EP1091739A1 - Procede pour inhiber l'expression de c-jun en utilisant les inhibiteurs de jak-3 - Google Patents

Procede pour inhiber l'expression de c-jun en utilisant les inhibiteurs de jak-3

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Publication number
EP1091739A1
EP1091739A1 EP99932145A EP99932145A EP1091739A1 EP 1091739 A1 EP1091739 A1 EP 1091739A1 EP 99932145 A EP99932145 A EP 99932145A EP 99932145 A EP99932145 A EP 99932145A EP 1091739 A1 EP1091739 A1 EP 1091739A1
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European Patent Office
Prior art keywords
cells
jun
alkyl
jak
halo
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EP99932145A
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German (de)
English (en)
Inventor
Fatih M. Uckun
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Parker Hughes Institute
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Uckun Faith M
Parker Hughes Institute
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Publication of EP1091739A1 publication Critical patent/EP1091739A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/94—Nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the protooncogene c-jun is the cellular counterpart of the v-jun oncogene of avian sarcoma virus 17.
  • C-jun expression is activated in response to a diverse set of DNA-damaging agents including ara-C, UV radiation, topoisomerase II inhibitors, alkylating agents, and ionizing radiation.
  • DNA-damaging agents including ara-C, UV radiation, topoisomerase II inhibitors, alkylating agents, and ionizing radiation.
  • c-jun may have important regulatory functions for cell cycle progression, proliferation, and survival. See Ryder, K., Lau, L. F., and Nathans, D. "A gene activated by growth factors is related to the oncogene v-jun," Proc Natl Acad Sci USA.
  • C-jun encodes the nuclear DNA-binding protein, JUN, that contains a leucine-zipper region involved in homo- and heterodimerization.
  • JUN protein dimerizes with another JUN protein or the product of c-fos gene and forms the activating protein- 1 (AP-1) transcription factor.
  • AP-1 activating protein- 1
  • JUN- JUN homodimers and JUN-FOS heterodimers preferentially bind to a specific heptameric consensus sequence found in the promoter region of multiple growth regulatory genes. Alterations of c-jun protooncogene expression can therefore modulate the transcription of several growth-regulators affecting cell proliferation and differentiation. See Ryder, K., Lau, L. F., and Nathans, D.
  • C-jun plays a pivotal role in Ras-induced transformation and has also been implicated as a regulator of apoptosis when de novo protein synthesis is required.
  • C-jun induction is required for ceramide-induced apoptosis and stress-induced apoptosis after UV exposure or other forms of DNA damage. This induction is thought to be triggered by activation of JUN-N-terminal kinases (JNKs) (also known as stress-activated protein kinases) which leads to enhanced c-jun transcription by phosphorylation of JUN at sites that increases its ability to activate transcription.
  • JNKs JUN-N-terminal kinases
  • JNK1 a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain
  • Cell. 76: 1025-37, 1994 Chen, Y. R, Wang, X., Templeton, D., Davis, R. J., and Tan, T. H. "The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation," JBiol Chem. 271: 31929-36, 1996.
  • PTK Protein tyrosine kinases
  • the invention provides a method comprising inhibiting c-jun expression in cells (e.g. mammalian or avian) by contacting the cells (in vitro or in vivo) with a substance that inhibits the activity of Janus family kinase 3 (JAK-3).
  • cells e.g. mammalian or avian
  • JNK-3 Janus family kinase 3
  • the invention also provides a therapeutic method for preventing or treating a pathological condition in a mammal (e.g. a human) wherein c-jun activation is implicated and inhibition of its expression is desired comprising administering to a mammal in need of such therapy, an effective amount of a substance that inhibits the activity of JAK-3.
  • a mammal e.g. a human
  • the invention also provides novel compounds of formula I as well as processes and intermediates useful for their preparation.
  • the invention also provides substances that are effective to inhibit JAK-3 for use in medical therapy (preferably for use in treating conditions that result from exposure to radiation or to chemical agents that cause DNA damage), as well as the use of substances that inhibit JAK-3 for the manufacture of a medicament for the treatment of a condition that is associated with exposure to radiation, or to chemical agents that cause DNA damage.
