EP0592634A1 - Inhibiteur de proteine-kinase c - Google Patents

Inhibiteur de proteine-kinase c

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Publication number
EP0592634A1
EP0592634A1 EP93908891A EP93908891A EP0592634A1 EP 0592634 A1 EP0592634 A1 EP 0592634A1 EP 93908891 A EP93908891 A EP 93908891A EP 93908891 A EP93908891 A EP 93908891A EP 0592634 A1 EP0592634 A1 EP 0592634A1
Authority
EP
European Patent Office
Prior art keywords
pkc
arg
peptides
seq
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93908891A
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German (de)
English (en)
Inventor
Marie Teresa Diaz-Meco Conde
Jorge Moscat Guillen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glaxo Wellcome SA
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Glaxo Wellcome SA
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Filing date
Publication date
Application filed by Glaxo Wellcome SA filed Critical Glaxo Wellcome SA
Priority to EP93908891A priority Critical patent/EP0592634A1/fr
Publication of EP0592634A1 publication Critical patent/EP0592634A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11013—Protein kinase C (2.7.11.13)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • This invention relates to a novel method of treatment and in particular to novel peptide inhibitors of a specific protein kinase C isotype, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medicine.
  • PC-PLC phosphatidylcholine
  • Oocytes from Xenopus laevis are a suitable system for investigating the involvement of different enzymatic activities in relevant signal transduction mechanisms controlled by oncogenes (L J Korn et al.. Science 236, 840 (1987); J C Lacal et al.. Science 238, 533 (1987b)).
  • Xenopus oocytes undergo a maturation program following stimulation with either insulin or progesterone, and several lines of evidence indicate the specific involvement of ras p21 in the maturation signalling cascades activated by insulin/IGF- 1 : 1. microinjection of rgs p21 activates maturation in oocytes (C. Birchmeier et al..
  • microinjection of a neutralizing anti-ras p21 antibody blocks the maturation program induced by insulin but not by progesterone (L J Korn et aU1987) i ⁇ £ 2it).
  • X is H or one or more amino acids and J is OH or one or more amino acids, and pharmaceutically acceptable derivatives thereof, which peptides contain 3 to 15 amino acid residues.
  • the term "derivatives” includes salts and solvates of the peptides according to the invention and peptides according to the invention which contain one or more end group modified structures, with the proviso that the C-terminal amino acid residue is other than an alkyl ester of arginine.
  • End group modified structures include deletion, acylation (for example acetylation or benzoylation), alkylation, fatty acylation (for example myristoylation) or cyclisation of the N-terminal amine group and deletion, amidation (including mono- and dialkyiamidation), cyclisation or reduction of the C-terminal carboxy group.
  • N-acyl for example N-acetyl derivatives are preferred.
  • Suitable salts of the peptides of formula (I) include physiologically acceptable acid addition salts derived from inorganic and organic acids, such as hydrochlorides, hydrobromides, sulphates, nitrates, oxalates, phosphates, tartrates, acetates, citrates, trifluoroacetates, fumarates, maleates, succinates and sulphonates e.g. p-toluenesulphonates and methanesulphonates.
  • Suitable solvates of the peptides of formula (I) include, for example, hydrates.
  • the peptides according to the invention are specific inhibitors of protein kinase C isotype zeta ( ⁇ -PKC).
  • ⁇ -PKC protein kinase C isotype zeta
  • ⁇ -PKC protein kinase C isotype zeta
  • ⁇ -PKC protein kinase C isotype zeta
  • RRGARRWRK SEQ ID NO. 5
  • This specific sequence has been found to be perfectly (100%) conserved in ⁇ -PKC variants isolated from a number of different sources including rat brain ⁇ -PKC (Ono et al.. Proc.Natl.Acad.Sci.USA, 8 ⁇ , 3099-3103 (1989)) and Xenopus laevis ⁇ -PKC and ⁇ n-PKC (present work).
  • Preferred peptides of general formula (I) contain 3 to 9 amino acid residues, particularly 3 to 6 amino acid residues.
  • X is H, acetyl, Gly, Arg-Gly or Arg-Arg-Gly and J is OH, Trp, Trp-Arg or Trp-Arg-Lys.
  • Preferred peptides according to the invention include : Ala-Arg-Arg (SEQ ID NO. 1);
  • N-acetyl-Ala-Arg-Arg SEQ ID NO. 17; and pharmaceutically acceptable derivatives thereof.
  • the peptides of formula (I) contain at least three chiral centres. It is to be understood that formula (I) is intended to encompass all diastereoisomers of the peptides of the invention as well as mixtures thereof, including racemates.
  • the amino acids may be either of the natural L-form or of the D-form or a mixture e.g. a racemic mixture, of L- and D-forms. Nevertheless, the peptides of formula (I) preferably contain the natural L-forms of the amino acid residues contained therein.
  • the peptides according to the invention are prepared in substantially pure form and are substantially free of other peptides or amino acids. Preferably the peptides have a purity of 90% or higher, for example 95%, although a purity of 98% or higher (based on all peptides present) is preferred for clinical use.
  • novel peptides of this invention can be made by appropriate adaptation of conventional methods for peptide synthesis.
  • the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence.
  • N-protecting groups e.g. the carbobenzyloxy group or the t- butyloxycarbonyl group (BOC)
  • various coupling reagents e.g. dicyclohexylcarbodiimide ' or carbonyldimidazole
  • various active esters e.g. esters of N-hydroxypthalimide or N-hydroxysuccinimide
  • cleavage reagents e.g.
