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WO1997044467A1 - PROTEINE KINASE p38-2 ACTIVEE PAR MITOGENE ET MODES D'UTILISATION DUDIT PRODUIT - Google Patents

PROTEINE KINASE p38-2 ACTIVEE PAR MITOGENE ET MODES D'UTILISATION DUDIT PRODUIT Download PDF

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Publication number
WO1997044467A1
WO1997044467A1 PCT/US1997/008738 US9708738W WO9744467A1 WO 1997044467 A1 WO1997044467 A1 WO 1997044467A1 US 9708738 W US9708738 W US 9708738W WO 9744467 A1 WO9744467 A1 WO 9744467A1
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Prior art keywords
polypeptide
agent
kinase
substrate
cascade
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PCT/US1997/008738
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English (en)
Inventor
Bernd Stein
Maria X. H. Yang
David B. Young
Miguel S. Barbosa
Francesco Belardetti
M. A. Wilk-Blaszczak
Melanie Cobb
Original Assignee
Signal Pharmaceuticals, Inc.
University Of Texas Southwestern Medical Center At Dallas
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Priority claimed from US08/651,940 external-priority patent/US5948885A/en
Application filed by Signal Pharmaceuticals, Inc., University Of Texas Southwestern Medical Center At Dallas filed Critical Signal Pharmaceuticals, Inc.
Priority to JP54275097A priority Critical patent/JP2002515744A/ja
Priority to AU31397/97A priority patent/AU736316B2/en
Priority to CA002255579A priority patent/CA2255579A1/fr
Priority to EP97926689A priority patent/EP0914450A1/fr
Publication of WO1997044467A1 publication Critical patent/WO1997044467A1/fr

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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
    • 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)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to compositions and methods useful for the study of mitogen-activated protein kinase cascades and for treating conditions associated with such cascades.
  • the invention is more particularly related to a mitogen-activated protein kinase p38-2, and variants thereof that may be activated by bradykinin to stimulate phosphorylation and activation of substrates, such as ATF2.
  • the present invention is also related to the use of such polypeptides to identify antibodies and other agents that inhibit signal transduction via the p38-2 kinase cascade. Such agents may be used, for example, to reduce pain sensations.
  • MAPKs Mitogen-activated protein kinases
  • MAPKs are members of conserved signal transduction pathways that activate transcription factors, translation factors and other target molecules in response to a variety of extracellular signals.
  • MAPKs are activated by phosphorylation at a dual phosphorylation motif with the sequence Thr-X- Tyr by mitogen-activated protein kinase kinases (MAPKKs).
  • MAPKKs mitogen-activated protein kinase kinases
  • the physiological role of MAPK signaling has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAPK signaling, such as inflammatory diseases, autoimmune diseases and cancer.
  • MKK3 appears to be specific for p38 (i.e., does not activate JNK or ERK), while MKK4 activates both p38 and JNK (see Derijard et al., Science 267:682-685, 1995).
  • the third MAPKK, MEK6 appears to be a stronger and more specific in vivo stimulator of p38 phosphorylation (see U.S. Patent Application Serial Number 08/576,240).
  • These proteins appear to have utility in therapeutic methods for treating conditions associated with the p38 signal transduction pathway.
  • the present invention fulfills these needs and further provides other related advantages.
  • the present invention provides compositions and methods employing a mitogen-activated protein kinase (MAPK) p38-2, or a variant thereof.
  • MPK mitogen-activated protein kinase
  • the present invention provides polypeptides capable of activating a substrate of p38-2.
  • the polypeptides may comprise an amino acid sequence recited in SEQ ID NO:2, or a variant thereof.
  • the polypeptide is selectively activated by bradykinin.
  • the present invention also provides a polypeptide comprising the amino acid sequence recited in SEQ ID NO:2, modified at no more than 25% of the amino acid residues, such that said polypeptide is rendered constitutively inactive.
  • the present invention provides isolated DNA molecules encoding polypeptides as described above, as well as recombinant expression vectors comprising such DNA molecules and host cells transformed or transfected with such expression vectors.
  • the present invention provides methods for phosphorylating a substrate of p38-2, comprising contacting a polypeptide as described above with a substrate of p38-2, thereby phosphorylating the substrate of p38-2.
  • methods for activating a substrate of p38-2 in a patient comprising administering to a patient a polypeptide as described above in combination with a pharmaceutically acceptable carrier, thereby activating a substrate of p38-2.
  • the present invention provides methods for screening for an agent that modulates signal transduction via the p38-2 cascade.
  • the method comprises: (a) contacting a candidate agent with a polypeptide as described above, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact; and (b) subsequently measuring the ability of the candidate agent to modulate kinase activity of said polypeptide.
  • the method comprises: (a) contacting a candidate agent with a polynucleotide encoding a polypeptide according to either of claims 1 or 4, wherein the step of contacting is carried out under conditions and for a time sufficient to allow generation of the polypeptide and interaction between the polypeptide and the candidate agent; and (b) subsequently measuring the ability of the candidate agent to modulate p38-2 activity.
  • the present invention provides antibodies that bind to a polypeptide as described above.
  • methods are provided for treating a condition associated with the p38-2 cascade, comprising administering to a patient a therapeutically effective amount of an agent that modulates signal transduction via the p38-2 cascade.
  • an agent may modulate p38-2 kinase activity and/or may modulate phosphorylation of p38-2.
  • such methods may reduce a pain sensation in a patient.
  • the present invention provides methods and kits for detecting mitogen activated protein kinase kinase activity in a sample.
  • kits comprise p38-2 in combination with a suitable buffer.
  • Figure 1 presents the primary amino acid sequence of p38-2, and splice variants thereof, using standard one-letter codes.
