[go: up one dir, main page]

WO1998012217A1 - Heterodimer peptides with immunomodulatory effect - Google Patents

Heterodimer peptides with immunomodulatory effect Download PDF

Info

Publication number
WO1998012217A1
WO1998012217A1 PCT/SE1997/001551 SE9701551W WO9812217A1 WO 1998012217 A1 WO1998012217 A1 WO 1998012217A1 SE 9701551 W SE9701551 W SE 9701551W WO 9812217 A1 WO9812217 A1 WO 9812217A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
amino acid
side chains
peptide heterodimer
salt
Prior art date
Application number
PCT/SE1997/001551
Other languages
English (en)
French (fr)
Inventor
Håkan BERGSTRAND
Tomas Eriksson
Magnus Lindvall
Bengt Särnstrand
Original Assignee
Astra Aktiebolag (Publ)
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 Astra Aktiebolag (Publ) filed Critical Astra Aktiebolag (Publ)
Priority to AU44060/97A priority Critical patent/AU4406097A/en
Publication of WO1998012217A1 publication Critical patent/WO1998012217A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to non-antigen-specific immunomodulation, including both immunosuppression and immunostimulation.
  • the invention relates to immunomodulatory peptide heterodimers comprising cysteine amino acid residues, which peptides are capable of inducing an immunomodulatory response in mammals and thereby io a therapeutic effect, and uses thereof.
  • the immune system when it is working properly, protects the individual from infection i s and from growth of cancers. In order to carry out these functions, it must be able to recognise and mount an attack against foreign antigens (including cancer-specific antigens), but not against self antigens present on normal cells throughout the body.
  • Vaccines including single-protein antigens such as diptheria toxoid, are widely used to generate immunity against a specific antigen and thus a specific disease. Where general stimulation of the immune system is desired, this can sometimes be achieved with nonspecific agents such as adjuvants, interleukins, interferons, and colony stimulating factors.
  • the immune system loses its critical ability to distinguish self from non-self.
  • the resulting immunological assault on the individual's own tissues can take the form of autoimmune disease: for example, systemic lupus erythrematosis, Type 1 diabetes, or rheumatoid arthritis.
  • autoimmune disease for example, systemic lupus erythrematosis, Type 1 diabetes, or rheumatoid arthritis.
  • suppression rather than stimulation of the immune response is desirable.
  • Non-specific down-regulation of the immune response is typically achieved by treatment with corticosteroids, azathioprine, cyclosporine, tacrolimus (FK506), rapamycin, or mycophenolate mofetil.
  • Certain immunoglobulins, including the monoclonal antibody OKT3, have also been used for this purpose.
  • Suppression of immunity against a specific antigen called “tolerance induction”, may also be possible.
  • Methods that have been used for inducing tolerance against a particular antigen include intravenous or repeated topical administration of the antigen in dilute form, treatment with a very high dose of the antigen, and oral administration of the antigen..
  • immunomodulatory activity has been found to be immunoinhibitory or immunostimulatory in effect, on the basis of experiments described herein and furthermore, that the immunomodulatory activity has been shown to be indicative of having some therapeutic effect in the treatment of certain diseases, such as cancer and arthritis.
  • a further surprising finding is that the oral presentation of "naked" peptides of the invention did not require the inclusion of added transport agents.
  • the peptides of the invention do not need to be administered in association with transport agents such as delivery vehicles e.g. vesicular delivery systems which are designed to improve delivery to the mucosal epithelial cell lining of the gut.
  • the amount of peptide required to produce the therapeutic effect by oral delivery can be significantly lower than that required to produce a similar effect when the peptide is delivered systemically, eg by parenteral injection.
  • WO95/01990 purports to describe hemoregulatory penta- or hexapeptides comprising two monomeric peptides joined together by two or more divalent bridging units having cell poliferation regulatory activity.
  • the bridging units may be disulphide bridging units.
  • WO96/1 1943 describes peptides with immunomodulatory effect comprising 4 to 15 amino acids and represented by the general formula A-X-Y-Cys-Z-B (wherein X is Gly, Ala, He, Asp, Thr, Ser, Arg or Trp, Y is Pro, pipecolic acid or He and Z is selected from He, Phe, Pro, Ala, Tyr or Gly). Dimers of these monomers formed by intramolecular disulphide bonds resulting from oxidization of the cysteine residues are described.
  • EP-A-0687685 purports to describe physiologically active peptides for use as anti- inflammatory or anti-allergy drugs, which can be dimers bridged by a disulphide bond.
  • each A is independently selected from H, a protecting group e.g. ethyl, trityl (Trt), allyl, or t-butyl, or at least one amino acid residue independently selected from the group of amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, aromatic side chains, and sulphur containing side chains;
  • a protecting group e.g. ethyl, trityl (Trt), allyl, or t-butyl
  • at least one amino acid residue independently selected from the group of amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, aromatic side chains, and sulphur containing side chains;
  • R] is a residue of an amino acid selected from serine (Ser), glycine (Gly), alanine (Ala) and aspartic acid (Asp),
  • R 2 is a residue of an amino acid selected from proline (Pro), pipecolic acid (Pec), and isoleucine (He),
  • R 3 is a residue of an amino acid selected from phenylalanine (Phe), isoleucine (He), proline (Pro) and alanine (Ala),
  • x is 0 or 1 ;
  • each B is independently selected from the group consisting of OH, NH 2 , an oxygen or a nitrogen carrying a protecting group, such as ethyl, trityl (Trt), allyl or t-butyl, or at least one amino acid residue selected from the group of amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, aromatic side chains, and sulphur containing side chains; provided that when x is 0, B is OH, NH 2 , or an oxygen or a nitrogen carrying a protecting group, such as ethyl, trityl (Trt), allyl or t-butyl, the entire peptide sequence containing 3 to 30 amino acid residues; and the second monomer is a cysteine containing peptide different from the first peptide.