  • peptides of this invention are prepared by the well-known
  • a further aspect of the invention provides a process for preparing a peptide as described herein which comprises (a) binding the desired protected carboxy-terminal amino acid to a suitable solid support;
  • This procedure though using many of the same chemical reactions and blocking groups of classical peptide synthesis, provides a growing peptide chain anchored by its carboxyl terminus to a solid support, usually cross-linked polystyrene or styrene divinylbenzene copolymer.
  • This method conveniently simplifies the number of procedural manipulations since removal of the excess reagents at each step is effected simply by washing of the solid support.
  • the carboxyl terminal amino acid having its alpha-amino group suitably protected, is coupled to a solid support.
  • the alpha- amino protecting group for example by using trifluoroacetic acid in methylene chloride, the next step in the synthesis is ready to proceed.
  • Other standard cleaving reagents and conditions for the removal of specific amino protecting groups may be used, as described in the literature.
  • alpha-amino- and side-chain-protected amino acids are then coupled stepwise in the desired order to obtain an intermediate peptide connected to the solid support.
  • some of them may be coupled to one another prior to the addition to the growing solid-phase chain.
  • the selection of the appropriate coupling reagents is within the skill of the art. Common to chemical syntheses of peptides is the protection of the labile side-chain groups of the various amino acid moieties with suitable protecting groups at that site until the group is ultimately removed after the chain has been completely assembled.
  • the intermediate peptide is removed from the solid support by treatment with a reagent, such as liquid HF, which not only cleaves the peptide from the support but also cleaves all the remaining side-chain protecting groups.
  • a reagent such as liquid HF
  • the peptide can then be purified for example by gel permeation, HPLC (Rivier et al.. Peptides : Structure and Biological Function (1979) pp. 125-128), ion exchange gel and partition chromatography, countercurrent distribution or other known methods.
  • a peptide of the invention as a salt, for example as an acid addition salt
  • this may be achieved by treating the free base of the peptide with an appropriate acid, preferably with an equivalent amount of acid.
  • the peptides according to the invention have been found to be potent and specific inhibitors of ⁇ -PKC. Accordingly the peptides are useful as scientific tools for studying the role of ⁇ -PKC within biological systems, particularly its role in mitogenic signalling pathways, for example in the maturation of germ cells and the proliferation of somatic mammalian cells.
  • the present invention provides a method for inhibiting cellular ⁇ -PKC activity which comprises contacting cells capable of responding to ⁇ -PKC activation with an effective amount of an inhibitor of ⁇ -PKC.
  • ⁇ -PKC ⁇ -PKC
  • Useful ⁇ -PKC inhibitors preferably exhibit the potency and selectivity demonstrated herein for the preferred peptide according to the invention Ala-Arg-Arg (SEQ.ID.NO.1).
  • Suitable assay systems include, for example, phosphorylation assays using exogenous ⁇ -PKC isolated from a suitable tissue source, for example, rat brain, bovine brain or X.laevis oocytes, or using a source of recombinant ⁇ -PKC, such as that described by Ono et al (1989) loc.cit.
  • Alternative assay protocols may employ appropriate microinjection techniques such as those described herein, for example analysis of oocyte maturation pathways such as H1 -kinase and germinal vesicle breakdown, or analysis of mammalian cell proliferation, for example by assaying DNA synthesis.
  • Such methods are useful for in vitro studies of ⁇ -PKC activity, particularly its role in mitogenic signalling pathways, and as screening methods for the selection of novel ⁇ -PKC inhibitory agents.
  • Suitable in vivo screens may be established by conventional techiques employing transgenic animal e.g. mouse models.
  • a further aspect of the invention therefore provides a screening method for the selection of agents suitable for the prevention or treatment of pathological conditions mediated by ⁇ -PKC activity which comprises a) incubating a sample comprising the agent to be tested with an assay system capable of indicating inhibition and/or activation of ⁇ -PKC activity; b) determining whether and, if desired, the extent to which ⁇ -PKC activity is altered by said agent; and c) selecting agents determined to be potent and selective ⁇ -PKC inhibitors.
  • ⁇ -PKC is a critical step in the transduction of mitogenic signals in response to the insulin/ras p21 /PC-PLC specific pathway.
  • ⁇ -PKC may play a role in the cascade of events linking cell activation to viral genome transcription.
  • the in vivo significance of ⁇ -PKC had not hitherto been appreciated and thus the present applicants are the first to recognise that in vitro inhibitors of ⁇ -PKC may have in vivo utility in medicine for the diagnosis, prevention or treatment of pathological conditions mediated by ⁇ -PKC activity.
  • ⁇ -PKC inhibitors may be effective against a wide spectrum of tumours, hyperproliferative disorders such as psoriasis, and viral infections such as HIV.
  • tumor means both benign and malignant tumours, cancers, cancerous growths or neoplasms.
  • tumours include, but are not limited to, a wide spectrum of mammalian (including human) tumours such as carcinomas, adenocarcinomas, melanomas, sarcomas, lymphomas and leukemias.
  • mammalian tumours such as carcinomas, adenocarcinomas, melanomas, sarcomas, lymphomas and leukemias.
  • Specific examples include cancers of the oral cavity and pharynx (lip, tongue, mouth, pharynx), oesophagus, stomach, small intestine, large intestine, rectum, liver and biliary passages, pancreas, larynx, lung, bone, connective tissue, skin, colon, breast, cervix uteri, corpus endometrium, ovary, prostate, testis, bladder, kidney and other urinary tissues, eye, brain and central nervous system, thyroid and other endocrine glands, leukemias (lymphocytic, granuloc
  • tumours for example maxilofacial squamous cell carcinoma
  • ⁇ -PKC overexpression of ⁇ -PKC, by using a plasmid with a potent viral promoter in NIH 3T3 fibroblasts confers to these cells severe deregulation of their growth properties including a reduced serum dependence, lower doubling times and higher saturation densities, as well as colony formation in semi-solid medium.