  • the inset shows the values for the c-y ' ww/GAPDH transcript expression ratios as determined with a Bio Rad Storage Phosphor Imager and corresponding SI values [B].
  • Effect of the PTK inhibitor genistein on induction of c-jun mRNA Cells were treated with 30 mg/ml of genistein for 24 hours at 37°C prior to exposure to 20 Gy ionizing radiation, c-jun expression levels were determined as in [A].
  • Figure 2. Radiation-induced activation of c-jun in BTK " DT-40 cells. Two representative experiments (shown in [A] and [B]) showing induction of c-jun mRNA expression by ionizing radiation in wild type (WT) and BTK " DT-40 cells.
  • Poly (A) + RNA was isolated from non-irradiated cells as well as irradiated cells (20 Gy, with a 2 hours post-radiation recovery period). Northern blots of 2 mg of poly (A) + were hybridized with c-jun probe (top panel), (-actin probe (middle panel in [A] only), and GAPDH probe (bottom panel). The inset below each panel shows the relative expression of c-jun normalized for RNA load (c-jun/GAPDH ratio) and SI (fold induction over non-irradiated controls).
  • FIG. 3 Induction of c-jun mRNA expression by ionizing radiation in wild type and mutant DT-40 cell lines.
  • DT-40, BTK DT-40, SYK “ DT-40 (shown in [A]), as well as LYN " DT-40 and LYN “ SYK " DT 40 cells (shown in [B]) were irradiated with 20 Gy and poly (A) + RNA (in [A]) or total RNA (in [B]) was harvested after a 2 hour recovery period. RNA from non-irradiated cells was used as a control.
  • FIG. 1 JAK-3 Inhibitors.
  • FIG. 1 Sf21 cells infected with baculo virus expression vectors for JAK-1 JAK-2 or JAK-3 were subjected to immunoprecipitation with anti-JAK antibodies.
  • JAK-1 shown in B.l
  • JAK-2 shown in B.2
  • JAK-3 shown in B.3 and B.4 which illustrate results from 2 independent experiments
  • FIG. 5 Effects of a JAK-3 inhibitor on c-jun induction in irradiated DT-40 cells.
  • Cells were treated with the quinazoline derivative 4-(3 ' -Bromo-4 ' -hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline ( 100 mg/ml) for 24 hours at 37 °C prior to exposure to 20 Gy ionizing radiation.
  • c-jun expression levels were determined as outlined in Figures 1-3.
  • the term “inhibit” means to reduce by a measurable amount, or prevent entirely; and the phrase “inhibit c-jun activation” includes the inhibition of RNA production and the inhibition of the production of the protein encoded by the RNA.
  • JAK-3 Janus family kinase 3
  • C-jun expression can be activated by exposure to chemical agents that damage DNA such as ara-C, a topoisomerase II inhibitors, or alkylating agents. C-jun activation can also result from exposure to ultraviolet radiation or ionizing radiation.
  • inhibitors of JAK-3 can be used to inhibit c-jun expression resulting from exposure to radiation or exposure to chemical agents.
  • the methods of the invention can be carried out in vitro. Such in vitro methods are also useful for studying the biological processes associated with cell response to DNA damaging agents.
  • the methods of the invention can also be carried out in vivo. Such methods can also be used to study the biological processes associated with cell response to DNA damaging agents, as well as for treating pathological conditions in mammals (e.g. humans) that result from exposure to DNA-damaging agents.
  • Pathological conditions that result from exposure to DNA- damaging agents include conditions that result from oxidative stress, such as tissue or organ (e.g. heart, liver, or kidney) damage, inflammation, and hair loss, as well as the negative effects that are produced by oxygen free radicals during chemotherapy. Oxidative stress may result from exposure to external agents, or may result from internal processes.
  • JAK-3 inhibitors are also useful for treating conditions resulting from the action of internally generated oxygen free radicals, such as aging and amyelotrophic lateral sclerosis (ALS).
  • the JAK-3 inhibitors may be administered prophylactically, i.e. prior to exposure to the DNA-damaging agent, or the JAK-3 inhibitors may be administered after exposure to the DNA damaging agent.