  • a protecting group such as ethyl, trityl (Trt)
  • Amino acid residues of A and B independently selected from amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, and sulphur containing side chains.
  • Suitable amino acids may be independently selected from the groups comprising naturally and non- naturally occurring amino acid residues.
  • Naturally occurring amino acid residues include isoleucine (He), leucine (Leu), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), lysine (Lys), phenyl alanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), methionine (Met), valine (Val) and histidine (His).
  • Naturally occurring amino acid residues means those amino acid residues which are found in peptides and/or proteins of living organisms.
  • the skilled addressee will also appreciate that such naturally occurring amino acid residues may be present in peptides of the invention in chemically modified forms eg including added protecting groups such as ethyl, trityl (Trt), allyl, t-butyl and the like.
  • protecting groups such as ethyl, trityl (Trt), allyl, t-butyl and the like.
  • any protecting group(s) which may be present on the peptides of the invention should be such so as not to substantially interfere with the immunomodulatory properties thereof and hence therapeutic effect thereof.
  • amino acid units making up the peptides according to the invention may be in L- and D- form. Generally the amino acid units will be in the L- form.
  • the peptide heterodimers of the invention take the form of dimers consisting of two dissimilar peptide monomers linked to each other by way of covalent bonds (e.g. sulphur to sulphur bonds).
  • the peptide monomers are linked one to the other through at least one bond between at least a cysteine amino acid residue of the first monomer and at least a cysteine amino acid residue of the second monomer.
  • Dimers of the present invention may be in parallel form, i.e. two peptide monomers aligned parallel one to the other such that both the peptide monomers are readable in one direction eg from the N- terminal to C-terminal direction.
  • the peptide monomers making up the dimer may or may not be the same length.
  • the peptide monomers are the same length and the N- and C- terminal amino acid residues of one monomer are located adjacent to the N- and C- terminal amino acid residues of the second peptide monomer.
  • a dimer of the present invention may be in anti-parallel form. That is to say, a first monomer read from the N-terminal amino acid residue to the C-terminal amino acid residue is aligned against a second monomer which is read from the C-terminal amino acid residue to the N- terminal amino acid residue ie in the opposite direction to that of the first peptide monomer; the two peptide monomers being linked through covalent bonds as described above for parallel or anti-parallel dimers of the invention.
  • a heterodimer can be composed of two peptide monomers of the same length, differing, for example, in the substitution of an amino acid residue having the L- form to an amino acid residue having the D- form.
  • the lengths of the peptide monomers making up the dimer may be different.
  • at least one of the monomers is of formula (I) and generally both are of formula (I).
  • peptides of the invention by way of, for example, oral administration, intra-tracheal, nasal or parenteral administration gives rise to a measurable modulated immune response, as indicated in the examples herein.
  • Epithelial cell lining is defined as being the cell lining and associated cells thereto which covers the internal and external surfaces of the body, including the lining of vessels and other small cavities.
  • the epithelial cell lining is regarded as being at least one cell layer in depth and as many as several cell layers deep.
  • Cells included within the ambit of "epithelial cell lining” also includes those cells and specialised lymphoid tissues which are located in or associated with the said epithelial cell lining and which influence the immune response such as T- lymphocytes, B- lymphocytes, enterocytes, NK-cells, monocytes, dendritic cells and cells comprising mucosal associated lymphoid tissue (MALT), such as Peyer's patches and the like.
  • MALT mucosal associated lymphoid tissue
  • the peptides of the invention may be absorbed by the epithelial cell lining in a passive or active sense.
  • the peptides may be absorbed on the cell surface, or actively or passively taken up by cells located on the lumen surface side of the epithelial cell lining , or they may pass inbetween cells located on the lumen surface side of the epithelial cell lining and are taken up by cells located deeper in the epithelial cell lining eg T-lymphocytes or Peyer's patches.
  • peptides of the invention initiate an immune response by interacting with cell surface receptors found in or on the membranes of certain specialised cells located in the epithelial cell lining, such as on enterocytes, and intra-epithelial lymphocytes, without physically penetrating the epithelial cell lining.
  • peptides of the invention may interact with, bind to, pass through or penetrate the epithelial cell lining.
  • the peptides of the invention are preferably administered by oral, nasal, or intra-tracheal administration in oral, nasal or intra-tracheal dosage forms. It has been found that the amount of a peptide of the invention required to produce a given therapeutic effect when orally administered can be significantly lower than that required to produce the same effect via other types of administration, such as parenteral administration.
  • an oral dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the gastrointestinal tract in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease.
  • an oral dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the gastrointestinal tract in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease wherein the amount of the at least one orally administered peptide needed to induce an observable level of modulated immune response in a mammal is less than the amount of the same at least one peptide administered parenterally and needed to achieve a similar observable level of modulated immune response in the said mammal.
  • a nasal dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the nasal passages in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease.
