the test compound using different animals In each experimental session a total of three rats for each time point was used
- Plasma and serum samples were immediately frozen and stored at - 20°C
- GH serum levels were measured by a commercial RIA kit
- hGRF plasma levels were measured by a commercial RIA kit for hGRF(l-44)
- GRF- 1 PEG 1 st peak' corresponds to [Lys(MPEG 5 ,ooo-CH 2 -CO-Nle-CO) 21 -hGRF(l-29)-NH 2
- GPF-1PEG 2 ,nd peak corresponds to [Lys(MPEG 5 ,ooo-CH 2 -CO-Nle-CO) 12 -hGRF(l-29)-NH 2
- GPF- 2PEG corresponds to [Lys(MPEG 5 ,ooo-CH 2 -CO-Nle-CO) 12 ' 21 -hGRF(l-29)-NH 2
- GPF-3PEG corresponds to N ⁇ -(MPEG 5 ,ooo-CH 2 -CO-Nle-CO)[Lys(MPEG 5 ,ooo -CH 2 - CO-Nle-CO) 1Z21 -hGRF(l-29)-NH
- Fig 11 A and 1 IB the blood levels of GH and GRF at different time-points up to 48 hours in rats treated with 400 ⁇ g i v of GRF- 1 PEG 1 st and 2 nd peak, GRF-2PEG and GRF-3PEG (DMSO preparations) are reported
- EXAMPLE 8 Solid-Phase Synthesis Of Site-Protected hGRF(l-29 -NH 7 Derivatives As Starting Compounds In The Pegylation Process
- FMOC-Lys(Alloc)-OH was purchased from Perseptive Biosystems (Framingham, MA) in bulk and the cartridges filled in house All amino acids used were of the L-configuration
- Resins the primary resins used for the hGRF analogs were PAL-PEG-PS (Peptide Amide Linker - Polyethylene Glycol - Polystyrene) resins
- PAL-PEG-PS Peptide Amide Linker - Polyethylene Glycol - Polystyrene
- the PAL-PEG-PS supports purchased from PerSeptive Biosystems, consistently show superior results in purity and yield of crude product
- a low substitution resin of 0 16 mmol/g was used for all derivatives
- Lower substitution resins are commonly used for long, difficult sequences to ensure better coupling by decreasing steric hindrance and ⁇ -sheet formation in the growing peptide chains
- Step 4 Couple for 45 minutes with Amino Acid activated with HBTU/NMM in DMF
- an extra capping step can be inserted after coupling, which uses 70% Acetic Anhydride in DMF for 20 minutes to acetylate any uncoupled sites on the peptide-resin, resulting in truncated sequences rather than deletion sequences in the final crude product
- the cleavage cocktail used for removing side-chain protecting groups and releasing the peptide from the resin is a standard mixture used for peptides containing
- peptide-resin 100 mg -1 g of peptide-resin is placed into a 20 ml glass vessel and cooled in an ice bath The cleavage cocktail is prepared and also cooled in an ice bath, then added to the peptide-resin for a final volume of approx 10 ml
- the solution is vacuum filtered through a medium-to-course porosity filter into approx 30 ml of cold MTBE
- the reaction vessel is washed with 1 ml TFA and filtered through the same filter funnel into the cold MTBE
- the entire suspension is then transferred to a 50 ml centrifuge tube and centrifuged for approx 10 minutes at 2,000 rpm at room temperature
- the supernatant is aspirated, the precipitate re-suspended in 40 ml cold MTBE and centrifuged again This step is repeated once more
- the final supernatant is aspirated and the precipitate is purged with nitrogen to evaporate most of the remaining ether
- Lyophilized crude peptides are prepared by dissolving 50-100 mg of peptide in 200 ml of aqueous 0 1 % TFA The peptide solution is then loaded directly onto the preparative column through the "A" buffer reservoir line and the gradient program started