  • the peptides of formula (I) are effective against a wide spectrum of tumours, hyperproliferative disorders and viral infections in mammals, including humans, and may be used for the treatment of cancer, hyperproliferative disorders such as psoriasis, and viral infections such as HIV.
  • the invention thus further provides peptides of formula (I) and their physiologically acceptable derivatives for use as active therapeutic agents, in particular for the treatment of conditions whose underlying aetiology is associated with ⁇ -PKC activity in animals (especially humans).
  • a peptide of formula (I) or a physiologically acceptable derivative thereof for use in the treatment of cancer for example maxilofacial squamous cell carcinoma.
  • a method for inhibiting the activity of ⁇ -PKC in a mammal including a human comprising administration of an effective amount of a peptide of formula (I) or a physiologically acceptable derivative thereof.
  • a peptide of formula (I) or a physiologically acceptable derivative thereof for the manufacture of a medicament for inhibiting ⁇ -PKC activity in a mammal.
  • a peptide of the invention may be administered to a patient as the raw chemical, but it is preferable to present the active ingredient as a pharmaceutical formulation.
  • the invention accordingly provides a pharmaceutical formulation comprising a peptide of formula (I) or a physiologically acceptable derivative thereof together with one or more physiologically acceptable carriers and, optionally, other therapeutic and/or prophylactic ingredients.
  • the carriers must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions include those suitable for oral, rectal, nasal, topical, implant or parenteral (including intramuscular, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • the formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate).
  • binding agents e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g. lactose, microcrystalline cellulose or calcium phosphate
  • lubricants e.g. magnesium stearate, talc or silica
  • disintegrants e.g. potato starch or sodium starch glycollate
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond oil, oily esters or ethyl alcohol); and preservatives (e.g. methyl or propyl- ⁇ -hydroxybenzoates or sorbic acid).
  • suspending agents e.g. sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agents e.g. lecithin or acacia
  • non-aqueous vehicles e.g. almond oil, oily esters or ethyl alcohol
  • preservatives e.g
  • the pharmaceutical compositions may take the form of buccal or sub-lingual tablets, drops or lozenges formulated in conventional manner.
  • the peptides may be formulated as creams, gels, ointments or lotions or as a transdermal patch.
  • Such compositions may for example be formulated with an aqueous or oily base with the addition of suitable thickening, gelling, emulsifying, stabilising, dispersing, suspending, and/or colouring agents.
  • the peptides of the invention may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the peptides may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example as a sparingly soluble salt.
  • the peptides of the invention may be formulated for parenteral administration by injection, conveniently intravenous, intramuscular or subcutaneous injection, for example by bolus injection or continuous intravenous infusion.
  • Formulations for injection may be presented in unit dosage form e.g. in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
  • the peptides of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glyceride.
  • the peptides of the invention may be used, for example, as a liquid spray, as a powder or in the form of drops.
  • the peptides according to the invention are conveniently delivered in the form of an aerosol spray presentation from pressurised packs or a nebulise r, with the use of a suitable propeliant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propeliant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a peptide of the invention and a suitable powder base such as lactose or starch.
  • compositions described above may be presented in a conventional manner associated with controlled release forms.
  • compositions according to the invention are suitable for intranasal, topical or parenteral administration-
  • amount of a peptide of formula (I) required for use in treatment will vary not only with the particular peptide selected, but also with the route of administration, the nature of the condition being treated and the age, weight and condition of the patient and will ultimately be at the discretion of the attendant physician or veterinarian.
  • a suitable dose will be in the range of from about 1 to about 500mg per day, preferably in the range of 20 to 200mg per day, most preferably in the range of 50 to 120mg per day.
  • a suitable daily dose for use in prophylaxis will generally be in the range of 0.1 mg to ⁇ Omg.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.
  • the peptide is conveniently administered in unit dosage form.
  • a convenient unit dosage formulation contains the active ingredient in an amount of from 0.1 to about 500mg.
  • the peptides of the present invention may also be used in combination with other therapeutic agents, for example, other anticancer agents.
  • other therapeutic agents for example, other anticancer agents.
  • the compounds of the invention may be employed together with known anticancer agents.
  • the invention thus provides, in a further aspect, a combination comprising a peptide of formula (I) as defined herein together with another therapeutically active agent, in particular an anticancer agent.
  • compositions comprising a combination as defined above together with a pharmaceutically acceptable carrier therefor comprise a further aspect of the invention.
  • the active compounds When peptides of formula (I) are used in combination with a second therapeutic agent, the active compounds may be administered either sequentially or simultaneously by any of the routes described above.
  • Suitable therapeutic agents for use in the combinations defined above include, for example alkylating agents such as cyclophosphamide, antimetabolites such as methotrexate, mitotic inhibitors such as vinblastine, antitumour antibiotics such as adriamycin, endocrine therapy such as tamoxifen, flutamide, goserelin acetate and medroxyprogesterone acetate, radiotherapy and ⁇ mmunotherapy.