  • Figures 2A and 2B are autoradiograms that depict Northern blot analyses of the expression of human p38-2 ( Figure 2A) and p38 ( Figure 2B) mRNA in selected human tissues. The position of RNA size markers in kb is shown on the left.
  • Figure 3 is an autoradiogram that shows the size of in vitro translated HA-tagged p38-2, as determined by SDS-PAGE. The position of protein size markers in kDa is shown on the left.
  • Figure 4 is an autoradiogram presenting the relative levels of p38-2 kinase activity in COS cells transiently infected with epitope tagged p38-2 (lanes 1 to 7) and treated for 45 minutes with UV (250 nm, 120 J/m 2 ; lane 2), anisomycin (50 ng/ml; lane 3), or NaCl (200 ⁇ M; lane 4), or cotransfected with 1000 ng of the empty expression vector Sr ⁇ 3 (lane 5), the expression vector for the constitutively active mutant MEK6(DD) (lane 6) or the MAPK TAK1 ⁇ N (lane 7).
  • Figure 5 presents the nucleotide and amino acid sequence of a native p38-2 polypeptide.
  • Figure 6 is an immunoblot showing the levels of p38 and BRK detected in NG108-15 cells with polyclonal antibodies.
  • the endogenous p38 and BRK proteins are shown in the lanes identified as lysate.
  • antibodies were raised against the full length p38 protein, and recombinant p38 was used as a standard to identify the species that migrates as a band of 38 kD.
  • antibodies were raised against a unique small peptide derived from BRK, and BRK protein generated by in vitro transcription and translation in the present of 35 S-methionine migrates as a band of about 40 kD.
  • Figure 7 is an autoradiogram showing the results of an immunoprecipitation assay to evaluate the level of BRK in NG108-15 cells following treatment with bradykinin.
  • Lanes 1-5 show the levels in untreated cells at 1 , 2, 5, 15 and 30 minutes, respectively.
  • Lanes 6-10 show the levels in cells treated with 1 ⁇ M bradykinin for 1, 2, 5, 15 and 30 minutes, respectively.
  • Figures 8A-D show the leakage- and capacitance-subtracted current traces observed in NG108-15 cells in the presence and absence of inhibitors.
  • Figures 9A and 9B show peak I Ca V - voltage relations in two NG108-15 cells before (open circles) and during (closed circles) application of bradykinin (0.1 ⁇ M) after intracellular dialysis of SKF 106978 ( Figure 9 A; 20 ⁇ M) or SB2O3580 ( Figure 9B; 20 ⁇ M).
  • Figure 9C presents the time course of the peak I Ca v during perfusion with SB203580 (20 ⁇ M) and application of bradykinin and Leu-Enk (both at 0.1 ⁇ M). Between ) and 20 minutes, I Ca V was activated every 30 s. In the remaining portion of the time course, I Ca V was activated every 10 s. Data were acquired at 10 kHz.
  • Figure 10A shows the activation of I K BK by BK (0.1 ⁇ M) obtained from two NG108-15 cells, after intrapipette dialysis with SB203580 (20 ⁇ M, panel A,) or SKF 106978 (20 ⁇ M, panel A 2 ). Data were acquired at 100 Hz.
  • Figure 10B is a graph showing a summary of the I K BK responses to BK (0.1 ⁇ M). The graph displays means and standard deviations.
  • Figure IOC presents a proposed pathway for the inhibition of I Ca V by BK.
  • the present invention is generally directed to compositions and methods for modulating (i.e., stimulating or inhibiting) signal transduction via MAP kinase cascades, and for treating conditions associated with such cascades.
  • the present invention is directed to compositions comprising a MAP kinase p38-2 or a polypeptide variant thereof, and to the use of such compositions for activating substrates of p38-2 and for identifying modulators of p38-2 activity.
  • the term "p38-2 polypeptide” encompasses native p38-2 sequences, as well as variants thereof.
  • a p38-2 polypeptide is selectively activated by bradykinin.
  • a p38-2 polypeptide When active, a p38-2 polypeptide is generally capable of activating at least one substrate of p38-2 (e.g., ATF-2, MAPKAP kinase 2, MAPKAP kinase 3, MNKl, PHAS-1 and/or Sapl-a).
  • a substrate of a p38-2 polypeptide is said to be "activated” if its biological or enzymatic activity increases by a statistically significant amount.
  • Variants of a native p38-2 sequence are modified such that the ability of the variant to phosphorylate substrates is not substantially diminished.
  • the present invention also encompasses compositions and methods for modulating p38-2 activity.
  • compositions that inhibit p38-2 activity may inhibit phosphorylation of p38-2, or may inhibit the ability of p38-2 to phosphorylate a substrate.
  • p38-2 cascade refers to any signal transduction pathway that involves p38-2, and such a cascade may include any compound that modulates p38-2 activity or acts as a substrate for p38-2.
  • p38-2 polypeptide variants within the scope of the present invention may contain one or more substitutions and/or modifications, such that the ability of the variant to phosphorylate substrates (such as ATF2, MAPKAP kinase 2 and MAPKAP kinase 3) is not substantially diminished.
  • a variant contains substitutions and/or modifications at no more than 25% of the amino acid residues, more preferably at no more than 20% of the amino acid residues and most preferably at no more than 10% of residues. Such substitutions, which are preferably conservative, may be made in non-critical and/or critical regions of the native protein. Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the activity of the polypeptide. In particular, variants may contain additional amino acid sequences at the amino and/or carboxy termini. Such sequences may be used, for example, to facilitate purification or detection ofthe polypeptide.
  • p38-2 polypeptides may be selectively activated by bradykinin.