  • a nasal dosage form comprising at least one immunomodulatory peptide according to the invention, the peptide being absorbable by the epithelial cell lining of the nasal passages in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease wherein the amount of the nasally administered peptide needed to induce an observable level of modulated immune response in a mammal is less than the amount of the same peptide administered parenterally and needed to achieve a similar observable level of modulated immune response in the said mammal.
  • an intra-tracheal dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the lung in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease.
  • an intra-tracheal dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the lung in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease wherein the amount of the at least one intra-tracheally administered peptide needed to induce an observable level of modulated immune response in a mammal is less than the amount of the same at least one peptide administered parenterally and needed to achieve a similar observable level of modulated immune response in the said mammal.
  • the peptide monomers of formula (I) contain 3 to 30 amino acid residues in the sequence.
  • x is 1 and thus the or each peptide sequence is from 4 amino acid residues up to 30 amino acid residues in length.
  • the peptide sequence is from 4 amino acid residues to about 20 amino acid residues in length. More preferably, the peptide sequence is from 4 to 15 amino acids in length (e.g. 4 to 10 or 4 to 9), and, most preferably, from 4 to 7 amino acids in length.
  • the peptide sequences can be 4, 5, 6, 7, or 8 amino acid residues in length, with or without protecting groups.
  • the peptides of the invention may or may not be associated with transport agents as defined herein.
  • the peptides of the invention are administered in a "naked" form ie free from added transport agents.
  • Added transport agents are those with which the peptides of the invention are intentionally placed in contact or in association either before, during or immediately after administration and which may serve to improve absorption and/or improve the stability of the peptide.
  • a physiologically active peptide according to the invention free from added transport agents.
  • R> to R 3 may suitably be residues of the amino acids selected from the following:
  • R t selected from Ser, Gly,
  • R 2 is Pro
  • amino acid residue or sequence of amino acid residues can include naturally occurring amino acid residues, such as those described hereinabove or analogues thereof or can include non-naturally occurring amino acid residues, such as synthetic amino acid residues and analogues thereof, or amino acid residues or sequences of amino acid residues including both naturally occurring amino acid residues and/or analogues thereof and non- naturally occurring amino acid residues and/or analogues thereof.
  • Heterodimers according to the invention formed by linking of two dissimilar monomers of formula (I) through a covalent sulphur-sulphur bond of one cysteine amino acid residue of the first monomer and one cysteine amino acid residue of the second monomer may be represented by the formula (II)
  • A, R R 2 , R 3 , B and x are as defined above and A', R,', R 2 ', R 3 ', B' and x' are as defined for A, R l 5 R 2 , R 3 , B and x except that at least one of them has a different meaning from that of A, R,, R 2 , R 3 , B and x.
  • a or B comprise e.g. more than one cysteine amino acid residues then of course further bonds between the peptide monomers may be formed.
  • the heterodimers according to the invention may be prepared by methods known per se. Dimers may be obtained by oxidation of the corresponding monomers.
  • the starting first monomer of formula (I) can be made synthetically, for example by chemical means, or through the use of recombinant DNA technology. Alternatively, they can be isolated from polypeptides or proteins and the like. For example the starting monomers can be made by a chemical process in which individual amino acid residues or fragments of the monomers are joined to form peptide bonds and wherein protecting groups are employed at the beginning and/or end of the process.
  • compositions of formula (I) or physiologically functional derivatives thereof are also included within the ambit of the invention.
  • peptides of the invention will include within their ambit variants of the formula (I) which contain one or more modifications of the peptide backbone and which retain the immunomodulatory properties according to the invention.
  • Such modificiations have been reviewed for example by A.F. Spatola “Chemistry and Biochemistry of Amino Acids, Peptides and Proteins”; B. Weinstein, Ed; Marcel Dekker, New York, 1983, Vol 1, Chapter 5; Robert A Wiley et al, "Peptidomimetics derived from natural products” Medicinal Research Reviews, Vol 13, No.
  • the peptides of the invention can be administered with or without transport agents.
  • peptides of the invention are administered orally, intra-tracheally, nasally, or systemically free from added transport agents. More preferably, the peptides of the invention are administered intra-tracheally, nasally, or orally. Most preferably, the peptides of the invention are administered orally.
  • Transport agents includes added means for delivery such as vesicular delivery systems, micro particles, liposomes, and like systems which are designed to carry drugs ( eg peptides) to the epithelial cell lining or endothelial cell lining.
  • Transport agents also includes chemicals or additional peptide sequences which may form an association with, or are fused to, or are complexed with the peptides and which help to maintain physiological integrity of peptide sequences of the invention, for example, presenting the peptides in a prepro- or pro- form or fusing the peptides to carrier proteins, eg glucosyl transferase, or complexed to chemical agents, such as cyclodextrins and the like.
  • peptides of the invention are administered to the recipient as free peptides along with the usual adjuvants, excipients and diluents commonly found in pharmaceutical formulations.
  • peptides of the invention can be delivered by oral or systemic administration in simple oral or systemic formulations comprising adjuvants, diluents and excipients commonly employed in oral and systemic dosage forms.
  • the peptides are administered in an oral dosage form free from added transport agents.