  • alkylating agents such as cyclophosphamide
  • antimetabolites such as methotrexate
  • mitotic inhibitors such as vinblastine
  • antitumour antibiotics such as adriamycin
  • endocrine therapy such as tamoxifen, flutamide, goserelin acetate and medroxyprogesterone acetate
  • radiotherapy and ⁇ mmunotherapy include, for example alkylating agents such as cyclophosphamide, antimetabolites such as methotrex
  • the dose of each active compound may vary from that when the compound is used alone.
  • the dose of each active compound may be the same or different to that employed when the compound is used alone.
  • Appropriate doses will be readily appreciated by those skilled in the art.
  • the present invention provides antisense oligonucleotides and derivatives thereof corresponding to the DNA coding for ⁇ - PKC, preferably the regulatory domain of ⁇ -PKC DNA, or a degenerate equivalent thereof.
  • Preferred antisense oligonucleotides are fifteen-mer antisense oligonucleotides corresponding to the beginning of the coding region of ⁇ -PKC, preferably of mammalian ⁇ -PKC, especially human ⁇ -PKC.
  • An example of a suitable antisense oligonucleotide according to the invention isr
  • antisense oligonucleotides are preferably modified on the backbone to phosphorothioates to reduce nuclease degradation.
  • the antisense oligonucleotides and equivalents thereof according to the invention may be prepared in conventional manner as described hereinafter.
  • the antisense oligonucleotides according to the present invention have been found to be potent and specific inhibitors of ⁇ -PKC and have utility both as in vitro scientific tools and in vivo for the treatment of pathological conditions mediated by ⁇ -PKC activity, in particular tumours, hyperproliferative disorders such as psoriasis, and viral infections such as HIV.
  • Suitable pharmaceutical compositions containing the antisense oligonucleotides according to the invention and dosages thereof are as described for the peptides according to the invention as hereinbefore described.
  • Figure 1 The involvement of E-protein kinase C in the activation of H1 -kinase.
  • Stage VI oocytes either untreated or microinjected with 1 ng of purified bovine brain PKC (Nishizuka (1988), Science 334, 61 ) were microinjected with either buffer control (speckled bars) or with 5 ⁇ M (final concentration into the oocyte) of a mixture of the three peptides corresponding to the pseudosubstrate region of PKC isotypes ⁇ , ⁇ , and ⁇ (Peptide A, SEQ ID NO. 6), ⁇ (Peptide D, SEQ ID NO. 7) and ⁇ (Peptide E, SEQ ID NO. 8) (striped bars) or with the peptide corresponding to the pseudosubstrate region of ⁇ PKC (Peptide Z, SEQ ID NO.
  • oocytes were microinjected with 25 ⁇ U of B. cereus PC-PLC or with 20ng of transforming v-H-ras p21 , or they were incubated in the presence of PMA (100ng/ml), insulin (1 ⁇ M) or progesterone (1 ⁇ M). The reactions were stopped by 2h after stimulation, and H1 -kinase activity was measured following precipitation of extracts with p13 suc1 linked to agarose beads.
  • the pseudosubstrate peptides have the following sequences : (A) peptide specific for PKC isotypes ⁇ , ⁇ , ⁇ : RKGALRQKN (SEQ ID NO.
  • control level of H1 -kinase was 76 fmol/min/oocyte and was not affected by microinjection of pseudosubstrates. Essentially identical results were obtained in three other experiments.
  • Oocyte extracts were incubated with 10 ⁇ g of anti- ⁇ -PKC antibody and immunocomplexes were recovered with protein G-agarose. Immunprecipitates were assayed for the autophosphorylation kinase activity of ⁇ -PKC in a mixture of 300 ⁇ M [ ⁇ - 32 P]ATP with or without 50 ⁇ g/ml phosphatidyiserine (PS). Incubations were carried out with different concentrations of peptides A or Z (described in the legend to Figure 1). Reactions were stopped by 45 min and proteins resolved by SDS-PAGE. No kinase activity was detected when oocyte extracts were incubated with protein G-agarose in the absence of antibody. Essentially identical results were obtained in three independent experiments.
  • Figure 4 mmunoblot analyses of ⁇ -PKC levels in extracts from RNA-microiniected oocvtes.
  • RNAs synthesized in vitro from a plasmid harboring a fragment from the regulatory domain of ⁇ -PKC were microinjected into stage VI oocytes. Forty-eight hr following microinjection of RNAs or distilled water, oocytes were homogenized and extracts were resolved by SDS-PAGE, electroblotted and incubated with antibodies specific for PKC isotypes ⁇ , ⁇ and ⁇ (A) or ⁇ (B). Essentially identical results were obtained in three independent experiments.
  • Stage VI oocytes were microinjected with water (empty bars) or with 25 ng of either sense (striped bars) or antisense (black bars) synthetic RNAs.
  • Another set of oocytes were microinjected with water (empty bars) or with 150 ng per oocyte of either sense (striped bars), antisense (black bars) or nonsense (speckled bars) oligonucleotides specific for ⁇ -PKC.
  • oocytes were microinjected with 20ng of transforming V-H-JSS p21 or with 25 ⁇ U of B.cereus PC-PLC, or incubated in the presence of insulin (1 ⁇ M) or progesterone (1 ⁇ M). Afterwards, H1 kinase activity was determined in the extracts as described under Experimental Procedures, when oocytes displayed a 50% induction of GVBD. Results are mean + SD of three independent experiments with incubations in duplicate.
  • Figure 6 A comparison of sequences corresponding to the different pseudosubstrates.