  • Bradykinin BK inhibits neurotransmitter voltage-dependent calcium currents (I Ca V )-
  • I Ca V neurotransmitter voltage-dependent calcium currents
  • G, 3 and Racl/Cdc42 G proteins
  • selectively activated refers to a strong stimulation (i.e., at least three fold) of kinase activity of a p38-2 polypeptide under conditions that produce at most only a modest stimulation (i.e., between about 1.5 and 3 fold) of JNK and/or ERK activity and little or no measurable activation (i.e., less than 1.5 fold) of type 1 and/or type 3 p38 kinases.
  • the selective activation of a p38-2 polypeptide by bradykinin may be evaluated using immunoprecipitation assays and/or measurements of I Ca v . Measurements of I Ca v may be performed using a standard whole-cell patch-clamp technique.
  • At least two neurotransmitters modulate the amplitude of I Ca V in NG108-15 cells.
  • the effect of bradykinin, but not that of leu-enkephalin, is sensitive to the block of p38-2 activity.
  • These transmitters may be applied via a capillary tubing positioned near the NG108-15 cells.
  • the I Ca v can be recorded on magnetic tape or on computer disk for subsequent analysis.
  • Substitutions and/or modifications may also render the polypeptide constitutively active or inactive.
  • Constitutively active polypeptides display the ability to stimulate substrate phosphorylation in the absence of stimulation, as described below. Such polypeptides may be identified using the representative assays for p38-2 kinase activity described herein.
  • Constitutively inactive proteins are those which are unable to phosphorylate a substrate even when stimulated as described below. Proteins modified so as to be constitutively active or inactive may generally be used in replacement therapy for treatment of a variety of disorders, as discussed in more detail below.
  • DNA sequences encoding a native p38-2 polypeptide may be prepared by amplification from a suitable cDNA library, using polymerase chain reaction (PCR) and methods well known to those of ordinary skill in the art.
  • PCR polymerase chain reaction
  • an adapter- ligated cDNA library prepared from a cell line or tissue that expresses p38-2 (such as skeletal muscle or heart) may be screened using a 5' specific forward primer and an adapter-specific primer.
  • a 1.6 kb cDNA identified using a human cDNA library has the sequence provided in SEQ ID NO: 1 and Figure 5.
  • the encoded p38-2 polypeptide shown in SEQ ID NO:2 and Figure 1, has a predicted size of 364 amino acids, with a molecular weight of about 42 kD as determined by calculation and SDS- polyacrylamide gel electrophoresis.
  • p38-2 is 73% identical to its closest homolog ⁇ 38 (see, e.g., Han et al., Science 265:808-811 , 1994; Lee et al., Nature 572:739-746, 1994), and all kinase subdomains characteristic for MAP kinase family members are conserved.
  • Polypeptides of the present invention may be prepared by expression of recombinant DNA encoding the polypeptide in cultured host cells.
  • the host cells are bacteria, yeast, baculovirus-infected insect cells or mammalian cells.
  • the recombinant DNA may be cloned into any expression vector suitable for use within the host cell, using techniques well known to those of ordinary skill in the art.
  • An expression vector may, but need not, include DNA encoding an epitope, such that the recombinant protein contains the epitope at the N- or C-terminus.
  • Epitopes such as glutathione-S transferase protein (GST), HA (hemagglutinin)-tag, FLAG and Histidine- tag may be added using techniques well known to those of ordinary skill in the art.
  • DNA sequences expressed in this manner may encode a native p38- 2 polypeptide, or may encode alternate splice variants, portions or other variants of p38-2.
  • DNA molecules encoding variants of p38-2 may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides.
  • any such changes should not diminish the ability of the variant to stimulate phosphorylation of substrates such as ATF2 (see, e.g., Gupta et al., Science 267:389-393, 1995), MAPKAP kinase 2 (see, e.g., Rouse et al., Cell 7S:1027-1037, 1994 Ben Levy et al., EMBO J.:l 4:5920-6930, 1995) or MAPKAP kinase 3 (see e.g., McLaughlin et al., J. Biol. Chem. 277:8488-8492, 1996).
  • ATF2 see, e.g., Gupta et al., Science 267:389-393, 1995
  • MAPKAP kinase 2 see, e.g., Rouse et al., Cell 7S:1027-1037, 1994 Ben Levy et al., EMBO J.:l 4:
  • non-critical regions are regions of the native sequence that do not substantially change the properties of p38-2.
  • Non-critical regions may be identified by modifying the p38-2 sequence in a particular region and assaying the ability ofthe resulting variant in a kinase assay, using a suitable substrate, as described herein.
  • Modifications may also be made in critical regions of p38-2, provided that the resulting variant substantially retains the ability to stimulate substrate phosphorylation.
  • the effect of any modification on the ability of the variant to stimulate substrate phosphorylation may generally be evaluated using any assay for p38-2 kinase activity, such as the representative assays described herein.
  • Expressed polypeptides of this invention are generally isolated in substantially pure form.
  • the polypeptides are isolated to a purity of at least 80% by weight, more preferably to a purity of at least 95% by weight, and most preferably to a purity of at least 99% by weight.
  • purification may be achieved using, for example, the standard techniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis, and affinity chromatography.
  • p38-2 polypeptides for use in the methods of the present invention may be native, purified or recombinant.
  • p38-2 polypeptides may be used to identify agents, which may be antibodies or drugs, that modulate (preferably inhibit) signal transduction via the p38-2 cascade. Modulation includes the suppression of expression of p38-2 when it is overexpressed, as well as suppression of phosphorylation of p38-2 or the inhibition of the ability of activated (i.e., phosphorylated) p38-2 to phosphorylate a substrate.
  • a modulating agent may modulate the kinase activity of one or more MAPKKs, such as MEK6, thereby inhibiting p38-2 activation.