  • Mucosal associated lymphoid tissue is also found in the epithelial cell linings of the gastrointestinal tract, ie, oesophagous, stomach, duodenum, ileum, and colon; bronchiole linings in the lung; and in the linings of the nasal passages. Without the intention of being bound by theory, it is thought that the peptides of the invention interact with MALT and thereby set in train a sequence of immunomodulating events which results in a therapeutic effect against certain diseases.
  • the immunomodulatory response can be immunoinhibitory or immunostimulatory in effect.
  • the immunomodulatory response has been shown to be indicative for therapy against cancer.
  • the peptides of the invention having an immunomodulatory effect are indicated as being advantageous in the treatment of cancers of mesenchymal origin such as sarcoma, eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma or chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma or mesotheliosarcoma; leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma or Hodgkins disease; sarcomas like leiomysarcoma
  • Peptides of the invention are indicated on the basis of their activity for the treatment of malignancies such as melanoma, mammary carcinoma, gastrointestinal carcinoma such as colonic carcinomas, glioma, bladder carcinoma and squamous cell carcinoma of the neck and head region.
  • peptides according to the invention are indicated for therapy in the treatment of acute and/or chronic infections associated with autoimmune disease and autoimmune disease per se such as non-obese diabetes, systemic lupus erythematosus, sclerodermia, Sjogren's syndrome, dermatomyositis or multiple sclerosis, rheumatoid arthritis, artheriosclerosis, and psoriasis, asthma, rhinitis, fibrosis, chronic bronchitis, hepatitis, post- infectious anergy, acquired immune deficiency diseases such as AIDS, HIV and post traumatic immunological anergy.
  • acute and/or chronic infections associated with autoimmune disease and autoimmune disease per se such as non-obese diabetes, systemic lupus erythematosus, sclerodermia, Sjogren's syndrome, dermatomyositis or multiple sclerosis, rheumatoid arthritis, arthe
  • the peptides according to the present invention may, if appropriate, be used together with a traditional therapy regime, such as with methotrexate (MTX).
  • MTX methotrexate
  • peptides according to the present invention may be advantageously employed as adjuvants in various forms of vaccine preparations and in formulations designed to inhibit rejection of organs in transplants.
  • a method of inducing a modulated immune response in a mammal which comprises administering to the epithelial cell lining of the mammal a dose of a peptide according to the invention, enough to induce said modulated immune response and thereby a therapeutic effect.
  • a method of inducing a modulated immune response in a mammal which comprises 1 ) identifying a mammal in need of modulation of its immune response and 2) administering to at least one epithelial cell lining of the mammal a dose of a peptide according to the invention, enough to induce said immunomodulatory response and thereby a therapeutic effect.
  • the epithelial cell lining to which the peptide is administered is the epithelial cell lining of the gastroinestinal tract.
  • the peptide is administered to the MALT.
  • a method of inducing a modulated immune response in a mammal which comprises administering to MALT of the mammal a dose of a peptide according to the invention, said peptide being free from added transport agents and being sufficient to induce said modulated immune response and thereby a therapeutic effect.
  • a peptide according to the invention in the preparation of a medicament suitable for the treatment of disease.
  • Particular forms of cancer which may be treated with peptides of the invention are listed hereinabove.
  • the peptides may be used in combination with surgery pre- or more preferably, post- operationally.
  • a peptide according to the invention free from added transport agents in the preparation of a medicament suitable for the treatment of disease, in particular cancer and rheumatoid arthritis.
  • a physiologically active peptide according to the invention preferably free from added transport agents, for use in therapy, for example, in cancer or rheumatoid arthritis therapy.
  • a peptide of the invention for use in therapy, for example in cancer therapy or rheumatoid arthritis therapy.
  • a suitable effective dose of peptides of the invention generally lies in the range of from about 0.0001 ⁇ mol/kg to about lOOO ⁇ mol/kg body weight, preferably from about 0.003 to about 300 ⁇ mol/kg body weight, e.g.
  • the total dose may be given as a single dose or multiple doses, e.g two to six times per day.
  • the dose range would be about 2.25 ⁇ mol/kg/day to 225 ⁇ mol/kg/day and a typical dose could be about 100 ⁇ mol of peptide.
  • discrete multiple doses are indicated treatment might typically be 25 ⁇ mol of a peptide of the o invention given up to 4 times per day.
  • peptides of the invention may be given on alternate days or even once or twice a week. The skilled addressee will appreciate that an appropriate administrative regimen would be at the discretion of the physician or veterinary practitioner.
  • Formulations of the present invention for medical use, comprise a peptide of the invention or a salt thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • the carrier(s) should be pharmaceutically acceptable in the sense of being 0 compatible with the other ingredients of the formulation and substantially non-deleterious to the recipient thereof.
  • free acid addition salts e.g. hydro-halo salts
  • base salts are encompassed within the ambit of the invention.
  • the salts will be pharmaceutically acceptable. 5
  • Suitable acid addition salts include those formed from hydrochloric, hydrobromic, nitric, perchloric, sulphuric, citric, tartaric, phosphoric, lactic, benzoic, glutamic, oxalic, aspartic, pyruvic, acetic, succinic, fumaric, maleic, oxaloacetic, isethionic, stearic, phthalic, methanesulphonic, p-toluene sulphonic, benzenesulphonic, lactobionic, glucuronic and trifluoracetic acids.
  • Suitable base salts include inorganic base salts such as alkali metal (e.g.
  • salts will be pharmaceutically acceptable.
  • alkaline earth metal e.g. calcium
  • organic base salts e.g. phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine and diethanolamine salts
  • amino acid salts e.g. lysine and arginine.
  • the salts will be pharmaceutically acceptable.
  • the present invention therefore, further provides a pharmaceutical formulation comprising a peptide of the invention together with a pharmaceutically acceptable carrier therefor.