- Purified lyophilized peptide samples are prepared by dissolving 0 2 - 1 0 mg of peptide in aqueous 0 1% TFA to a concentration of 0 5 - 1 0 mg/ml
- Sinapinic Acid 10 mg/ml in 50% ACN/0 1% TFA
- Peptide samples are prepared at 1 - 20 ⁇ mol cone in 50% ACN/0 1% TFA 0 5 ⁇ ⁇ l of matrix solution, followed by 0 5 ⁇ l of peptide sample, is applied to analysis plate wells and allowed to dry The analysis plate is loaded into the machine and the samples scanned and analyzed using a Reflector Delayed-Extraction method optimized for peptides. For each sample, a cumulative data signal from 32 - 128 laser shots is collected and analyzed Each run includes a sample well with a standard peptide for calibration
- the peptide-resin was cleaved with a mixture of TFA 1,2-ethanedithiol thioanisole water [10 0 5 0 5 0 5 (v/v) ] for 2 hrs, and the peptide isolated by precipitation in MTBE to give 240 mg of crude peptide Purification by preparative reverse-phase HPLC with a Vydac C18 column (22 x 250 mm) resulted in 60 mg of purified product (>95% by analytical HPLC) MALDI-TOF mass spec Calculated 3523 8, Observed 3524.2
- N ⁇ -isopropyl group was added by reductive alkylation of the peptide-resin using sodium cyanoborohydride and the corresponding ketone (acetone) as described by Hocart, et al , 1987 880 mg peptide-resin (approx 70 ⁇ mols) was swelled in 5 ml DCM for 30 mins, then 10 mmol (174 ⁇ l) acetone in 7 ml MeOH/ 1% HO Ac added and the mixture swirled intermittently for 2 hrs at ambient temperature 2 mmols (129 mg) sodium cyanoborohydride in 12 ml MeOH/1% HO Ac was then added, the mixture - swirled intermittently for 2 hrs, then allowed to sit overnight (15 hrs) Qualitative ninhyd ⁇ n monitoring indicated a completed reaction (no blue color) The peptide-resin was cleaved with a mixture of TFA 1,2-ethanedithiol thioani
Abstract
Description
Claims
Priority Applications (24)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
UA2000063885A UA73716C2 (en) | 1997-12-03 | 1998-01-12 | Site-specific preparation of grf conjugated with polyethylene glycol |
BR9815159-2A BR9815159A (en) | 1997-12-03 | 1998-12-01 | Preparation of site-specific polyethylene glycol-grf conjugates |
HU0100507A HUP0100507A3 (en) | 1997-12-03 | 1998-12-01 | Polyethylene glycol-grf conjugates, site-specific preparation thereof, pharmaceutical compositions comprising thereof and their use |
SK824-2000A SK8242000A3 (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-grf conjugates |
EP98959898A EP1037587B1 (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-grf conjugates |
DE69813291T DE69813291T2 (en) | 1997-12-03 | 1998-12-01 | MANUFACTURING SITE-SPECIFIC POLYETHYLENE GLYCOL-GRF CONJUGATES |
EEP200000319A EE200000319A (en) | 1997-12-03 | 1998-12-01 | Site-Specific Method for the Preparation of hGRF-PEG Conjugates, hGRF-PEG Conjugates, Intermediates, Use, and Pharmaceutical Composition |
PL341139A PL192082B1 (en) | 1997-12-03 | 1998-12-01 | Method for oriented production of hGRF-PEG conjugate, hGRF-PEG conjugate and its application, [Lys (alloc)¹²ˑ²¹] - hGRF compound and [N α-isopropyl- Tyr ¹, Lys (Alloc)¹²]- hGRF compound as well as pharmaceutical compound as such |
AU15633/99A AU755285B2 (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-GRF conjugates |