  • H1 -kinase Stage VI oocytes, prepared following standard procedures, were microinjected with either buffer control or with 1 ⁇ M (final concentration into the oocyte) of peptides Z, Z1 , Z2, Z3, Z4 or A4 (SEQ ID NOS. 5, 4, 3, 2, 1 or 9 respectively). Afterwards, oocytes were either microinjected with 25 ⁇ U of EL cereus PC-PLC, or 20ng of ⁇ as p21 . or they were incubated with insulin (1 ⁇ M) or progesterone (1 ⁇ M). Subsequently, reactions were stopped by 2h after stimulation, and H1 -kinase activity was measured as described in the legend to Figure 1. The control level of H1 -kinase was 80 fmol/min/oocyte and was not affected by microinjection of peptides. Essentially identical results were obtained in three other experiments.
  • Oocytes were prepared following standard procedures (Garcia de Herreros et al. (1991) loc cit.). Briefly, ovaries from Xenopus laevis frogs (Blades Biologicals, UK) were incubated with 2mg/ml of collagenase (Boehringer Mannheim, Germany) for 45 min in modified Barth Solution (MBS) without Ca 2+ (110mM NaCl, 2mM KCI, 1mM MgCi , 1mM CaCl 2 , 2mM NaHC0 3 , 10mM HEPES, pH 7.5). After extensively washing, stage VI oocytes were selected and incubated overnight at 20°C.
  • MBS modified Barth Solution
  • a probe was generated by amplification of a DNA fragment directly on a lambda-ZAP library from rat brain by the polymerase chain reaction with the following oligonucleotides:
  • Hybridizations were carried out in 50% formamide at 42°C and filters washed at 65 ⁇ C with 0.1 x SSC, 0.1% SDS. Positive signals were picked and phages purified. Inserts were subcloned into pBluescript plasmid. Clones were analyzed by DNA-sequencing with fmofM DNA Sequencing System (Promega, Madison).
  • bacteriophage T3 or T7 RNA polymerases were used to synthesize sense and antisense RNAs in the presence of the cap analogue GpppG by using 10 ⁇ g of linearized DNA as template (mCAPTM mRNA capping Kit; Stratagene, CA.
  • oligonucleotides identical or complementary to the beginning (starting at the initiation codon) of the coding region of Xenopus ⁇ -PKC were synthesized and modified on the backbone to phosphorothioates (Operon Technologies, Alameda, C.
  • a nonsense oligonucleotide with a random sequence of nucleotides identical to those present in the antisense oligonucleotide was synthesized.
  • PC-PLC isolated from Bacillus cereus PC-PLC was isolated from cultures of Bacillus cereus SE-1 essentially -as described previously (Larrodera et al. (10) loc. cit.): Garcia de Herreros et al (11 loc. cit). Following this protocol the enzyme preparation was purified to complete homogeneity as confirmed by SDS-PAGE followed by silver-staining. The specific activity of the purified enzyme was 1.5 U/ ⁇ g.
  • AuroProbeTM BL plus system Amersham, Int.
  • an anti- peptide antibody generated against the peptide ILKKDVVIQDDDVE (SEQ ID NO. 12) corresponding to aminoacids 381-394 of ⁇ -PKC was used.
  • an anti-peptide antibody generated using the peptide corresponding to aminoacids 577-592 of ⁇ -PKC was used.
  • These antibodies were purchased from Givco BRL (Gaithersburg, MD).
  • oocytes Groups of 20 oocytes were cultured at 20°C in modified Barth solution, and germinal vesicle (nuclear) breakdown (GVBD) was assessed by the appearance of a white spot in the animal pole. In some cases, nuclear breakdown was confirmed by dissection of trichloroacetic acid (10%)-fixed oocytes (Garcia de
  • extracts (1 -2mg/assay) were assayed for 10min at 3 ⁇ OC in a final reaction volume of 50 ⁇ l containing 20mM HEPES (pH 7.0), 5mM ⁇ -mercaptoethanol, 10mM MgCl 2 , 100 ⁇ M [ ⁇ - 32 P] (2- 5dpm/fmol), 0.2 ⁇ g of heat-stable inhibitor of cAMP-dependent protein kinase, and 0.6mg/ml of Sigma type III— S calf thymus histone.
  • Immunoprecipitation and autophosphorylation assay Oocyte extracts were incubated with 10 ⁇ g of anti- ⁇ -PKC antibody and immunocomplexes were recovered with protein G-agarose. Immunoprecipitates were assayed for the autophosphorylation kinase activity of ⁇ -PKC in a mixture of 300 ⁇ M [ ⁇ - 32 P]ATP with or without 80nM Ca 2+ either in the absence or in the presence of 50 ⁇ g/ml phosphatidylserine (PS). Incubations were carried out with different concentrations of peptides A or Z (SEQ ID NOS 6 or 5 respectively). Reactions were stopped by 45min and proteins resolved by SDS-PAGE. No kinase activity was detected when oocyte extracts were incubated with protein G-agarose in the absence of antibody. Essentially identical results were obtained in three independent experiments.
  • the peptides of Examples 1 to 6 (SEQ ID NOS 1 to 5 and 17 respectively) were all synthesised using the Merrifield solid support method and were analysed by amino acid analysis and reverse phase HPLC. All peptides had an amino acid analysis as expected according to the structure and were shown to be at least 95% homogeneous (single peak was obtained by reverse phase HPLC). Peptide concentrations were calculated from the amino acid analysis data.
  • Example 7 PKC activity in X. laevis oocvtes and bovine brain
  • PKC has a sequence conserved in the pseudosubstrate region of PKC isotypes ⁇ , ⁇ and ⁇ , and therefore should be a good candidate inhibitor of these isotypes (Osada et aL J. Biol. Chem. 265, 22434 (1990)).