  • any pharmaceutical formulation comprising a peptide of the invention can include more than one peptide of the invention.
  • a pharmaceutical formulation may comprise at least two peptides of the invention or a cocktail of peptides of the invention.
  • a method for the preparation of a pharmaceutical formulation comprising bringing into association one or more peptides of the invention, or a physiologically functional derivative thereof, and a pharmaceutically acceptable carrier therefor.
  • the peptides of the invention and physiologically functional derivatives thereof may be administered by any route appropriate to the condition to be treated, suitable routes including oral, intra-tracheal, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, intraperitoneal, and epidural). It will be appreciated that the route may vary with, for example, the condition of the recipient.
  • Preferred formulations are those suitable for oral, nasal or intra-tracheal administration. Most preferred formulations are those suitable for oral administration.
  • Formulations for topical administration in the mouth include lozenges comprising the peptide(s) in a flavoured basis, usually sucrose and acacia and tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth washes comprising the peptide(s) in a suitable liquid carrier.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, lozenges comprising the peptide(s) in a flavoured base, usually sucrose and acacia and tragacanth; pastilles comprising the active ingredient(s) in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth washes comprising the active ingredient(s) in a suitable liquid carrier.
  • Each formulation generally contains a predetermined amount of the active peptide(s); as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like.
  • a tablet may be made by compression or moulding, optionally with one or more accessor ⁇ ' ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active peptide(s) in a free-flowing form such as a powder or granules, optionally mixed with a binder, (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent.
  • a binder eg povidone, gelatin, hydroxypropylmethyl cellulose
  • lubricant e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose
  • disintegrant e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered peptide(s) moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.
  • a syrup may be made by adding the active peptide(s) to a concentrated, aqueous solution of a sugar, for example, sucrose, to which may also be added any necessary ingredients.
  • a sugar for example, sucrose
  • Such accessory ingredient(s) may include flavourings, an agent to retard crystallisation of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol, for example, glycerol or sorbitol.
  • the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives, (including antioxidants) and the like.
  • accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives, (including antioxidants) and the like.
  • Emulgents and emulsion stabilisers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl mono- stearate and sodium lauryl sulphate.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired therapeutic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a blend of branch-chained esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft parafin and or liquid parafin or other mineral oils can be used.
  • Formulations for rectal administration may be presented in any suitable form e.g. as a suppository with a suitable base comprising peptide(s) of the invention in admixture with a neutral fatty base, for example cocoa butter, or, for example in admixture with a salicylate, or in the form of solutions and suspensions.
  • a suitable base comprising peptide(s) of the invention in admixture with a neutral fatty base, for example cocoa butter, or, for example in admixture with a salicylate, or in the form of solutions and suspensions.
  • formulations in the form of gelatin rectal capsules comprising active peptide(s) of the invention in admixture with vegetable oil(s) or paraffin oil can be used.
  • Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns. Where the particle size relates to an active substance in particle form per se, the particle size may be in the range of from 2 to 500 microns.
  • Coarse powder formulations can be administered by rapid inhalation through the nasal passage from a container of the powder held up close to the nose.
  • Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops include aqueous or oily solutions of the active ingredient.
  • peptides of the invention may be formulated in pressurised metered dose inhalers or dry powder inhalers for oral or nasal inhalation or in liquid formulations for nebulisation.
  • the active peptide(s) is micronised or otherwise processed to a particle size suitable for inhalation therapy (mass median diameter ⁇ 10 ⁇ m).
  • the micronised peptide(s) can be suspended in a liquefied propellant or a mixture of liquefied propellants. Such propellants can also, but not necessarily act as solvents. In either case, the micronised peptide(s) can be filled into a container equipped, for example with a metering valve.
  • Suitable propellants include those commonly employed in the art, such as, hydrofluoroalkanes (HFAs).
  • HFAs hydrofluoroalkanes
  • the HFA propellants can be present in any mixture which is appropriate for delivering peptide(s) of the invention to MALT.
  • suitable HFAs for. use in the invention include tetrafluoroethane (eg propellant 134a (Hoechst)) and heptafluoropropane (eg propellant 227 (Hoechst)).
  • surfactants can also be present in such formulations, for example, sorbitan trioleate, lecithin, oleic acid and the like, the use of surfactants being to increase the physical stability of the peptide(s) preparation.
  • the formulation can also contain solvents, such as ethanol, to improve the solubility of the peptide(s) in the chosen propellant.
  • Active peptides of the invention may be delivered through inhaling devices suitable for dry powder inhalation, such as portable inhaler devices and the like.
  • the active peptide(s) of the invention can be used either alone or in combination with a carrier, such as lactose, mannitol, or glucose.
  • a carrier such as lactose, mannitol, or glucose.
  • the selection of carrier is not critical, provided that the physiological action of the peptide(s) of the invention is substantially unimpaired.