DK98959898T DK1037587T3 (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-GRF conjugates |
CA002312004A CA2312004C (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-grf conjugates |
AT98959898T ATE236607T1 (en) | 1997-12-03 | 1998-12-01 | PREPARATION OF SITE-SPECIFIC POLYETHYLENE GLYCOL-GRF CONJUGATES |
NZ504570A NZ504570A (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of hGRF-PEG with PEG units covalently bound to Lys12, Lys21 or amino terminal |
IL13655198A IL136551A0 (en) | 1997-12-03 | 1998-12-01 | Preparation of peg-grf conjugates |
JP2000522885A JP2001524505A (en) | 1997-12-03 | 1998-12-01 | Method for site-specific preparation of polyethylene glycol-GRF conjugate |
SI9830400T SI1037587T1 (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-grf conjugates |
KR1020007005736A KR100561768B1 (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-grf conjugates |
EA200000601A EA004269B1 (en) | 1997-12-03 | 1998-12-01 | Method for site-specific preparation of conjugates of human growth hormone releasing factor and polyethylene glycol, conjugates, prepared according to that method, use in production of a medicament thereof |
NO20002664A NO327238B1 (en) | 1997-12-03 | 2000-05-24 | Method for the site-specific preparation of hGRF-PEG conjugates, GRF-PEG conjugates, use thereof for the preparation of a medicament and a pharmaceutical composition comprising the same. |
IL136551A IL136551A (en) | 1997-12-03 | 2000-06-04 | Preparation of peg-grf conjugates |
US09/587,460 US6528485B1 (en) | 1997-12-03 | 2000-06-05 | Site-specific preparation of polyethylene glycol-grf conjugates |
HK01104960A HK1034203A1 (en) | 1997-12-03 | 2001-07-16 | Site-specific preparation of polyethylene glycol-grf conjugates |
US10/299,790 US6869932B2 (en) | 1997-12-03 | 2002-11-20 | Site-specific preparation of polyethlene glycol-GRF conjugates |
US11/011,617 US7317002B2 (en) | 1997-12-03 | 2004-12-15 | Site-specific preparation of polyethylene glycol-GRF conjugates |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97121264A EP0922446A1 (en) | 1997-12-03 | 1997-12-03 | Solution-phase site-specific preparation of GRF-PEG conjugates |
EP97121264.2 | 1997-12-03 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/587,460 Continuation US6528485B1 (en) | 1997-12-03 | 2000-06-05 | Site-specific preparation of polyethylene glycol-grf conjugates |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1999027897A1 WO1999027897A1 (en) | 1999-06-10 |
WO1999027897A9 true WO1999027897A9 (en) | 1999-09-30 |
Family
ID=8227732
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1998/007748 WO1999027897A1 (en) | 1997-12-03 | 1998-12-01 | Site-specific preparation of polyethylene glycol-grf conjugates |
Country Status (30)
Country | Link |
---|---|
US (3) | US6528485B1 (en) |
EP (2) | EP0922446A1 (en) |
JP (2) | JP2001524505A (en) |
KR (1) | KR100561768B1 (en) |
CN (1) | CN1149967C (en) |
AR (1) | AR017780A1 (en) |
AT (1) | ATE236607T1 (en) |
AU (1) | AU755285B2 (en) |
BG (1) | BG64815B1 (en) |
BR (1) | BR9815159A (en) |
CA (1) | CA2312004C (en) |
DE (1) | DE69813291T2 (en) |
DK (1) | DK1037587T3 (en) |
EA (1) | EA004269B1 (en) |
EE (1) | EE200000319A (en) |
ES (1) | ES2194376T3 (en) |
HK (1) | HK1034203A1 (en) |
HU (1) | HUP0100507A3 (en) |
IL (2) | IL136551A0 (en) |
NO (1) | NO327238B1 (en) |
NZ (1) | NZ504570A (en) |
PL (1) | PL192082B1 (en) |