  • compositions that inhibit p38-2 activity by inhibiting p38-2 phosphorylation may include one or more agents that inhibit or block MAPKK activity, such as an antibody that neutralizes a MAPKK, a competing peptide that represents the substrate binding domain of a MAPKK or the dual phosphorylation motif of p38-2, an antisense polynucleotide or ribozyme that interferes with transcription and/or translation of a MAPKK, a molecule that inactivates a MAPKK by binding to the kinase, a molecule that binds to p38-2 and prevents phosphorylation by a MAPKK or a molecule that prevents transfer of phosphate groups from the kinase to the substrate.
  • modulating agents may be identified by combining a test compound with an activated p38-2 polypeptide, or a polynucleotide encoding such a polypeptide, in vitro or in vivo, and evaluating the effect of the test compound on the p38-2 kinase activity using, for example, a representative assay described herein.
  • An increase or decrease in kinase activity can be measured by adding a radioactive compound, such as [ ⁇ 32 P]-ATP, to the mixture of components, and observing radioactive incorporation into a suitable substrate for p38-2, to determine whether the compound inhibits or stimulates kinase activity.
  • a candidate agent may be included in a mixture of active p38-2 polypeptide and substrate (such as ATF2), with or without pre-incubation with one or more components of the mixture.
  • Activation of p38-2 may be achieved by any of a variety of means. Typically, activation involves the addition of a MAP kinase kinase, which may in turn be activated via stimulation as described above.
  • a suitable amount of antibody or other agent for use in such an assay ranges from about 0.1 ⁇ M to about 10 ⁇ M.
  • the effect of the agent on p38-2 kinase activity may then be evaluated by quantitating the incorporation of [ 32 P]phosphate into ATF2, and comparing the level of incorporation with that achieved using activated p38-2 without the addition of a candidate agent.
  • the incorporation of phosphate into ATF2 may be measured using an antibody specific for phosphorylated substrate, using well known techniques.
  • a polynucleotide encoding the kinase may be inserted into an expression vector and the effect of a composition on transcription of the kinase measured, for example, by Northern blot analysis.
  • a p38-2 polypeptide may be used for phosphorylating and activating a substrate of p38-2.
  • a substrate may be phosphorylated in vitro by incubating a p38-2 polypeptide with a substrate and ATP in a suitable buffer (described in more detail below) for 30 minutes at 30°C.
  • a suitable buffer described in more detail below
  • Any compound that can be phosphorylated by p38-2, such as ATF2, MAPKAP kinase 2 and MAPKAP kinase 3 may be used as a substrate.
  • the amounts of the reaction components may range from about 0.1 ⁇ g to about 10 ⁇ g of p38-2 polypeptide, from about 0.1 ⁇ g to about 10 ⁇ g of substrate, and from about 10 nM to about 500 nM of ATP.
  • Phosphorylated substrate may then be purified by binding to GSH-sepharose and washing. The extent of substrate phosphorylation may generally be monitored by adding [ ⁇ - 32 P]ATP to a test aliquot, and evaluating the level of substrate phosphorylation as described below.
  • p38-2 polypeptides, modulating agents as described above and/or polynucleotides encoding such polypeptides and/or modulating agents may also be used to modulate p38-2 activity in a patient.
  • a "patient” may be any mammal, including a human, and may be afflicted with a condition associated with the p38-2 cascade or may be free of detectable disease. Accordingly, the treatment may be of an existing disease or may be prophylactic.
  • Conditions associated with the p38-2 cascade include any disorder which is etiologically linked to MAP kinase activity, including cardiovascular disease, immune-related diseases (e.g., inflammatory diseases, autoimmune diseases, malignant cytokine production or endotoxic shock), cell growth- related diseases (e.g., cancer, metabolic diseases, abnormal cell growth and proliferation or cell cycle abnormalities) and cell regeneration-related diseases (e.g., cancer, degenerative diseases, trauma, environmental stress by heat, UV or chemicals or abnormalities in development and differentiation).
  • immune-related diseases e.g., inflammatory diseases, autoimmune diseases, malignant cytokine production or endotoxic shock
  • cell growth- related diseases e.g., cancer, metabolic diseases, abnormal cell growth and proliferation or cell cycle abnormalities
  • cell regeneration-related diseases e.g., cancer, degenerative diseases, trauma, environmental stress by heat, UV or chemicals or abnormalities in development and differentiation.
  • the high expression of p38-2 in heart tissue suggests an important role for p38-2 in cardiovascular diseases.
  • Immunological-related cell proliferative diseases appropriate for treatment with p38-2 polypeptides include osteoarthritis, ischemia, reperfusion injury, trauma, certain cancers and viral disorders, and autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, and other acute phase responses.
  • pain is also a condition associated with the p38-2 cascade, and that pain may be treated by inhibiting p38-2 kinase activity.
  • Bradykinin is a major mediator of pain, acting on the peripheral endings of dorsal root ganglion neurons.
  • One of bradykinin's actions in these cells is the inhibition of I Cg v , which allows a faster rate of action potential generation (the electrophysical substrate of the pain sensation). Pain within a patient may be reduced by inhibiting p38-2 kinase activity, without the vascular side effects of bradykinin antagonists and without the addictive risks of opioids.
  • Treatment may include administration of a p38-2 polypeptide and/or a compound which modulates p38-2 activity.
  • one or more polypeptides (and/or modulating agents) are generally formulated as a pharmaceutical composition.
  • a pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention.
  • a pharmaceutical composition may additionally contain preservatives and/or other additives such as, for example, antimicrobial agents, anti-oxidants, chelating agents and/or inert gases, and/or other active ingredients.
  • a pharmaceutical composition may comprise a polynucleotide encoding a p38-2 polypeptide, and/or modulating agent, such that the polypeptide and/or modulating agent is generated in situ, in combination with a physiologically acceptable carrier.
  • the polynucleotide may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, bacterial and viral expression systems, as well as colloidal dispersion systems, including liposomes.