  • Other additives may also be included in powder formulations as required e.g. to maintain stability etc. Again, such additives should be such so as not to substantially interfere with the physiological and hence therapeutic effect of the peptide(s) of the invention.
  • the inhaling device can be of any type known in the art, such as a single 5 dose inhaler having a predetermined dose or a multi-dose inhaler wherein the dose is measured by a metering unit within the inhaler or is delivered from an assembly of predetermined doses.
  • Formulations suitable for parenteral administration conveniently comprise a sterile aqueous io preparation of the active compound which is preferably isotonic with the blood of the recipient.
  • Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable acid addition salt of a peptide(s) of the invention that is isotonic with the blood of the recipient.
  • Useful formulations also comprise concentrated solutions or solids containing peptide(s) of the invention which upon dilution with an appropriate solvent give a solution for parenteral administration as above.
  • N- and C- terminals of peptides of the invention herein described that the N-terminal is in the amino form (NH2) and that the C-terminal is in the carboxyl form (-COOH).
  • Peptide monomers used were prepared using standard solid phase sequentional coupling techniques on a Millipore 9050 automatic peptide synthesizer (for further information about this technique see for example Jones, J. The Chemical Synthesis of Peptides, pp 132- 30 156, first edition, Oxford University Press, 1991 and R. Epton (ed) Innovation and Perspectives in Solid Phase Synthesis, SPCC (UK) Ltd 1990). Penicillamine was purchased from Bachem.
  • Oxidation of the thiol monomers into disulfide heterodimers was accomplished using general oxidation techniques (e.g. Andreu, D et al. Methods in Molecular Biology, Vol 35: Peptide Synthesis Protocols edited by M.W. Pennington and B.M. Dunn Humana Press Inc., Totowa, NJ.).
  • the C-terminal aminoacid was purchased coupled to a resin which consisted of a crosslinked polystyrene backbone grafted with polyethyleneglycol chains, functionalized with the linker p-hydroxymethyl phenoxyacetic acid (TentaGel S PHB-Aaa-Fmoc, Sheppard, R.C., Williams, B.J. Acid-labile Resin Linkage Agents for Use in Solid Phase Peptide Synthesis. Int. J. Peptide Protein Res. 1982, 20, 451-454) from RAPP Polymere. Isf-Fmoc-protected L-amino acid pentafluorophenyl esters used were purchased from Bachem and Biosearch.
  • DMF and 20% piperidine/DMF in peptide reagent quality was purchased from Biosearch.
  • the coupling reagent 1-hydroxybenzotriazole (HOBT) came from Fluka. Synthesis were performed on a Millipore 9050 Plus PepSynthesizer.
  • amino acid units are in L- form unless otherwise specified.
  • N ⁇ -Fmoc-Aaa-OH derivative was used which was coupled to aminoacids on the resin with HOBT (0.9 mmol) and DIPCDI (0.9 mmol).
  • the activated amino acids were added to the column and recycled 30 min each.
  • the synthesizer ended the synthesis with a deblocking of the N-terminal Fmoc-group and a final wash with DMF.
  • the resulting peptide on the resin was transferred to a sintered glas funnel where it was washed twice with MeOH (2 x 10 ml) and three times with CH 2 C1 2 (3 x 10 ml).
  • the resin was allowed to dry under vacuum over night after which the peptide was side chained deprotected and cleaved from the resin using ethanedithiol/TFA 5/95 (20 ml) at room temperature for 3 h.
  • the resin was filtered off and washed with 3 x 10 ml of acetic acid.
  • the combined acidic fraction was evaporated after which the residue was triturated 3 times with ether.
  • the crude peptide was dissolved in H 2 O/CH 3 CN 1/1 and lyophilized.
  • the resulting material was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5C18 25 cm x 20 mm id reversed phase column (0.1% TFA/CH 3 CN - 0.1% TFA/H 2 O 90 - 10, 10 ml/min, 220 nm).
  • the combined HPLC fraction was lyophilized giving the monomer- Leu Leu Phe Gly Pro Cys He (SEQ. ID. NO. 1) or leu Leu Phe Gly Pro Cys He (SEQ. ID. NO. 3) respectively, each of which was used to prepare the heterodimer.
  • Example 2 Leu Leu Phe Gly Pec Cys He (SEQ. ID. NO. 4)
  • the unprotected cysteine on one of the monomers was activated with ditihiopyridine resulting in the S-pyridyl derivative H-Phe- Cys(S-Pyr)-Leu-Gly-Pro-Cys(Acm)-Pro-OH (SEQ. ID. NO. 18).
  • This derivative was reacted with the second peptide chain resulting in the first disulphide.
  • the second disulphide bond was accomplished using the protocol of Ruiz-Gayo in which a onepot deprotection and oxidation of the Acm protected cysteine with iodine in 80% aqueous acetic acid resulted in a crude product which was purified on HPLC.
  • the ability of the peptides according to the invention to modulate immune responses can be illustrated by their effect in the delayed type hypersensitivity (DTH) test in mice.
  • the DTH test is used to illustrate immunomodulation, the protocol for which is described, for example, by Carlsten H., et al (1986) Int. Arch. Allergy Appl. Immunol 81 :322, herein incorporated by reference.
  • the peptides were tested at one or more of the following dosages: 0.0003 ⁇ mol/kg, 0.003 ⁇ mol/kg, 0.03 ⁇ mol/kg, 0.3 ⁇ mol/kg, and 3.0 ⁇ mol/kg.
  • mice Male and female Balb/c mice were obtained from Bomholtsgaard (Denmark) with a weight of 18-20 grams each. 4-Ethoxymethylene-2-phenyloxazoIin-5-one (OXA) (Sigma Chemicals) was used as the antigen in the DTH test.
  • OXA 4-Ethoxymethylene-2-phenyloxazoIin-5-one
  • mice were sensitized, Day 0, by epicutaneous application of 150 ⁇ l of an absolute ethanol-acetone (3:1) solution containing 3% OXA on the shaved abdomen.
  • the intensity of the DTH reactions was measured according to the method described by van Loveren H., et al (1984) J. Immunol. Methods 67: 31 1 and expressed according to the formula: T t24/48 -T t0 ⁇ m units, where tO, t24 and t48 represent the ear thickness at time 0, +24hrs or +48 hrs after challenge respectively, in individual tests (T).
  • T the intensity of the DTH reactions was measured according to the method described by van Loveren H., et al (1984) J. Immunol. Methods 67: 31 1 and expressed according to the formula: T t24/48 -T t0 ⁇ m units, where tO, t24 and t48 represent the ear thickness at time 0, +24hrs or +48 hrs after challenge respectively, in individual tests (T).
  • T the results are expressed as the mean +/- S.E.M..
  • the level of significance between means of the groups is obtained by Student's two-tailed t-test
  • MOLECULE TYPE peptide