PT (1) | PT1037587E (en) |
SI (1) | SI1037587T1 (en) |
SK (1) | SK8242000A3 (en) |
TR (1) | TR200001615T2 (en) |
TW (1) | TWI254641B (en) |
UA (1) | UA73716C2 (en) |
WO (1) | WO1999027897A1 (en) |
ZA (1) | ZA9811068B (en) |
Families Citing this family (46)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20010055581A1 (en) | 1994-03-18 | 2001-12-27 | Lawrence Tamarkin | Composition and method for delivery of biologically-active factors |
US7229841B2 (en) * | 2001-04-30 | 2007-06-12 | Cytimmune Sciences, Inc. | Colloidal metal compositions and methods |
US6407218B1 (en) * | 1997-11-10 | 2002-06-18 | Cytimmune Sciences, Inc. | Method and compositions for enhancing immune response and for the production of in vitro mabs |
AU2001295589B2 (en) * | 2000-10-05 | 2005-10-13 | Ares Trading S.A. | Regioselective liquid phase pegylation |
EP1234583A1 (en) * | 2001-02-23 | 2002-08-28 | F. Hoffmann-La Roche Ag | PEG-conjugates of HGF-NK4 |
US20030171285A1 (en) * | 2001-11-20 | 2003-09-11 | Finn Rory F. | Chemically-modified human growth hormone conjugates |
EP1465933B1 (en) * | 2002-01-16 | 2007-08-29 | Biocompatibles UK Limited | Polymer conjugates |
US7998705B2 (en) * | 2002-08-06 | 2011-08-16 | FUJIFILM Diosynth Biotechnologies U.S.A., Inc | Increased dynamic binding capacity in ion exchange chromatography by addition of polyethylene glycol |
BR0314619A (en) * | 2002-09-18 | 2005-08-02 | Univ Montreal Ct Hospitalier Chum | Ghrh's analogs |
GB0301014D0 (en) * | 2003-01-16 | 2003-02-19 | Biocompatibles Ltd | Conjugation reactions |
AU2003206158A1 (en) * | 2003-01-18 | 2004-08-13 | Pegsphere Co., Ltd. | Peptides having protected amines of untargeted sites, methods for production thereof and of specifically conjugated peg peptides using the same |
EP1644408A1 (en) * | 2003-07-15 | 2006-04-12 | Barros Research Institute | Eimeria tenella antigen for immunotherapy of coccidiosis |
WO2005010163A2 (en) * | 2003-07-15 | 2005-02-03 | Barros Research Institute | Compositions and methods for immunotherapy of human immunotherapy of human immunodeficiency virus (hiv) |
US20050175583A1 (en) * | 2003-12-02 | 2005-08-11 | Lawrence Tamarkin | Methods and compositions for the production of monoclonal antibodies |
US20050175584A1 (en) * | 2004-01-28 | 2005-08-11 | Paciotti Giulio F. | Functionalized colloidal metal compositions and methods |
CN100355784C (en) * | 2004-02-12 | 2007-12-19 | 江苏恒瑞医药股份有限公司 | Method for preparing polyethylene glycol-modified alpha-interferon 1b |
US20050261475A1 (en) * | 2004-02-13 | 2005-11-24 | Harvard Medical School | Solid-phase capture-release-tag methods for phosphoproteomic analyses |
US6986264B1 (en) * | 2004-07-15 | 2006-01-17 | Carrier Corporation | Economized dehumidification system |
PL2363414T3 (en) | 2004-11-12 | 2022-09-05 | Bayer Healthcare Llc | Site-directed modification of FVIII |
US20070027301A1 (en) * | 2005-07-26 | 2007-02-01 | Gabriella Cristobal-Lumbroso | Polymers with pendant poly(alkyleneoxy) substituent groups and their use in personal care applications |
US20070238656A1 (en) * | 2006-04-10 | 2007-10-11 | Eastman Kodak Company | Functionalized poly(ethylene glycol) |
WO2007130453A2 (en) | 2006-05-02 | 2007-11-15 | Allozyne, Inc. | Non-natural amino acid substituted polypeptides |
US20080096819A1 (en) * | 2006-05-02 | 2008-04-24 | Allozyne, Inc. | Amino acid substituted molecules |