  • Appropriate nucleic acid expression systems contain the necessary polynucleotide sequences for expression in the patient (such as a suitable promoter and terminating signal). DNA may also be "naked,” as described, for example, in Ulmer et al., Science 259:1745-1749 (1993).
  • the retroviral vector is a derivative of a murine or avian retrovirus including, but not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • a retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a gene that encodes the ligand for a receptor on a specific target cell (to render the vector target specific).
  • retroviral vectors can be made target specific by inserting a nucleotide sequence encoding a sugar, a glycolipid, or a protein. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.
  • Viral vectors are typically non-pathogenic (defective), replication competent viruses, which require assistance in order to produce infectious vector particles.
  • This assistance can be provided, for example, by using helper cell lines that contain plasmids that encode all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR, but that are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsulation.
  • helper cell lines include (but are not limited to) ⁇ 2, PA317 and PA 12.
  • a retroviral vector introduced into such cells can be packaged and vector virion produced.
  • the vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUV large unilamellar vesicles
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:11, 1981).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques 6:882, 1988).
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity and may be, for example, organ-specific, cell-specific and/or organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticuloendothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • compositions may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity or transdermally. Between 1 and 6 doses may be administered daily.
  • a suitable dose is an amount of polypeptide or polynucleotide that is sufficient to show improvement in the symptoms of a patient afflicted with a condition associated with the p38-2 cascade.
  • Such improvement may be detected based on a determination of relevant cytokine levels (e.g., IL-1, IL-6 and/or IL-8), by monitoring inflammatory responses (e.g., edema, transplant rejection, hypersensitivity) or through an improvement in clinical symptoms associated with the condition (e.g., pain).
  • relevant cytokine levels e.g., IL-1, IL-6 and/or IL-8
  • monitoring inflammatory responses e.g., edema, transplant rejection, hypersensitivity
  • clinical symptoms associated with the condition e.g., pain
  • the amount of polypeptide present in a dose, or produced in situ by DNA present in a dose ranges from about 1 ⁇ g to about 250 ⁇ g per kg of host, typically from about 1 ⁇ g to about 60 ⁇ g. Suitable dose sizes will vary with the size of the patient, but will typically range from about 10 mL to about 500 mL for 10-60 kg animal.
  • the present invention also provides methods for detecting the level of mitogen activated protein kinase kinase (such as MEK6) activity in a sample.
  • the level of MAPKK activity may generally be determined by evaluating the ability of the sample to phosphorylate a p38-2 polypeptide, thereby rendering the p38-2 polypeptide active (i.e., capable of phosphorylating in vivo substrates such as ATF2).
  • a kinase assay may be performed substantially as described in Derijard et al.. Cell 76:1025-1037, 1994 and Lin et al., Science 268:2X6-290, 1995, with minor modifications.
  • a sample may be incubated with p38-2 and [ ⁇ - 32 P]ATP in a suitable buffer (such as 20 mM HEPES (pH 7.6), 5 mM MnCl 3 , 10 mM MgCl 2 , 1 mM dithiothreitol) for 30 minutes at 30°C.
  • a suitable buffer such as 20 mM HEPES (pH 7.6), 5 mM MnCl 3 , 10 mM MgCl 2 , 1 mM dithiothreitol
  • a suitable buffer such as 20 mM HEPES (pH 7.6), 5 mM MnCl 3 , 10 mM MgCl 2 , 1 mM dithiothreitol
  • a suitable buffer such as 20 mM HEPES (pH 7.6), 5 mM MnCl 3 , 10 mM MgCl 2 , 1 mM dithiothreitol
  • Proteins
  • this assay may also be performed with unlabeled phosphate, using an antibody specific for phosphorylated substrate (i.e., the antibody binds to phosphorylated substrate, and does not bind significantly to unphosphorylated substrate, such that the antibody can be used to distinguish between the two forms) and standard methods.
  • an antibody specific for phosphorylated substrate i.e., the antibody binds to phosphorylated substrate, and does not bind significantly to unphosphorylated substrate, such that the antibody can be used to distinguish between the two forms
  • a coupled in vitro kinase assay may be performed using a substrate for p38-2, such as ATF2, with or without an epitope tag.
  • ATF2 for use in such an assay may be prepared as described in Gupta et al., Science 267:389-393, 1995.
  • the p38-2 following phosphorylation of p38-2 as described above, isolation of the protein by binding to GSH-sepharose and washing with 20 mM HEPES (pH 7.6), 20 mM MgCl 2 , the p38-2 (0.1-10 ⁇ g) may be incubated with ATF2 (0.1-10 ⁇ g) and [ ⁇ - 32 P]ATP (10-500 nM) in a buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl 2 . It should be noted that alternative buffers may be used and that buffer composition can vary without significant effect on kinase activity. Reactions may be separated by SDS-PAGE, visualized by autoradiography and quantitated using any of a variety of known techniques.
  • Activated p38-2 will be capable of phosphorylating ATF2 at a level of at least 5% above background using this assay.
  • a kinase assay may be performed as described above, except that a MAPKK, such as MEK6 (rather than a sample) is generally employed and the candidate modulating agent is added to the incubation mixture.
  • the candidate agent may be preincubated with MAPKK before addition of ATP and p38-2 polypeptide.
  • the p38-2 may be preincubated with the candidate agent before the addition of MAPKK.
  • Further variations include adding the candidate agent to a mixture of MAPKK and ATP before the addition of p38-2, or to a mixture of p38-2 and ATP before the addition of MAPKK, respectively. All these assays can further be modified by removing the candidate agent after the initial preincubation step. In general, a suitable amount of antibody or other candidate agent for use in such an assay ranges from about 0.1 ⁇ M to about 10 ⁇ M. The effect of the agent on phosphorylation of p38-2 may then be evaluated by quantitating the incorporation of [ 32 P]phosphate into p38, as described above, and comparing the level of incorporation with that achieved using MAPKK without the addition of the candidate agent.