  • Peptides D, E and Z (SEQ ID NOS 7, 8 and 5 respectively) have sequences identical to the PKC pseudosubstrate regions of isoenzyme types ⁇ , ⁇ and ⁇ respectively, which significantly differ from that of isotypes ⁇ , ⁇ or ⁇ (Osada ej_aj. (1990 loc. cit - see legend to Figure 1).
  • Oocytes were therefore microinjected with a mixture of three peptides corresponding to the conserved pseudosubstrate regions of PKC isotypes ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , thought to be good candidate inhibitors of these isotypes, after which they were microinjected with 25 ⁇ U of B. cereus PC-PLC, or with 20ng of transforming v-H-ras p21 , or they were incubated with insulin (1 ⁇ M) or progesterone (1 ⁇ M). Control oocytes were microinjected with bovine brain PKC and incubated, afterwards, with 100ng/ml of PMA.
  • This probe encompasses the regulatory portion of the enzyme, including the cysteine-rich domain. From the sequence of this clone it is clear that the Xenopus ⁇ -PKC displays a 92% identity at the aminoacid level to its rat brain homologue. All the important signatures of the enzyme, including the cysteine-rich and ATP-binding domains are perfectly well conserved. Interestingly, the sequence corresponding to the pseudosubstrate region is 100% identical at the aminoacid level to that from its rat brain homologue.
  • ⁇ nPKC PKC isozyme
  • Results from Figure 1 demonstrate that oocytes microinjected with a synthetic peptide having a sequence identical to the PKC isotype ⁇ pseudosubstrate region (SEQ ID NO.5) did not respond to the activation with insulin, jas p21 , or PC-PLC although they did respond to the addition of progesterone.
  • the ability of PMA to stimulate H1 -kinase in PKC-microinjected oocytes was not affected by the microinjection of this peptide.
  • ⁇ -PKC is a specific critical step in mitogenic signal transduction in response to insulin/ra ⁇ p21 /PC-PLC.
  • the fact that the progesterone - activated maturation program is not affected by this peptide is a good control of the specificity of its effect on the insulin pathway.
  • microinjection of transforming v-H-ras p21 , or B. cereus PC-PLC promote a potent maturation response comparable to that produced by the addition of insulin or progesterone.
  • Addition of PMA does not induce GVBD unless oocytes were previously microinjected with purified bovine brain PKC, consistent with the data on H1 kinase.
  • microinjection of peptide A inhibited PMA-induced maturation in PKC-microinjected oocytes but produced little or no effect on the induction of GVBD in response to insulin/ras p21/PC-PLC or progesterone.
  • oocytes Groups of 20 oocytes were cultured at 20°C in modifed Barth solution following different treatments and germinal vesicle (nuclear) breakdown (GVBD) was assessed by the appearance of a white spot in the animal pole, in some cases, nuclear breakdown was confirmed by dissection of trichloroacetic acid (10%)-fixed oocytes.
  • Oocytes were either microinjected with buffer control or with 1 ng of purified bovine PKC. Afterwards, corresponding oocytes were microinjected with either water, or peptides A or Z (5 ⁇ M final concentration into the oocyte), respectively.
  • PKC RNAs were microinjected with 25 ⁇ U of B.cereus PC- PLC, or with 20ng of transforming v-H-ras p21 , or they were incubated with insulin (1 ⁇ M) or progesterone (1 ⁇ M).
  • Results from Figure 5A clearly demonstrate that ⁇ -PKC- depleted oocytes responded less efficiently to the activation with insulin, ⁇ as p21 , or PC-PLC although they gave a full response to the addition of progesterone. This strongly suggests that ⁇ -PKC is a specific critical step in maturation signal transduction in response to insulin/ras p21/PC- PLC.
  • oligonucleotides are a potential method utilized to inhibit the expression of proteins in oocytes (Sumikawa and Miledi, Proc. Natl. Acad. Sci. USA 85,1302 (1988)). Therefore, as an independent strategy to deplete ⁇ -PKC levels in oocytes we synthesized a 15-mer antisense oligonucleotide starting at the initiation codon of ⁇ -PKC as well as the corresponding sense and nonsense controls. To overcome degradation by nucleases, the oligonucleotides were modified on the backbone to phosphorothioates (Matsukura ej_aL, Proc. Natl. Acad, Sci.
  • Oocytes were microinjected with 150ng of either sense (SEQ ID NO 15), antisense (SEQ ID NO 14) or nonsense oligonucleotides.
  • SEQ ID NO 15 sense
  • SEQ ID NO 14 antisense
  • nonsense oligonucleotides nonsense oligonucleotides
  • oocytes were microinjected with 25 ⁇ U of B.cereus PC-PLC, or with 20ng of transforming v-H-ras p21 , or they were incubated with insulin (1 ⁇ M) or progesterone (1 ⁇ M).
  • Results from Figure 5B clearly demonstrate that the ability of ⁇ -PKC-depieted oocytes to respond to insulin, jas p21 , or PC-PLC was significantly impaired although they did respond normally to the addition of progesterone.
  • Microinjection of sense or nonsense oligonucleotides did not affect the activation of H1 kinase by any of the stimuli tested in this study.
  • microinjection of transforming v-H-ras p21 , or B.cereus PC-PLC promote a potent maturation response comparable to that produced by the addition of insulin or progesterone.
  • depletion of ⁇ -PKC levels by microinjection of its antisense RNA dramatically inhibited GVBD induction in response to microinjection of transforming v-H-ras p21 , B.cereus PC-PLC or to the addition of insulin.