  • JAK-3 inhibitors useful in the methods of the invention include all compounds capable of inhibiting the activity of JAK-3, it being well known in the art how to measure a compounds ability to inhibit JAK-3, for example, using standard tests similar to the test described hereinbelow in Example 2 under the heading "Effects of a JAK-3 inhibitor on radiation-induced c-jun activation in DT40.cells.”
  • JAK-3 inhibitors that are useful in the methods of the invention include compounds of formula I:
  • X is HN, R n N, S, O, CH 2 , or R personallyCH;
  • R n is hydrogen, (C r C 4 )alkyl, or (C,-C 4 )alkanoyl;
  • R ⁇ R- 8 re eacn independently hydrogen, hydroxy, mercapto, amino, nitro,
  • R> and R 10 are each independently hydrogen, (C r C 4 )alkyl, (C,-C 4 )alkoxy, halo, or (C,-C 4 )alkanoyl; or R, and R 10 together are methylenedioxy; or a pharmaceutically acceptable salt thereof.
  • halo is fluoro, chloro, bromo, or iodo.
  • Alkyl, alkanoyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl” embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to.
  • (C 1 -C 4 )Alkyl includes methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, and sec-butyl;
  • (C,-C 4 )alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, and sec-butoxy;
  • (C,-C 4 )alkanoyl includes acetyl, propanoyl and butanoyl.
  • R Rs are each independently hydrogen, mercapto, amino, nitro, (C,- C 4 )alkyl, (C,-C 4 )alkoxy, (C,-C 4 )alkylthio, or halogen.
  • R g and R 10 are each independently hydrogen, (C,-C 4 )alkyl, halo, or (C,-C 4 )alkanoyl; or R, and R 10 together are methylenedioxy; or a pharmaceutically acceptable salt thereof.
  • JAK-3 inhibitors that are useful in the methods of the invention also include compounds of formula I as described in U.S. Patent Application Serial Number 09/087,479 (entitled Quinazolines For Treating Brain Tumor; filed 28 May 1998).
  • Preferred JAK-3 inhibitors include 4-(4'-hydroxylphenyl)-amino-
  • Substances that inhibit JAK-3 can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • the Substances may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the Substance may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • the Substance may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of the Substance.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the Substance may be incorporated into sustained-release preparations and devices. The Substances may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the Substance can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the Substance which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • a polyol for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like
  • vegetable oils nontoxic glyceryl esters, and suitable mixtures thereof.
  • suitable mixtures thereof can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the Substance in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the Substances may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • a dermatologically acceptable carrier which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the Substances can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Examples of useful dermatological compositions which can be used to deliver the Substances to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
  • Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
  • the concentration of the Substance in a liquid composition, such as a lotion will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.
  • the concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
  • the amount of the Substance required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
  • the Substance is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
  • the Substance should be administered to achieve peak plasma concentrations of from about 0.5 to about 75 ⁇ M, preferably, about 1 to 50 ⁇ M, most preferably, about 2 to about 30 ⁇ M. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the Substance, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the Substance. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the Substance.
  • the Substance 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 sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
  • 4,5-Dimethoxy-2-nitrobenzoic acid (3) was treated with thionyl chloride and then reacted with ammonia to give 4,5-dimethoxy-2-nitrobenzamide (4) as described by F. Nomoto et al. Chem. Pharm. Bull. 1990, 38, 1591-1595.
  • the nitro group in compound (4) was reduced with sodium borohydride in the presence of copper sulfate (see C.L. Thomas Catalytic Processes and Proven Catalysts Academic Press, New York (1970)) to give 4,5-dimethoxy-2-aminobenzamide (5) which was cyclized by refluxing with formic acid to give 6,7-dimethoxyquinazoline-4(3H)-one (6).
  • Compound (6) was refluxed with phosphorus oxytrichloride to provide the common synthetic precursor (7).