  • p38-2 activity may also be measured in whole cells transfected with a reporter gene whose expression is dependent upon the activation of an appropriate substrate, such as ATF2.
  • appropriate cells i.e., cells that express p38-2
  • expression of the luciferase gene depends upon activation of ATF2 by p38-2, which may be achieved by the stimulation of MAPKK with an activator or by cotransfection with an expression vector that produces a constitutively active variant of MAPKK, such as MEK6.
  • Candidate modulating agents may be added to the system, as described above, to evaluate their effect on the p38-2 cascade.
  • a whole cell system may employ only the transactivation domain of ATF2 fused to a suitable DNA binding domain, such as GHF-1 or GAL4.
  • the reporter system may then comprise the GH-luciferase or GAL4-luciferase plasmid.
  • Candidate modulating agents may then be added to the system to evaluate their effect on ATF2-specif ⁇ c gene activation.
  • the present invention also provides methods for detecting the level of p38-2 polypeptide in a sample.
  • the level of p38-2, or nucleic acid encoding p38-2 may generally be determined using a reagent that binds to p38-2, or to DNA or RNA encoding p38-2.
  • a reagent that binds to p38-2, or to DNA or RNA encoding p38-2.
  • standard hybridization and/or PCR techniques may be employed using a nucleic acid probe or a PCR primer. Suitable probes and primers may be designed by those of ordinary skill in the art based on the p38-2 cDNA sequence provided in SEQ ID NO:l .
  • the reagent is typically an antibody, which may be prepared as described below.
  • the antibody may be immobilized on a solid support such that it can bind to and remove the polypeptide from the sample.
  • the bound polypeptide may then be detected using a second antibody that binds to the antibody/peptide complex and contains a detectable reporter group.
  • a competitive assay may be utilized, in which polypeptide that binds to the immobilized antibody is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample.
  • reporter groups for use in these methods include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • enzymes e.g., horseradish peroxidase
  • substrates e.g., cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • Antibodies encompassed by the present invention may be polyclonal or monoclonal, and may be specific for p38-2 and/or one or more variants thereof.
  • Preferred antibodies are those antibodies that inhibit or block p38-2 activity in vivo and within an in vitro assay, as described above.
  • antibodies and other agents having a desired effect on p38-2 activity may be administered to a patient (either prophylactically or for treatment of an existing disease) to modulate the activation of p38-2 in vivo.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • an immunogen comprising the polypeptide is initially injected into a suitable animal (e.g., mice, rats, rabbits, sheep and goats), preferably according to a predetermined schedule inco ⁇ orating one or more booster immunizations, and the animals are bled periodically.
  • a suitable animal e.g., mice, rats, rabbits, sheep and goats
  • Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
  • Monoclonal antibodies specific for p38-2 or a variant thereof may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:51 1-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal.
  • the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • kits for detecting p38-2 and p38-2 kinase activity, as well as MAPKK kinase activity are provided.
  • kits may be designed for detecting the level of p38-2 or nucleic acid encoding p38-2, or may detect kinase activity of p38-2 or MAPKK in a direct kinase assay or a coupled kinase assay, in which both the level of phosphorylation and the kinase activity of p38-2 may be determined.
  • MAPKK or p38-2 kinase activity may be detected in any of a variety of samples, such as eukaryotic cells, bacteria, viruses, extracts prepared from such organisms and fluids found within living organisms.
  • the kits of the present invention comprise one or more containers enclosing elements, such as reagents or buffers, to be used in the assay.
  • a kit for detecting the level of p38-2, or nucleic acid encoding p38-2 typically contains a reagent that binds to the p38-2 protein, DNA or RNA.
  • the reagent may be a nucleic acid probe or a PCR primer.
  • the reagent is typically an antibody.
  • kits also contain a reporter group suitable for direct or indirect detection of the reagent (i.e., the reporter group may be covalently bound to the reagent or may be bound to a second molecule, such as Protein A, Protein G, immunoglobulin or lectin, which is itself capable of binding to the reagent).
  • Suitable reporter groups include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. Such reporter groups may be used to directly or indirectly detect binding of the reagent to a sample component using standard methods known to those of ordinary skill in the art.
  • Kits for detecting p38-2 activity typically comprise a p38-2 substrate in combination with a suitable buffer. p38-2 activity may be specifically detected by performing an immunoprecipitation step with a p38-2-specific antibody prior to performing a kinase assay as described above.
  • the substrate provided may be a substrate that is phosphorylated only by p38-2 (i.e., is not phosphorylated by p38).
  • a kit for detecting MAPKK kinase activity based on measuring the phosphorylation of p38-2 generally comprises a p38-2 polypeptide in combination with a suitable buffer.
  • a kit for detecting MAPKK kinase activity based on detecting p38-2 activity generally comprises a p38-2 polypeptide in combination with a suitable p38-2 substrate, such as ATF2.
  • a kit may additionally comprise a suitable buffer and/or material for purification of p38 after activation and before combination with ATF2.
  • kits for use in detecting phosphorylation of p38-2 and/or kinase activity (e.g., antibody specific for phosphorylated substrate, which may be used to distinguish phosphorylated substrate from unphosphorylated substrate) may also be provided.
  • Such kits may be employed in direct or coupled kinase assays, which may be performed as described above.
  • p38-2 or a variant thereof may be used to identify one or more native upstream kinases (i.e., kinases that phosphorylate and activate p38-2 in vivo).
  • a p38-2 polypeptide may be used in a yeast two-hybrid system to identify proteins that interact with p38-2.