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
PCT/SE1997/001551 1996-09-23 1997-09-15 Heterodimer peptides with immunomodulatory effect WO1998012217A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU44060/97A AU4406097A (en) 1996-09-23 1997-09-15 Heterodimer peptides with immunomodulatory effect

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9603465-7 1996-09-23
SE9603465A SE9603465D0 (sv) 1996-09-23 1996-09-23 New compounds

Publications (1)

Publication Number Publication Date
WO1998012217A1 true WO1998012217A1 (en) 1998-03-26

Family

ID=20403987

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1997/001551 WO1998012217A1 (en) 1996-09-23 1997-09-15 Heterodimer peptides with immunomodulatory effect

Country Status (3)

Country Link
AU (1) AU4406097A (sv)
SE (1) SE9603465D0 (sv)
WO (1) WO1998012217A1 (sv)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10533016B2 (en) 2015-01-09 2020-01-14 Revolution Medicines, Inc. Compounds that participate in cooperative binding and uses thereof
EP3126373B1 (en) 2014-04-03 2020-03-04 Amgen Inc. Method for preparing amg 416
US10907188B2 (en) 2016-04-12 2021-02-02 Ginkgo Bioworks, Inc. Compositions and methods for the production of compounds
US10948495B2 (en) 2015-10-01 2021-03-16 Revolution Medicines, Inc. Methods and reagents for analyzing protein-protein interfaces
US10989710B2 (en) 2010-12-28 2021-04-27 Revolution Medicines, Inc. Identifying new therapeutic agents
US11479797B2 (en) 2016-10-28 2022-10-25 Ginkgo Bioworks, Inc. Compositions and methods for the production of compounds
US11566007B2 (en) 2019-11-04 2023-01-31 Revolution Medicines, Inc. Ras inhibitors
US11608346B2 (en) 2019-11-04 2023-03-21 Revolution Medicines, Inc. Ras inhibitors
US11690915B2 (en) 2020-09-15 2023-07-04 Revolution Medicines, Inc. Ras inhibitors
US11739074B2 (en) 2019-11-04 2023-08-29 Revolution Medicines, Inc. Ras inhibitors
US12202845B2 (en) 2021-10-08 2025-01-21 Revolution Medicines, Inc. Ras inhibitors
US12252497B2 (en) 2021-05-05 2025-03-18 Revolution Medicines, Inc. Ras inhibitors

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021273A2 (de) * 1993-03-23 1994-09-29 Harald Tschesche VERWENDUNG VON ANGIOGENIN UND/ODER α1-MICROGLOBULIN UND/ODER KOMPLEMENTFAKTOR D ZUR BEGRENZUNG DER SEKRETION VON PROTEINEN
WO1996001644A1 (en) * 1994-07-11 1996-01-25 Athena Neurosciences, Inc. Inhibitors of leukocyte adhesion
WO1996011943A1 (en) * 1994-10-14 1996-04-25 Astra Aktiebolag New peptides with immunomodulatory effects
WO1996030397A1 (en) * 1995-03-24 1996-10-03 Astra Aktiebolag New peptides with immunomodulatory effects