  • PTK Inhibitors Cells (2 x 10 6 /ml) were treated for 24 hours at 37 °C with either (1) the PTK inhibitory isoflavone genistein (Calbiochem, La Jolla, CA) at 111 mM (30 mg/ml) concentration or (2) the Janus family kinase, 3 (JAK-3)-specific PTK inhibitor
  • Primer sequences were determined based upon the sequence of chicken c-jun (GenBank accession code CHKJUN). Two primers: 5'-ACTCTGCACC CAACTACAACGC-3' (SEQ. ID NO: 1) and 5'-CTTCTACCGT CAGCTTTACGCG-3' (SEQ LD NO: 2) were used for amplification. Amplification was performed with a mix of Taq polymerase and a proofreading polymerase (eLONGase:7 ⁇ polymerase plus Pyrococcus species GB-D polymerase, Gibco BRL, Grand Island, NY) on an thermocycler, Ericomp Delta II cycler, using a hot start.
  • a proofreading polymerase eLONGase:7 ⁇ polymerase plus Pyrococcus species GB-D polymerase, Gibco BRL, Grand Island, NY
  • PCR products were subsequently cloned into the cloning vector, PCR 2.1 (Invitrogen, San Diego, CA).
  • An insert of the proper size (506 basepair) was identified as chicken c-jun by sequence analysis using PRISM dye terminator cycle sequencing (AmpliTaq ® DNA Polymerase, FS) and analyzed on an automated sequencer, ALF express sequencer (Pharmacia Biotech, Piscataway, NJ).
  • a 538 base pair chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe was generated by reverse transcription and subsequent PCR amplification (RT-PCR) from chicken RNA with the following primers: 5'-AGAGGTGCTGCCCAGAACATCATC-3' (SEQ LD NO: 3) and 5'-GTGGGGAGACAGAAGGGAACAGA-3' (SEQ LD NO: 4).
  • a 413 bp chicken B-actin probe was generated by RT-PCR amplification from chicken RNA with the following primers: 5 '-GCCCTCTTCCAGCATCTTTCTT-3 ' (SEQ ID NO: 5) and 5'-TTTATGCGCATTTATGGGTT-3' (SEQ LD NO: 6).
  • the amplified cDNAs were cloned into PCR 2.1.
  • RNA isolation and Northern blot hybridization analysis Total RNA was extracted from approximately 2.5 x 10 7 cells with Trizol Reagent, a monophasic solution of phenol and guanidine isothiocyanate as described by Chomcznski, P. and Sacchi, N. "Single-step method of RNA isolation by guanidinium-thiocyanate-phenol-chloroform extraction," Anal. Biochem. 162: 156-159, 1987.
  • Poly (A) + RNA was isolated directly from 1-3 x 10 8 cells with an Invitrogen Fast Trak 2.0 mRNA isolation kit. In brief, cells were lysed in a sodium dodecyl sulfate (SDS) lysis buffer containing a proprietary mixture of proteases. The lysate was directly incubated with oligo-dT for absorption and subsequent elution of poly (A) + RNA.
  • SDS sodium dodecyl sulfate
  • SSC sodium citrate
  • the c-jun fragment was radiolabeled by random priming with [(- 32 P]-dCTP (3000 Ci/mM) [Amersham, Arlington Heights, LL] (40).
  • Northern blots were hybridized overnight at 42 °C in prehybridization/hybridization solution (50% formamide with proprietary blocking and background reduction reagents; Ambion, Austin, TX) for 16-24 hours and unbound probe was removed by washing to a final stringency of 0.1% SDS, 0.1XSSC (65 °C). The blots were analyzed both by autoradiography and using the BioRad Storage Phosphor Imager System
  • Exposure of DT-40 chicken lymphoma B-cells to ionizing radiation activates the c-jun protooncogene. Exposure of human lymphoma B-cells to 10-20 Gy-rays results in enhanced c-jun expression with a maximum response at 1-2 hours (Chae, H. P., Jarvis, L. J., and Uckun, F. M. Cancer Res. 53: 447-51 , 1993). It has also been reported that ionizing radiation triggers in DT-40 chicken lymphoma B-cells biochemical and biological signals similar to those in human lymphoma B-cells (Uckun, F. M., Waddick, K.