  • an expression library may be sequenced for cDNAs that phosphorylate p38-2.
  • This Example illustrates the cloning of a cDNA molecule encoding the human MAPK p38-2.
  • the EST sequence R72598 from a breast cDNA library displayed the highest similarity score.
  • a clone corresponding to the EST sequence R72598 was obtained from Research Genetics Inc., (Huntsville, AL), and the insert size was determined to be about 0.9 kb. Sequencing revealed that this clone encodes the 5' portion of a previously unknown gene and that the 3' end with the polyA tail was missing.
  • the 3 1 portion was obtained from a skeletal muscle cDNA library by RACE PCR using a gene specific forward primer and an adapter-based reverse primer.
  • the complete cDNA was obtained by fusion ligation of the 5' portion and the 3' portion using a common Kpnl site into pBluescript (Stratagene, La Jolla, CA), and verified by miniprep analysis.
  • Example 2 In vivo Expression of p38-2 This Example illustrates the expression of p38-2, as compared to p38, in various human tissues. Northern blots were performed using 2 ⁇ g of polyA + RNA isolated from
  • HA tagged p38-2 was in vitro transcribed and translated using the Promega TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) in the presence of 35S-methionine using SP6 polymerase and the template DNA 3xHA-p38- 2-SR ⁇ 3). Radioactive, in vitro-translated proteins were separated by SDS-PAGE and visualized by autoradiography (Figure 3).
  • Example 4 Activation of p38-2 bv Stress-inducing Agents This Example illustrates the activation of p38-2 by a variety of stimulators ofthe MAPK pathway.
  • SR ⁇ 3 was constructed by adding sequence encoding three copies of a 10 amino acid hemagglutinin (HA) epitope to the N-terminus of p38-2 and ligating the resulting cDNA into the expression vector SR ⁇ 3.
  • HA hemagglutinin
  • COS cells were transiently transfected with HA-p38-2-SR ⁇ 3 (as described above) by the DEAE-Dextran method (Kawai and Nishizawa, Mol. Cell Biol. 4:1 172-1 174, 1984).
  • Cell lysates were used in an immune complex kinase assay with GST-ATF2 substrate, prepared as previously described (Gupta et al., Science 267:389- 39, 1995). The assay was generally performed as described previously (Derijard et al., Cell 76: 1025-1037, 1994; Lin et al., Science 265:286-290, 1995) with minor modifications. The concentration of [ ⁇ - 32 P]ATP was 50 nM, and 30 ⁇ g cell lysate was immunoprecipitated for 2 hours with the anti-HA antibody 12CA5 (Boehringer- Mannheim Co ⁇ ., Indianapolis, IN) and then incubated with 1 ⁇ g of recombinant substrate. Reactions were separated by SDS-PAGE, and the results are presented in Figure 4.
  • Example 5 Selective Activation of BRK bv Bradykinin This Example illustrates the ability of bradykinin to potently and selectively activate BRK.
  • An initial experiment was performed to identify the MAPK pathways represented in NG108-15 cells (ATCC Accession No. HB-12317). The cells were grown according to published methods (see Hamprecht et al., Methods in Enzymol. 709:316-347, 1985). Immunoblot analyses were performed on extracts using polyclonal antibodies raised against p38 and a small peptide unique to BRK. As shown in Figure 6, these kinases could be clearly detected.
  • bradykinin strongly stimulates BRK, only modestly stimulates JNK and ERK, and does not measurably activate type 1 p38 kinases.
  • Example 6 Effect of Inhibitors of ERK and BRK Kinase Activity on Inhibition of I Ca v by Bradykinin This Example illustrates the use of an inhibitor of BRK to block the inhibition of I Ca v by bradykinin.
  • Activation of ERK and p38 kinases can be selectively blocked by the compounds PD98059 (see Pang et al., J. Biol Chem. 270:13585-13588, 1995) and SB203580 (see Lee et al., Nature 372:739-746, 1994), respectively.
  • PD98059 Parke- Davis
  • SB203580 SmithKline Beecham
  • MAP kinase pathways play roles similar to those ofthe classical second messenger pathways.
  • ADDRESSEE SEED and BERRY LLP
  • GTACATCCAC TCGGCCGGGA TCATCCACCG GGACCTGAAG CCCAGCAACG TGGCTGTGAA 480
  • CAAGGCCCTC TTCCCGGGAA GCGACTACAT TGACCAGCTG AAGCGCATCA TGGAAGTGGT 720
  • GCTCACTTAC CAGGAAGTCC TCAGCTTCAA GCCCCCAGAG CCACCGAAGC CACCTGGCAG 1080
  • GGCCTGCACC CTTCCACAGC TGGCCTGGTT TCCTCGAGAG GCACCTCCCA CACTCCTATG 1200

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Abstract

L'invention concerne des compositions et des procédés permettant de traiter des maladies associées aux cascades de protéine kinase activée par mitogène. L'invention décrit en particulier la protéine kinase p38-2 activée par mitogène et des variantes polypeptides de ladite protéine qui stimulent la phosphorylation et l'activation de substrats tels que l'ATF2. Les polypeptides peuvent être utilisés, par exemple, pour identifier des anticorps ou d'autres agents qui inhibent la transduction de signal via la cascade de la kinase p38-2. Les polypeptides et les agents peuvent être utilisés dans une variété de procédés comme, par exemple, pour réduire la sensibilité à la douleur.