KR101304081B1 (en) * | 2006-08-04 | 2013-09-05 | 프로롱 파마슈티컬스, 엘엘씨 | Modified erythropoietin |
US20080083154A1 (en) * | 2006-10-05 | 2008-04-10 | Timothy M Gregory | Bait retention fish hook |
CA2707840A1 (en) | 2007-08-20 | 2009-02-26 | Allozyne, Inc. | Amino acid substituted molecules |
US20110014118A1 (en) * | 2007-09-21 | 2011-01-20 | Lawrence Tamarkin | Nanotherapeutic colloidal metal compositions and methods |
CN102203002A (en) * | 2007-09-21 | 2011-09-28 | 细胞免疫科学公司 | Nanotherapeutic colloidal metal compositions and methods |
CA2706700A1 (en) | 2007-11-08 | 2009-05-14 | Cytimmune Sciences, Inc. | Compositions and methods for generating antibodies |
KR20110091720A (en) * | 2008-11-04 | 2011-08-12 | 얀센 파마슈티카 엔.브이. | Crhr2 peptide agonists and uses thereof |
ES2587397T3 (en) | 2009-03-20 | 2016-10-24 | Hanmi Science Co., Ltd. | Procedure for preparing a site-specific conjugate of a physiologically active polypeptide |
US20100267636A1 (en) * | 2009-04-20 | 2010-10-21 | Theratechnologies Inc. | Use of cytochrome p450-metabolized drugs and grf molecules in combination therapy |
WO2010121351A1 (en) * | 2009-04-20 | 2010-10-28 | Theratechnologies Inc. | Use of (hexenoyl trans-3)hgrf(1-44)nh2 and ritonavir in combination therapy |
US10370245B2 (en) | 2009-06-22 | 2019-08-06 | Sanford-Burnham Medical Research Institute | Methods and compositions using peptides and proteins with C-terminal elements |
BR112012010661A2 (en) | 2009-11-04 | 2016-11-22 | Janssen Pharmaceutica Nv | method for treating heart failure with stressecopine-like peptides |
WO2012118778A1 (en) | 2011-02-28 | 2012-09-07 | Sanford-Burnham Medical Research Institute | Truncated car peptides and methods and compositions using truncated car peptides |
KR20140027284A (en) | 2011-04-21 | 2014-03-06 | 쎄러테크놀로지스 인코포레이티드 | Growth hormone releasing factor (grf) analogs and uses thereof |
EP2715291A4 (en) | 2011-05-31 | 2015-10-21 | Airware Inc | Re-calibration of ab ndir gas sensors |
US10179801B2 (en) | 2011-08-26 | 2019-01-15 | Sanford-Burnham Medical Research Institute | Truncated LYP-1 peptides and methods and compositions using truncated LYP-1 peptides |
WO2013190520A2 (en) | 2012-06-22 | 2013-12-27 | The General Hospital Corporation | Gh-releasing agents in the treatment of vascular stenosis and associated conditions |
US10064940B2 (en) | 2013-12-11 | 2018-09-04 | Siva Therapeutics Inc. | Multifunctional radiation delivery apparatus and method |
CA2986927C (en) | 2015-05-28 | 2023-12-19 | Institut National De La Recherche Scientifique | Inhibitors of prototypic galectin dimerization and uses thereof |
KR102520348B1 (en) * | 2016-04-19 | 2023-04-10 | 그리폰 파마수티컬스 인터내셔널 에스에이 | PEGylated Bioactive Peptides and Uses Thereof |
CN110317826B (en) * | 2019-05-22 | 2020-11-10 | 中国农业大学 | Application of substance for regulating content or activity of PvGRF9 in regulation of plant stem growth and development |
WO2021133407A1 (en) | 2019-12-27 | 2021-07-01 | Compagnie Generale Des Etablissements Michelin | Rubber mix with high specific surface area and high structure acetylene carbon black |
CA3206260A1 (en) | 2021-01-28 | 2022-08-04 | Ilse Roodink | Anti-sars-cov-2 spike glycoprotein antibodies and the therapeutic use thereof |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4563352A (en) * | 1982-10-04 | 1986-01-07 | The Salk Institute For Biological Studies | Human pancreatic GRF |