  • DT-40 chicken lymphoma B-cells show a similar c-jun response to ionizing radiation
  • DT-40 cells were irradiated with 5,10,15 or 20 Gy and examined total RNA harvested from cells 2 or 4 hours after radiation exposure for expression levels of 1.8 kb chicken c-jun transcripts by quantitative Northern blot analysis.
  • Cytoplasmic protein tyrosine kinases BTK, LYN, and SYK are not required for radiation induced c-jun activation.
  • BTK is abundantly expressed in lymphoma B-cells and its activation has been shown to be required for radiation-induced apoptosis of DT-40 cells (Uckun, F. M., Waddick, K. G., Mahajan, S., Jun, X., Takata, M., Bolen, J., and Kurosaki, T. Science. 273: 1096-100, 1996).
  • DT-40 cells rendered BTK-deficient by targeted disruption of the BTK genes do not undergo apoptosis after radiation exposure.
  • BTK BTK-deficient DT-40 cells
  • 20 Gy ionizing radiation did not fail to induce c-jun expression in BTK-deficient DT-40 cells in any of the three independent experiments performed.
  • ionizing radiation-induced increases in c-jun transcript levels do not depend upon the presence of BTK.
  • SYK is also abundantly expressed in DT-40 cells and is rapidly activated after ionizing radiation.
  • SYK might be the PTK responsible for radiation-induced increases in c-jun transcript levels.
  • 20 Gy ionizing radiation enhanced c-jun expression in SYK ' DT-40 cells rendered SYK-deficient by targeted gene disruption even though the stimulation indices observed in five independent experiments were lower than from those in wild-type cells (1.9 ⁇ 0.2, vs 2.9 ⁇ 0.4, p ⁇ 0.01).
  • SYK is not required for radiation-induced c-jun activation in DT-40 cells but it may participate in generation of an optimal signal.
  • DT-40 cells express high levels of LYN but do not express other members of the Src PTK family, including BLK, HCK, SRC, F YN, or YES at detectable levels (see Uckun, F. M., Waddick, K. G., Mahajan, S., Jun, X., Takata, M., Bolen, J., and Kurosaki, T. Science. 273: 1096-100, 1996; Kurosaki, T., Johnson, S. A., Pao, L., Sada, K., Yamamura, H., and Cambier, J. C. "Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling," J. Exp. Med.
  • LYN-deficient (LYN-) cells showed enhanced c-jun expression after irradiation, however the stimulation indices were lower than those in wild-type DT-40 ( Figure 3B). Since LYN and SYK have been shown to cooperate in the generation of other signals in B-cells (see Kurosaki, T. "Molecular mechanisms in B cell antigen receptor signaling," Curr Opin Immunol. 9: 309-18, 1997), the ability of ionizing radiation to induce c-jun expression in LYN ⁇ SYK- DT-40 cells, generated by targeted disruption of the syk gene in LYN " deficient DT-40 cells was examined.
  • LYN " SYK " DT-40 cells showed elevated c-jun transcript levels after irradiation, indicating that the c-jun response does not depend on either of these PTK, either alone or in cooperation. Similar to SYK, LYN is not required for radiation-induced c-jun activation in DT-40 cells but it may participate in generation of an optimal response.
  • STAT proteins are a family of DNA binding proteins that were identified during a search for interferon (JFK) a- or g-stimulated gene transcription targets. There are presently seven STAT family members.
  • JFK interferon
  • the JAK family of cytoplasmic protein kinases were originally demonstrated to also function in LFN signaling, and are now known to participate in a broad range of receptor-activated signal cascades.
  • STAT activation suggests that in unstimulated cells, latent forms of STATs are predominantly localized within the cytoplasm. Ligand binding induces STAT proteins to associate with intracellular phosphotyrosine residues of transmembrane receptors. Once STATs are bound to receptors, receptor-associated JAK kinases phosphorylate the STAT proteins. STAT proteins then dimerize through specific reciprocal SH2-phosphotyrosine interactions and may form complexes with other DNA-binding proteins. STAT complexes translocate to the nucleus and interact with DNA response elements to enhance transcription of target genes. Signaling events regulating apoptotic responses have been shown to utilize STAT proteins.