  • a good control of the specificity of this effect is that GVBD induction in response to progesterone was not affected by ⁇ -PKC depletion.
  • oocytes either control or with ⁇ -PKC levels depleted by microinjection of antisense RNA were cultured at 20°C in modified Barth solution following different treatments, and germinal vesicle (nuclear) breakdown (GVBD) was assessed by the appearance of a white spot in the animal pole. In some cases, nuclear breakdown was confirmed by dissection of trichloroacetic acid (10%)-fixed oocytes.
  • GVBD germinal vesicle (nuclear) breakdown
  • Peptide Z1 (SEQ ID NO. 5) was microinjected into mouse NIH-3T3 fibroblasts growing in the presence of 10% fetal calf serum, and DNA synthesis was determined as described in the legend to Table 4.
  • a nine- aminoacid peptide A (SEQ ID No. 6) with a sequence corresponding to the pseudosubstrate region of PKC isotypes ⁇ , ⁇ and ⁇ (see Figure 1) was also microinjected in parallel cultures.
  • Results from Table 4 demonstrate that microinjection of peptide Z1 (SEQ ID NO 5) dramatically inhibits DNA synthesis in proliferating NIH-3T3 fibroblasts, while peptide A (SEQ ID NO. 6) produced little or no effect.
  • Peptides A or Z (SEQ ID NOs 6 or 5 respectively) were microinjected along with a marker antibody into the cytoplasm of growing NIH-3T3 fibroblasts about 8-1 Oh following serum refeeding (beginning of S phase). Immediately after microinjection, bromo-deoxyuridine (Amersham International), an analogue of thymidine, was diluted
  • Example 18 -PKC pseudosubstrates inhibit growth of Swiss - 3T3 fibroblasts
  • Results from Table 5 demonstrate that microinjection of peptides based on the sequence of the pseudosubstrate region of ⁇ -PKC dramatically inhibited DNA synthesis in serum-stimulated Swiss 3T3 fibroblasts whereas peptides corresponding to the pseudosubstrate region of PKC isotypes ⁇ , b and ⁇ inhibited DNA synthesis much less potently.
  • a dose-response curve of tripeptides ALR (SEQ.ID.NO.9) and ARR (SEQ.ID.NO.1 ) is shown in Figure 8.
  • SEQ.ID.NO.9 ARR
  • Figure 8 A dose-response curve of tripeptides ALR (SEQ.ID.NO.9) and ARR (SEQ.ID.NO.1 ) is shown in Figure 8.
  • the concentration of peptides into the microinjection pipette was 8 ⁇ M. A 20 to 50-fold dilution occurs once the molecules enter the cell.
  • the growth properties of NIH-3T3 fibroblast cell lines overexpressing ⁇ - PKC were determined.
  • the cDNA of ⁇ -PKC was subcloned into a mammalian expression vector under the control of a potent transcriptional promoter (pRcCMV-lnvitrogen, USA) and a comparison of the growth rate of ⁇ -PKC- overexpressing clones (pRcCMV ⁇ 4 and pRcCMV ⁇ 6) was carried out under low- serum conditions (0.5% FCS), using a ras-transformed cell line as a positive control and the corresponding negative controls.
  • Results from Fig.9 show that clones pRcCMV ⁇ 4 and pRcCMV ⁇ displayed a dramatically increased degree of growth at 0.5% serum which is similar to that of the ⁇ a ⁇ transformed cell line. This indicates that overexpression of ⁇ -PKC decreases the requirement of mitogens for cell proliferation. Furthermore, according to the data from TABLE 7 both transfectants displayed lower doubling times and higher saturation densities as compared to controls.
  • the growth in monolayers was measured by plating 4 x 10 4 cells/culture dish
  • a sensitive autophosphorylation assay to determine ⁇ -PKC activity was developed by deletion of the regulatory domain of cloned rat brain ⁇ -PKC using conventional recombinant DNA techniques.
  • the cDNA of the permanently active ⁇ -PKC mutant thus produced was subcloned into the PMALc2 plasmid to obtain a recombinant fusion protein (MBP- ⁇ PKCdel).
  • MBP- ⁇ PKCdel recombinant fusion protein
  • Bacterial cultures containing this plasmid were induced and the protein purified by affinity chromatography on an amylose-sepharose column.
  • ⁇ -Protein kinase C activity was determined as described hereinbefore using myelin basic protein as phosphate acceptor.
  • Typical assay conditions were final reaction volume 40 * ⁇ l, enzyme 2 ⁇ l, buffer (HEPES 50mM, MgCI 2 10mM, CaCI 2 1 mM, EGTA 1 mM, ATP 10 ⁇ M final concentrations), ATP- ⁇ - 32 P 1 ⁇ Ci, myelin basic protein 2 ⁇ g, potential inhibitor e.g. peptide 1 ⁇ M.
  • Incubations at 30°C may be stopped after e.g. 10 mins by boiling in SDS sample buffer and the sample fractionated e.g. in SDS- PAGE. Gels were dried and exposed for two days.
  • Permanently activated ⁇ -PKC assays are at least ten times more sensitive than autophosphorylation assays described herein utilising native ⁇ -PKC. Typical results representative of three independent experiments are shown in Table 8.
  • PKC isoenzymes ⁇ , ⁇ and ⁇ are not involved in the maturation pathway activated by insulin/ras p21 /PC-PLC. Neither ⁇ nor ⁇ PKC isotypes appear to be involved, since no detectable amounts of these subspecies were found in stage VI oocytes. Consistent with these data is the fact that screening the oocyte cDNA library utilized to clone the Xenopus homologue of ⁇ -PKC with probes specific for isotypes ⁇ or ⁇ , reveal the complete absence of these PKC isotypes in Xenopus oocytes.