DE69026306T2 (en) * | 1989-05-27 | 1996-10-17 | Sumitomo Pharma | Process for the production of polyethylene glycol derivatives and modified proteins. |
NZ238748A (en) * | 1990-06-29 | 1993-09-27 | Hoffmann La Roche | Growth hormone releasing factor (grf) analogues |
JP3051145B2 (en) * | 1990-08-28 | 2000-06-12 | 住友製薬株式会社 | Novel polyethylene glycol derivative modified peptide |
JPH05507939A (en) * | 1991-04-09 | 1993-11-11 | エフ・ホフマン―ラ ロシユ アーゲー | Analogs of growth hormone releasing factor |
EP0518295A3 (en) * | 1991-06-14 | 1993-09-01 | Millipore Corporation | Allyl side chain protection in peptide synthesis |
FR2687681B1 (en) * | 1992-02-20 | 1995-10-13 | Transgene Sa | POLYETHYLENEGLYCOL-HIRUDINE CONJUGATES, THEIR PREPARATION PROCESS AND THEIR USE FOR THE TREATMENT OF THROMBOSES. |
US5932462A (en) * | 1995-01-10 | 1999-08-03 | Shearwater Polymers, Inc. | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
WO1997017367A1 (en) * | 1995-11-03 | 1997-05-15 | Theratechnologies Inc. | Method of grf peptides synthesis |
-
1997
- 1997-12-03 EP EP97121264A patent/EP0922446A1/en not_active Withdrawn
-
1998
- 1998-01-12 UA UA2000063885A patent/UA73716C2/en unknown
- 1998-12-01 DE DE69813291T patent/DE69813291T2/en not_active Expired - Lifetime
- 1998-12-01 CN CNB988117592A patent/CN1149967C/en not_active Expired - Fee Related
- 1998-12-01 EP EP98959898A patent/EP1037587B1/en not_active Expired - Lifetime
- 1998-12-01 AU AU15633/99A patent/AU755285B2/en not_active Expired
- 1998-12-01 AT AT98959898T patent/ATE236607T1/en active
- 1998-12-01 BR BR9815159-2A patent/BR9815159A/en not_active Application Discontinuation
- 1998-12-01 NZ NZ504570A patent/NZ504570A/en unknown
- 1998-12-01 EA EA200000601A patent/EA004269B1/en not_active IP Right Cessation
- 1998-12-01 KR KR1020007005736A patent/KR100561768B1/en not_active IP Right Cessation
- 1998-12-01 PL PL341139A patent/PL192082B1/en not_active IP Right Cessation
- 1998-12-01 IL IL13655198A patent/IL136551A0/en unknown
- 1998-12-01 CA CA002312004A patent/CA2312004C/en not_active Expired - Lifetime
- 1998-12-01 WO PCT/EP1998/007748 patent/WO1999027897A1/en active IP Right Grant
- 1998-12-01 HU HU0100507A patent/HUP0100507A3/en unknown
- 1998-12-01 ES ES98959898T patent/ES2194376T3/en not_active Expired - Lifetime
- 1998-12-01 TR TR2000/01615T patent/TR200001615T2/en unknown
- 1998-12-01 SI SI9830400T patent/SI1037587T1/en unknown
- 1998-12-01 DK DK98959898T patent/DK1037587T3/en active
- 1998-12-01 JP JP2000522885A patent/JP2001524505A/en active Pending
- 1998-12-01 EE EEP200000319A patent/EE200000319A/en unknown
- 1998-12-01 SK SK824-2000A patent/SK8242000A3/en unknown
- 1998-12-01 PT PT98959898T patent/PT1037587E/en unknown
- 1998-12-02 AR ARP980106098A patent/AR017780A1/en active IP Right Grant
- 1998-12-03 ZA ZA9811068A patent/ZA9811068B/en unknown
-
1999
- 1999-01-26 TW TW088101139A patent/TWI254641B/en not_active IP Right Cessation
-
2000
- 2000-05-24 NO NO20002664A patent/NO327238B1/en not_active IP Right Cessation
- 2000-05-29 BG BG10448A patent/BG64815B1/en unknown
- 2000-06-04 IL IL136551A patent/IL136551A/en not_active IP Right Cessation
- 2000-06-05 US US09/587,460 patent/US6528485B1/en not_active Expired - Lifetime
-
2001
- 2001-07-16 HK HK01104960A patent/HK1034203A1/en not_active IP Right Cessation
-
2002