  • JAKs were immunoprecipitated with appropriate antibodies (anti- JAK-1 : (HR-785), cat# sc-277, rabbit polyclonal IgG affinity purified, 0.1 mg/ml, Santa Cruz Biotechnology; anti- JAK-2: (C-20)-G, cat # sc-294-G, goat polyclonal IgG affinity purified, 0.2 mg/ml, Santa Cruz Biotechnology; anti-JAK-3: (C-21), cat # sc-513, rabbit polyclonal IgG affinity purified, 0.2 mg/ml, Santa Cruz Biotechnology), and kinase assays were performed following a 1 hour exposure of the immunoprecipitated Jaks to the quinazoline compounds, as described by Uckun, F.
  • Electrophoretic Mobility Shift Assays were performed to examine the effects of both compounds on cytokine-induced STAT activation. Specifically, 32Dcl l/IL2R ⁇ cells (gift from James Lhle, St. Jude Children's Research Hospital) were exposed at 8 x 10 6 /ml in RPMI supplemented with FBS to the JAK-3 inhibitors at a final concentration of 10 ⁇ g ml in 1% DMSO) for 1 hour and subsequently stimulated with IL2 or IL3 as indicated.
  • ESAs Electrophoretic Mobility Shift Assays
  • lysis buffer 100 mM Tris-HCl pH 8.0, 0.5% NP-40, 10% glycerol, 100 mM EDTA, 0.1 mM NaVO3, 50 mM NaF, 150 mM Nacl, 1 mM DTT, 3 (g/ml Aprotinin, 2 g/ml Pepstatin A, 1 (g/ml Leupeptin and 0.2 mM PMSF). Lysates were precleared by centrifugation for 30 minutes.
  • JAK-3 inhibitors may be useful to prevent or treat diseases or conditions that result from exposure to DNA- damaging agents.
  • JAK-3 maps to human chromosome 19pl2-13.1. A cluster of genes encoding protooncogenes and transcription factors is also located near this region. JAK-3 expression has been demonstrated in mature B-cells as well as B-cell precursors. JAK-3 has also been detected in leukemic B-cell precursors and lymphoma B-cells. The physiological roles for JAK-3 have been borne out through targeted gene disruption studies in mice, the genetic analysis of patients with severe combined immunodeficiency, and biochemical studies of JAK-3 in cell lines. A wide range of stimuli result in JAK-3 activation in B-cells, including interleukin 7 and interleukin 4.
  • the B-cell marker CD40 constitutively associates with JAK-3 and ligation of CD40 results in JAK-3 activation which has been shown to be mandatory for CD40-mediated gene expression.
  • Constitutive activity of JAK-3 has been observed in v-abl transformed pre-B cells and coimmunoprecipitations show that v-abl physically associates with JAK-3 implicating JAK-3 in v-abl induced cellular transformation.
  • JAK-3 constitutive activity of JAK-3 has been observed in v-abl transformed pre-B cells and coimmunoprecipitations show that v-abl physically associates with JAK-3 implicating JAK-3 in v-abl induced cellular transformation.
  • Jak-STAT signaling induced by the v-abl oncogene Science 269, 1875-7, 1995.
  • JAKs Janus family kinase 3
  • JAK-3 inhibitors are useful for preventing or treating diseases or conditions that result from chemical-induced or radiation-induced c-jun activation.

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Abstract

L'invention concerne l'utilisation d'un composé qui inhibe l'activité de la kinase de la famille Janus (JAK-3) ou de son sel pharmaceutiquement acceptable, de même que la fabrication d'un médicament destiné à inhiber l'activation de c-jun dans des cellules mammaliennes ou aviaires.
EP99932145A 1998-06-30 1999-06-30 Procede pour inhiber l'expression de c-jun en utilisant les inhibiteurs de jak-3 Withdrawn EP1091739A1 (fr)

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US9115098P 1998-06-30 1998-06-30
US91150P 1998-06-30
PCT/US1999/014923 WO2000000202A1 (fr) 1998-06-30 1999-06-30 Procede pour inhiber l'expression de c-jun en utilisant les inhibiteurs de jak-3

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JP2004504259A (ja) 2004-02-12

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