PCT/US1997/008738 1996-05-20 1997-05-20 PROTEINE KINASE p38-2 ACTIVEE PAR MITOGENE ET MODES D'UTILISATION DUDIT PRODUIT WO1997044467A1 (fr)

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AU31397/97A AU736316B2 (en) 1996-05-20 1997-05-20 Mitogen-activated protein kinase p38-2 and methods of use therefor
CA002255579A CA2255579A1 (fr) 1996-05-20 1997-05-20 Proteine kinase p38-2 activee par mitogene et modes d'utilisation dudit produit
EP97926689A EP0914450A1 (fr) 1996-05-20 1997-05-20 PROTEINE KINASE p38-2 ACTIVEE PAR MITOGENE ET MODES D'UTILISATION DUDIT PRODUIT

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US6319921B1 (en) 1999-01-19 2001-11-20 Boerhinger Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compound as antiinflammatory agents
US6358945B1 (en) 1999-03-12 2002-03-19 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as anti-inflammatory agents
US6476023B1 (en) 1999-03-12 2002-11-05 Boehringen Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as anti-inflammatory agents
US6525046B1 (en) 2000-01-18 2003-02-25 Boehringer Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as antiinflammatory agents
US6608060B1 (en) 1996-12-18 2003-08-19 Vertex Pharmaceuticals Incorporated Inhibitors of p38
US6608052B2 (en) 2000-02-16 2003-08-19 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as anti-inflammatory agents
US6753426B2 (en) 1999-07-09 2004-06-22 Boehringer Ingelheim Pharmaceuticals, Inc. Polymorph and process for preparing same
US7041669B2 (en) 2002-02-25 2006-05-09 Boehringer Ingelheim Pharmaceuticals, Inc. 1,4-benzofused urea compounds useful in treating cytokine mediated diseases
US7122360B1 (en) 1998-06-24 2006-10-17 University Of Dundee Polypeptides, polynucleotides and uses thereof
US7291499B2 (en) 2001-07-16 2007-11-06 Fuyuki Ishikawa Transformed cell, method of screening anti-aging agent and anti-aging agent
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide

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US8338412B2 (en) 1996-12-18 2012-12-25 Vertex Pharmaceuticals Incorporated Inhibitors of P38
US6147080A (en) * 1996-12-18 2000-11-14 Vertex Pharmaceuticals Incorporated Inhibitors of p38
US6608060B1 (en) 1996-12-18 2003-08-19 Vertex Pharmaceuticals Incorporated Inhibitors of p38
US7365072B2 (en) 1996-12-18 2008-04-29 Vertex Pharmaceuticals Incorporated Inhibitors of p38
US7951809B2 (en) 1996-12-18 2011-05-31 Vertex Pharmaceuticals Incorporated Inhibitors of p38
US6376214B1 (en) 1997-02-18 2002-04-23 Smithkline Beecham Corporation DNA encoding a novel homolog of CSBP/p38 MAP kinase
US6350856B1 (en) 1997-02-18 2002-02-26 Smithkline Beecham Corporation Cytokine suppressive anti-inflammatory drug binding protein
EP0859054A1 (fr) * 1997-02-18 1998-08-19 Smithkline Beecham Corporation Protéine fixatrice de médicaments
US7122360B1 (en) 1998-06-24 2006-10-17 University Of Dundee Polypeptides, polynucleotides and uses thereof
US6333325B1 (en) 1999-01-19 2001-12-25 Boehringer Ingelheim Pharmaceuticals, Inc. Method of treating cytokine mediated diseases or conditions
US6372773B1 (en) 1999-01-19 2002-04-16 Boehringer Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as antiinflammatory agents
US6329415B1 (en) 1999-01-19 2001-12-11 Boehringer Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as antiinflammatory agents
US6506748B2 (en) 1999-01-19 2003-01-14 Boehringer Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as antiinflammatory agents
US6319921B1 (en) 1999-01-19 2001-11-20 Boerhinger Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compound as antiinflammatory agents
US6242453B1 (en) 1999-02-22 2001-06-05 Boehringer Ingelheim Pharmaceuticals, Inc Polycyclo heterocyclic derivatives as antiinflammatory agents
US7019006B2 (en) 1999-03-12 2006-03-28 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as anti-inflammatory agents
US6656933B2 (en) 1999-03-12 2003-12-02 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as anti-inflammatory agents
US6358945B1 (en) 1999-03-12 2002-03-19 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as anti-inflammatory agents
US6476023B1 (en) 1999-03-12 2002-11-05 Boehringen Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as anti-inflammatory agents
US6660732B2 (en) 1999-03-12 2003-12-09 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as anti-inflammatory agents
US7026476B2 (en) 1999-03-12 2006-04-11 Boehringer Ingelheim Pharmaceuticals, Inc. Intermediate arylamine compounds
US6835832B2 (en) 1999-07-09 2004-12-28 Boehringer Ingelheim Pharmaceuticals, Inc. Process for synthesis of heteroaryl-substituted urea compounds useful as antiinflammatory agents
US6894173B2 (en) 1999-07-09 2005-05-17 Boehringer Ingelheim Pharmaceuticals, Inc. Intermediates useful for synthesis of heteroaryl-substituted urea compounds, and processes of making same
US6774233B2 (en) 1999-07-09 2004-08-10 Boehringer Ingelheim Pharmaceuticals, Inc. Process for synthesis of heteroaryl-substituted urea compounds useful as antiinflammatory agents
US6753426B2 (en) 1999-07-09 2004-06-22 Boehringer Ingelheim Pharmaceuticals, Inc. Polymorph and process for preparing same
US6525046B1 (en) 2000-01-18 2003-02-25 Boehringer Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as antiinflammatory agents
US6608052B2 (en) 2000-02-16 2003-08-19 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as anti-inflammatory agents
US7291499B2 (en) 2001-07-16 2007-11-06 Fuyuki Ishikawa Transformed cell, method of screening anti-aging agent and anti-aging agent
US7041669B2 (en) 2002-02-25 2006-05-09 Boehringer Ingelheim Pharmaceuticals, Inc. 1,4-benzofused urea compounds useful in treating cytokine mediated diseases
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide

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