- 2002-11-20 US US10/299,790 patent/US6869932B2/en not_active Expired - Lifetime
-
2004
- 2004-12-15 US US11/011,617 patent/US7317002B2/en not_active Expired - Fee Related
-
2009
- 2009-10-30 JP JP2009251100A patent/JP5431874B2/en not_active Expired - Lifetime
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1037587B1 (en) | Site-specific preparation of polyethylene glycol-grf conjugates | |
CA2737040C (en) | Polymer conjugates of therapeutic peptides | |
US7256258B2 (en) | Regioselective liquid phase pegylation | |
US6433135B1 (en) | PEG-LHRH analog conjugates | |
US20080085860A1 (en) | Selective Vpac2 Receptor Peptide Agonists | |
AU2001295589A1 (en) | Regioselective liquid phase pegylation | |
US20090118167A1 (en) | Selective Vpac2 Receptor Peptide Agonists | |
US20100009916A1 (en) | Selective vpac2 receptor peptide agonists | |
US6914121B2 (en) | PEG-LHRH analog conjugates | |
MXPA00005138A (en) | Site-specific preparation of polyethylene glycol-grf conjugates | |
CZ20002055A3 (en) | Selective preparation process of specific conjugates polyethylene glycol - GRF | |
US20210187077A1 (en) | GLP-1 Agonist Conjugates for Sustained Glycemic Control |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 136551 Country of ref document: IL Ref document number: 98811759.2 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL 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 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 | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL 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 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 3/14-5/14, DRAWINGS, REPLACED BY NEW PAGES 3/14-5/14; AFTER RECTIFICATION OF OBVIOUS ERRORS AS AUTHORIZED BY THE INTERNATIONAL SEARCHING AUTHORITY |
|
WWE | Wipo information: entry into national phase |
Ref document number: 504570 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15633/99 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/2000/005138 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020007005736 Country of ref document: KR |
|
ENP | Entry into the national phase |
Ref document number: 2312004 Country of ref document: CA Ref document number: 2312004 Country of ref document: CA Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 8242000 Country of ref document: SK |
|
WWE | Wipo information: entry into national phase |
Ref document number: PV2000-2055 Country of ref document: CZ Ref document number: 2000/01615 Country of ref document: TR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09587460 Country of ref document: US Ref document number: 1998959898 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 200000601 Country of ref document: EA |
|
WWP | Wipo information: published in national office |
Ref document number: 1998959898 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: PV2000-2055 Country of ref document: CZ |
|
WWP | Wipo information: published in national office |
Ref document number: 1020007005736 Country of ref document: KR |
|
WWG | Wipo information: grant in national office |
Ref document number: 15633/99 Country of ref document: AU |
|
WWG | Wipo information: grant in national office |
Ref document number: 1998959898 Country of ref document: EP |
|
WWG | Wipo information: grant in national office |
Ref document number: 1020007005736 Country of ref document: KR |