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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/14—Decongestants or antiallergics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • 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

• PKC protein kinase C
• the , ⁇ legal ⁇ 2 and ⁇ isoforms are Ca 2+ , phospholipid- and diacylglycerol-dependent and represent the classical isoforms of PKC, whereas the other isoforms are activated by phospholipid and diacylglycerol but are not dependent on Ca 2+ (House et al. Science, 23_&, 1726 (1987)).
• U.S. Patent Numbers 5,510,339 and 5,631,267 disclose the use of topical anesthetics, such as lidocaine and the like, to treat bronchial asthma and other eosinophil associated hypersensitivity diseases. Additionally, U.S. Patent Number 5,837,713 discloses the use of a synergistic combination of a topical anesthetic and a glucocorticoid to treat eosinophil associated pathologies.
• U.S. Patent Application Serial Number 08/985,613 discloses the use of a sulfonylurea receptor (SUR) binding agent to treat IL-5 mediated pathologies.
• SUR sulfonylurea receptor
• This application also discloses a method for inhibiting cytokine- induced eosinophil survival or activation with a sulfonylurea receptor binding agent, optionally in combination with one or more topical anesthetics and/or glucocorticoids.
• the application also discloses a method for treating a disease mediated by ' IL-5 with an agent that is able to modify (e.g., block) ATP- dependent potassium channels, or a protein with which an ATP-dependent potassium channel interacts (such as a SUR). dependent potassium channels, or a protein with which an ATP-dependent potassium channel interacts (such as a SUR).
• the present invention provides a method to treat an eosinophil-associated pathology in a mammal, comprising modulating the activity of PKC ⁇ in said mammal.
• PKC ⁇ modulation is not achieved by administering lidocaine or other topical anaesthetics as described in U.S. Patent Nos. 5,510,339, 5,631,267 and 5,837,713, supra.
• Figure 1 illustrates the specific binding of varying concentrations of lidocaine to both high and low density PKC ⁇ surfaces.
• Figure 2 illustrates the inhibition of eosinophil superoxide production by the PKC ⁇ -selective blocker rottlerin.
• Figure 3 illustrates the inhibition of eosinophil degranulation by rottlerin.
• hypersensitivity diseases and conditions associated with elevated levels of eosinophil activation and accumulation are amenable to treatment by the present therapy.
• These conditions include, but are not limited to, nasal inflammation, conjunctivitis, chronic eosinophilic pneumonia, allergic rhinitis, allergic sinusitis, allergic gastroenteropathy, eosinophilic gastroenteritis, atopic dermatitis, bullous pemphigoid, episodic angioedema associated with eosinophilia, ulcerative colitis, inflammatory bowel disease, vernal conjunctivitis, giant papillary conjunctivitis, and allergic conjunctivitis.
• PCK ⁇ activity can be modulated in a mammal (i.e., increased or decreased with respect to the level that would have existed in said mammal in the absence of intervention) using any suitable manner known in the art.
• PCK ⁇ activity can be modulated by administering an effective amount of a chemical agent that acts upon PKC ⁇ (e.g., an inhibitor), such as the specific inhibitor of the PKC ⁇ isozyme, rottlerin (CAS Registry No. 82-08-6), also known as mallotoxin.
• Rottlerin is available from commercial sources, including R.B.I. (Natick, MA., U.S.A.) and Calbiochem (California, U.S.A.).
• DEX dexniguldipine hydrochloride
• EPO erythropoietin
• PKC ⁇ protein kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinas
• human recombinant PKC ⁇ protein is also commercially available (BioMol Cat. No. SE-147).
• DNA encoding PKC ⁇ can be used to alter the amount of PKC ⁇ in a cell.
• DNA encoding any protein which interacts with PKC ⁇ , so as to modulate its activity (e.g., inhibit or increase its activity), is also envisioned.
• DNA encoding PKC ⁇ , or a PKC ⁇ modulating protein can be readily introduced into host cells (e.g., mammalian, bacterial, yeast or insect cells) by transfection with an expression vector comprising DNA encoding PKC ⁇ , a PKC ⁇ modulating protein, or comprising DNA complementary to DNA encoding PKC ⁇ or a PKC ⁇ modulating protein.
• host cells e.g., mammalian, bacterial, yeast or insect cells
• an expression vector comprising DNA encoding PKC ⁇ , a PKC ⁇ modulating protein, or comprising DNA complementary to DNA encoding PKC ⁇ or a PKC ⁇ modulating protein.
• This can be done by any procedure useful for the introduction into a particular cell (e.g., physical or biological methods), to yield a transformed cell having the recombinant DNA stably integrated into its genome, so that the DNA molecules, sequences, or segments, of the present invention are expressed by the host cell, as described by Sambrook et al., Molecular Cloning: A Laboratory
• Physical methods to introduce a preselected DNA into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
• Biological methods to introduce the DNA of interest into a host cell include the use of DNA and RNA viral vectors.
• the main advantage of physical methods is that they are not associated with pathological or oncogenic processes of viruses. However, they are less precise, often resulting in multiple copy insertions, random integration, disruption of foreign and endogenous gene sequences, and unpredictable expression. For mammalian gene therapy, it is desirable to use an efficient means of precisely inserting a single copy gene into the host genome.
• Viral vectors and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.
• Other viral vectors can be derived from poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.
• Antisense technology can also be used to alter the expression of PKC ⁇ in a mammal, for example, by administering to the mammal an effective amount of "antisense” mRNA transcripts or antisense oligonucleotides that encode PKC ⁇ which, when expressed from an expression cassette in a host cell, can alter PKC ⁇ expression.
• antisense means a sequence of nucleic acid which is the reverse complement of at least a portion of a RNA or DNA molecule that codes for PKC ⁇ .
• the introduced nucleic acid may be useful to modulate the expression of PKC ⁇ in mammals with an eosinophil-associated indication.
• the administration of an expression vector encoding PKC ⁇ peptide may increase the PKC ⁇ activity and thus be efficacious for diseases which are characterized by decreased levels of PKC ⁇ .
• the administration of an expression vector comprising antisense PKC ⁇ sequences may be useful to prevent or treat a disorder associated with increased PKC ⁇ expression.
• a dominant-negative mutant of PKC ⁇ may also specifically inhibit PKC ⁇ expression.
• Modulation of PKC ⁇ activity by the administration of antibodies which specifically react with PKC ⁇ is also contemplated.
• An anti-PKC ⁇ antibody is commercially available from BioMol (Cat. No. S A- 148). The invention will now be illustrated by the following non- limiting examples.
• Sensor chip CM5 (available from BIACORE AB, Uppsala, Sweden) was the surface of choice for this assay since it provides a versatile, flexible, robust surface which has high binding capacity and allows immobilization through primary amine coupling. Use of this surface in conjunction with a BIACORE® 2000 (BIACORE AB, Uppsala, Sweden) allows multi-channel analysis of four independent sensor surfaces termed flow cells.
• HBS-N (0.01 M HEPES pH 7.4, 0.15 M NaCl) buffer was degassed and filtered prior to use and employed as running buffer throughout these experiments. This buffer was employed in order to avoid any possible detergent effects on binding interactions.
• PKC ⁇ was diluted to 20 ⁇ g/ml in 10 mM Sodium Acetate, pH 4.5, using standard amine coupling procedures. The surface was derivatized through injection of a 1 : 1 EDC NHS mixture for 7 minutes, followed by injection of PKC ⁇ , followed by blocking of remaining activated carboxyl groups by 1 M ethanolamine, pH 8.5. The flow rate throughout was 10 ⁇ l/min. Low-density and high-density surfaces were prepared by controlled PKC ⁇ injections (6176.0 R.U.s and 15968.0 R.U.s respectively). A control surface was also prepared by exposing a separate flow cell to the activation and blocking steps.
• Lidocaine hydrochloride was dissolved in running buffer (HBS-N) and this stock solution diluted to concentrations of 5 ⁇ g/ml, 10 ⁇ g/ml, 25 ⁇ g/ml and 40 ⁇ g/ml. These solutions were then injected over prepared surfaces at a flow rate of 20 ⁇ l/min for two minutes ( Figure 1).
• PKC ⁇ coupled to the sensor chip surface, remained active to the binding of lidocaine following the immobilization procedure.
• Two different surface densities of PKC ⁇ were employed to show that binding of lidocaine to PKC ⁇ was specific. Comparison to the control surface also discriminates between bulk refractive index changes and specific binding interactions. This data demonstrates that lidocaine binds PKC ⁇ .
• Rottlerin a selective blocker of PKC ⁇ , was found to act like lidocaine by blocking superoxide production and inhibiting the activation of eosinophils.
• Inhibitors were added immediately before stimulation unless otherwise noted.
• a stock solution of rottlerin (Calbiochem) was diluted in HBSS so that the final concentration of DMSO or EtOH was less than 0.5%.
• the rate of eosinophil superoxide production was calculated using the linear part of the superoxide production curve (usually 20 to 50 minutes following IL-5 stimulation) and is presented as nanomoles of superoxide produced per minute (Figure 2).
• Supernatants from the superoxide assays were stored at -20°C and the degree of degranulation (EDN release) was assayed by RIA as described (Abu-Ghazaleh et al., J. Immunol.. 142. 2393-2400 (1989)) ( Figure 3).
• PKC ⁇ -selective blocker inhibited the rate of superoxide production and magnitude of degranulation in a concentration- dependent manner, with IC 50 values (0.7 ⁇ M and 0.6 ⁇ M respectively) for rottlerin consistent with inhibition of PKC ⁇ catalytic activity (3-6 ⁇ M).
• IC 50 values 0.7 ⁇ M and 0.6 ⁇ M respectively
• PKC ⁇ likely plays a central role in the regulation of NADPH oxidase activity and degranulation in eosinophils.
• agents that modulate the activity of PKC ⁇ are useful to treat diseases wherein activation of eosinophils is implicated (e.g., hypersensitivity diseases such as asthma).

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  • 2000-10-03 WO PCT/US2000/027277 patent/WO2001028543A1/en not_active Ceased
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