  • ⁇ -PKC is a critical step in the transduction of mitogenic signals funnelled by J2S p21 /PC-PLC specific pathway
  • MOLECULE TYPE Peptide SEQUENCE DESCRIPTION: Arg Lys Gly Ala Leu Arg Gin Lys Asn 1 5
  • MOLECULE TYPE Other nucleic acid (synthetic)
  • MOLECULE TYPE Other nucleic acid (synthetic)

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Abstract

Peptides répondant à la formule générale (I), dans laquelle X représente H ou un ou plusieurs acides aminés et J représente OH ou un ou plusieurs acides aminés, et leurs dérivés pharmaceutiquement acceptables, lesdits peptides contenant de 3 à 15 restes d'acides aminés. On a également prévu des oligonucléotides anti-sens et leurs dérivés correspondant à l'ADN codant pour la zeta-PKC, notamment l'oligonucléotide ayant la séquence GGTCCTGCTGGGCAT (SEQ ID NO 14). Les nouveaux peptides et oligonucléotides sont utilisables en médecine dans le traitement des troubles dont l'étiologie fondamentale est associée à l'activité de la zeta-PKC, par exemple les tumeurs, les troubles hyperprolifératifs et les infections virales. On décrit des procédés de préparation de ces nouveaux composés, des compositions pharmaceutiques contenant ceux-ci, et leur utilisation médicale.
EP93908891A 1992-04-06 1993-04-02 Inhibiteur de proteine-kinase c Withdrawn EP0592634A1 (fr)

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US5959096A (en) * 1992-03-16 1999-09-28 Isis Pharmaceuticals, Inc. Antisense oligonucleotides against human protein kinase C
US6339066B1 (en) 1990-01-11 2002-01-15 Isis Pharmaceuticals, Inc. Antisense oligonucleotides which have phosphorothioate linkages of high chiral purity and which modulate βI, βII, γ, δ, Ε, ζ and η isoforms of human protein kinase C
US5916807A (en) * 1992-03-16 1999-06-29 Isis Pharmaceuticals, Inc. Antisense oligonucleotides against human protein kinase C
US5681747A (en) * 1992-03-16 1997-10-28 Isis Pharmaceuticals, Inc. Nucleic acid sequences encoding protein kinase C and antisense inhibition of expression thereof
US6153599A (en) * 1992-03-16 2000-11-28 Isis Pharmaceuticals, Inc. Methoxyethoxy oligonucleotides for modulation of protein kinase C expression
US5948898A (en) * 1992-03-16 1999-09-07 Isis Pharmaceuticals, Inc. Methoxyethoxy oligonucleotides for modulation of protein kinase C expression
US5882927A (en) * 1992-03-16 1999-03-16 Isis Pharmaceuticals, Inc. Oligonucleotide inhibition of protein kinase C
US5885970A (en) * 1992-03-16 1999-03-23 Isis Pharmaceuticals, Inc. Antisense oligonucleotides against human protein kinase C
US5922686A (en) * 1992-03-16 1999-07-13 Isis Pharmaceuticals, Inc. Oligonucleotide modulation of protein kinase C
US6537973B1 (en) 1992-03-16 2003-03-25 Isis Pharmaceuticals, Inc. Oligonucleotide inhibition of protein kinase C
US6117847A (en) * 1992-03-16 2000-09-12 Isis Pharmaceuticals, Inc. Oligonucleotides for enhanced modulation of protein kinase C expression
US6235723B1 (en) 1992-03-16 2001-05-22 Isis Pharmaceuticals , Inc. Antisense oligonucleotide modulation of human protein kinase C-δ expression
AU673897B2 (en) * 1993-02-06 1996-11-28 Garvan Institute Of Medical Research Protein kinase C (iota)
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
US5648238A (en) * 1996-06-18 1997-07-15 Incyte Pharmaceuticals, Inc. Human protein kinase C inhibitor homolog
AU2002305269B2 (en) * 2001-04-27 2006-10-19 Cold Spring Harbor Laboratory Alleviation of the memory deficits and memory components of psychiatric dysfunctions by altering atypical PKM activity
DE60330985D1 (de) 2002-11-01 2010-03-04 Univ New York State Res Found Atypische protein-kinase-c-isoformen bei erkrankungen des nervensystems und krebs
US8592368B2 (en) 2003-12-19 2013-11-26 University Of South Florida JAK/STAT inhibitors and MAPK/ERK inhibitors for RSV infection
WO2006108270A1 (fr) * 2005-04-11 2006-10-19 Pharmagap Inc. Inhibiteurs de proteines kinases et leurs utilisations
JP2009502983A (ja) * 2005-08-05 2009-01-29 ファーマギャップ インコーポレイテッド タンパク質キナーゼｃイソフォームの抑制剤およびその使用
CA2659958A1 (fr) 2005-08-05 2007-02-15 Pharmagap Inc. Peptides cibles sur des isoforme c de proteine kinase et utilisation de ceux-ci
US7585865B2 (en) 2006-07-21 2009-09-08 The Penn State Research Foundation Protein kinase C zeta inhibition to treat vascular permeability
EP3333176A1 (fr) 2016-12-09 2018-06-13 Universite De Geneve Inhibiteurs peptidiques de protéine kinase c et leurs utilisations
EP3806880A1 (fr) 2018-06-12 2021-04-21 Université de Genève Inhibiteurs peptidiques de la protéine kinase c et leurs utilisations

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