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021273A2 (de) * 1993-03-23 1994-09-29 Harald Tschesche VERWENDUNG VON ANGIOGENIN UND/ODER α1-MICROGLOBULIN UND/ODER KOMPLEMENTFAKTOR D ZUR BEGRENZUNG DER SEKRETION VON PROTEINEN
WO1996001644A1 (en) * 1994-07-11 1996-01-25 Athena Neurosciences, Inc. Inhibitors of leukocyte adhesion
WO1996011943A1 (en) * 1994-10-14 1996-04-25 Astra Aktiebolag New peptides with immunomodulatory effects
WO1996030397A1 (en) * 1995-03-24 1996-10-03 Astra Aktiebolag New peptides with immunomodulatory effects

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11644460B2 (en) 2010-12-28 2023-05-09 Revolution Medicines, Inc. Identifying new therapeutic agents
US10989710B2 (en) 2010-12-28 2021-04-27 Revolution Medicines, Inc. Identifying new therapeutic agents
EP3126373B1 (en) 2014-04-03 2020-03-04 Amgen Inc. Method for preparing amg 416
US11377474B2 (en) 2014-04-03 2022-07-05 Amgen Inc. Method for preparing AMG 416 (etelcalcetide)
US11059830B2 (en) 2015-01-09 2021-07-13 Revolution Medicines, Inc. Compounds that participate in cooperative binding and uses thereof
US10533016B2 (en) 2015-01-09 2020-01-14 Revolution Medicines, Inc. Compounds that participate in cooperative binding and uses thereof
US11987590B2 (en) 2015-01-09 2024-05-21 Revolution Medicines, Inc. Compounds that participate in cooperative binding and uses thereof
US10948495B2 (en) 2015-10-01 2021-03-16 Revolution Medicines, Inc. Methods and reagents for analyzing protein-protein interfaces
US10907188B2 (en) 2016-04-12 2021-02-02 Ginkgo Bioworks, Inc. Compositions and methods for the production of compounds
US11447810B2 (en) 2016-04-12 2022-09-20 Ginkgo Bioworks, Inc. Compositions and methods for the production of compounds
US11479797B2 (en) 2016-10-28 2022-10-25 Ginkgo Bioworks, Inc. Compositions and methods for the production of compounds
US11608346B2 (en) 2019-11-04 2023-03-21 Revolution Medicines, Inc. Ras inhibitors
US11739074B2 (en) 2019-11-04 2023-08-29 Revolution Medicines, Inc. Ras inhibitors
US11952352B2 (en) 2019-11-04 2024-04-09 Revolution Medicines, Inc. Ras inhibitors
US11566007B2 (en) 2019-11-04 2023-01-31 Revolution Medicines, Inc. Ras inhibitors
US11690915B2 (en) 2020-09-15 2023-07-04 Revolution Medicines, Inc. Ras inhibitors
US12280113B2 (en) 2020-09-15 2025-04-22 Revolution Medicines, Inc. Ras inhibitors
US12252497B2 (en) 2021-05-05 2025-03-18 Revolution Medicines, Inc. Ras inhibitors
US12202845B2 (en) 2021-10-08 2025-01-21 Revolution Medicines, Inc. Ras inhibitors

Also Published As

Publication number Publication date
AU4406097A (en) 1998-04-14
SE9603465D0 (sv) 1996-09-23

Similar Documents

Publication Publication Date Title
US20020103136A1 (en) Conjugates useful in the treatment of prostate cancer
CA2265476A1 (en) Conjugates useful in the treatment of prostate cancer
EP0646127B1 (en) Polypeptide bombesin antagonists
AU705364B2 (en) New peptides with immunomodulatory effects
CZ203693A3 (en) Bradykinin antagonists
WO1998012217A1 (en) Heterodimer peptides with immunomodulatory effect
AU740597B2 (en) Conjugates useful in the treatment of prostate cancer
US6228373B1 (en) Peptides with immunomodulatory effects
WO1994021674A9 (en) Polypeptide bombesin antagonists
US6174858B1 (en) Conjugates useful in the treatment of prostate cancer
AU744652B2 (en) Conjugates useful in the treatment of prostate cancer
WO1998012219A1 (en) Methionine, penicillamine and cysteine-analogue containing peptides having immunomodulating activity
US6127333A (en) Conjugates useful in the treatment of prostate cancer
WO1998012218A1 (en) Tri- or polysulphide containing peptides having immunomodulating activity
WO1998012213A2 (en) Cysteine containing peptides
WO1998012216A1 (en) Methionine containing peptides having immunomodulatory effect
WO1998012214A1 (en) Cysteine analogue containing peptides having immunomodulatory effect
WO1998012215A1 (en) Penicillamine containing peptides having immunomodulatory effect
EP1261626A1 (en) Bombesin analogs for treatment of cancer
WO2004069857A2 (en) “peptides, compositions and uses thereof”
MXPA96005651A (en) New peptides with immune regulated effects
JPH09512787A (ja) ペプシンの放出を抑制するためのペプチド
CZ269199A3 (cs) Peptidy obsahující T-buněčný epitop specifický pro kolagen, jejich použití a farmaceutické přípravky obsahující takové peptidy

Legal Events

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

Ref document number: 09000087

Country of ref document: US

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 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 AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC

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

Ref country code: JP

Ref document number: 1998514577

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA