TREATMENT OF EOSINOPHIL ASSOCIATED PATHOLOGIES BY MODULATING PKC- delta ACTIVITY

Abstract: The invention provides methods to treat diseases associated with eosinophil activation, such as hypersensitivity diseases or conditions (e.g., asthma), by administrating an agent which modulates PKC delta activity.

Description: TREATMENT OF EOSTNOPHT ASSOCIATED PATHOT GTES BY MODULATING PKC-rielta ACTTVTTY Background of the Invention Protein kinases play a critical role in cellular development, differentiation and transformation. One of the largest gene families of non- receptor serine-threonine protein kinases is protein kinase C (PKC). Since the discovery of PKC more than a decade ago, a multitude of physiological signaling mechanisms have been ascribed to the enzyme. The PKC gene family consists presently of 11 genes which are divided into four subgroups: 1) classical PKCα, Pi, β 2 CP t an d β 2 are alternately spliced forms of the same gene) and γ; 2) novel PKCδ, e, η, and θ; 3) atypical PKCζ, λ, η and i ; and 4) PKCμ. The , β„ β 2 and γ 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. 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). Despite the above disclosures, there is a continuing need for methods to treat pathologies associated with eosinophil activation. In particular, there is a need for methods to treat eosinophil-associated pathologies such as hypersensitivity diseases or conditions (e.g., asthma) utilizing novel pathways. Summary of the Invention Applicant has discovered that compounds that inhibit the activity of PKCδ are useful to treat diseases associated with eosinophil activation, such as hypersensitivity diseases or conditions (e.g., asthma). Accordingly, the present invention provides a method to treat an eosinophil-associated pathology in a mammal, comprising modulating the activity of PKCδ in said mammal. Preferably, PKCδ 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. Brief Description of the Figures Figure 1 illustrates the specific binding of varying concentrations of lidocaine to both high and low density PKCδ surfaces. Figure 2 illustrates the inhibition of eosinophil superoxide production by the PKCδ-selective blocker rottlerin. Figure 3 illustrates the inhibition of eosinophil degranulation by rottlerin. Detailed Description of the Invention In addition to asthma, 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. According to the invention, PCKδ 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. For example, PCKδ activity can be modulated by administering an effective amount of a chemical agent that acts upon PKCδ (e.g., an inhibitor), such as the specific inhibitor of the PKCδ 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.). Other suitable chemical agents include dexniguldipine hydrochloride (DEX), which may affect PKCδ expression in cells (Proc. Annu. Meet. Am. Assoc. Cancer Res.. 36. A2598 (1995)). In addition, estrogen and erythropoietin (EPO) have also been reported to modulate PKCδ expression (Shanmugam et al, Mol. Cell. Endocrinology. 148. 109-118 (1999) and Cooper et al., Proc. Annu. Meet. Am. Assoc. Cancer Res.. 38, A2506 (1997)). Other methods to regulate the cellular expression of PKCδ can also be employed, such as gene therapy. The complete nucleotide coding sequence of PKCδ is available (see GenBank Accession No.: 5453969). Furthermore, human recombinant PKCδ protein is also commercially available (BioMol Cat. No. SE-147). DNA encoding PKCδ can be used to alter the amount of PKCδ in a cell. Alternatively, DNA encoding any protein which interacts with PKCδ, so as to modulate its activity (e.g., inhibit or increase its activity), is also envisioned. DNA encoding PKCδ, or a PKCδ 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 PKCδ, a PKCδ modulating protein, or comprising DNA complementary to DNA encoding PKCδ or a PKCδ 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 Manual. Cold Spring Harbor, NY (1989). 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 PKCδ in a mammal, for example, by administering to the mammal an effective amount of "antisense" mRNA transcripts or antisense oligonucleotides that encode PKCδ which, when expressed from an expression cassette in a host cell, can alter PKCδ expression. As used herein, the term "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 PKCδ. The introduction of PKCδ sense or antisense nucleic acid into a cell ex vivo or in vivo can result in a molecular genetic-based therapy directed to controlling the expression of PKCδ. Thus, the introduced nucleic acid may be useful to modulate the expression of PKCδ in mammals with an eosinophil-associated indication. For example, the administration of an expression vector encoding PKCδ peptide may increase the PKCδ activity and thus be efficacious for diseases which are characterized by decreased levels of PKCδ. Likewise, the administration of an expression vector comprising antisense PKCδ sequences may be useful to prevent or treat a disorder associated with increased PKCδ expression. In addition, the administration of a dominant-negative mutant of PKCδ may also specifically inhibit PKCδ expression. (Jain et al., J. Biol. Chem.. 274. 24392-24400 (1999)). Modulation of PKCδ activity by the administration of antibodies which specifically react with PKCδ is also contemplated. An anti-PKCδ antibody is commercially available from BioMol (Cat. No. S A- 148). The invention will now be illustrated by the following non- limiting examples. Example 1 The following procedure was carried out to assess the ability of lidocaine to bind PKCδ. Materials and Methods. 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. All experiments were carried out at 25°C and all reagents were used as supplied. Surface Preparation. PKCδ was diluted to 20 μg/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 PKCδ, followed by blocking of remaining activated carboxyl groups by 1 M ethanolamine, pH 8.5. The flow rate throughout was 10 μl/min. Low-density and high-density surfaces were prepared by controlled PKCδ 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. Binding Interactions. Lidocaine hydrochloride was dissolved in running buffer (HBS-N) and this stock solution diluted to concentrations of 5 μg/ml, 10 μg/ml, 25 μg/ml and 40 μg/ml. These solutions were then injected over prepared surfaces at a flow rate of 20 μl/min for two minutes (Figure 1). Results PKCδ. coupled to the sensor chip surface, remained active to the binding of lidocaine following the immobilization procedure. Two different surface densities of PKCδ were employed to show that binding of lidocaine to PKCδ was specific. Comparison to the control surface also discriminates between bulk refractive index changes and specific binding interactions. This data demonstrates that lidocaine binds PKCδ. Example 2 In the following assay, Rottlerin, a selective blocker of PKCδ, was found to act like lidocaine by blocking superoxide production and inhibiting the activation of eosinophils. Materials and Methods. Superoxide assays were performed in HBSS buffer supplemented with 10 mM HEPES. Cytochrome C was used to detect the production of extracellular superoxide formation as previously described (Bankers-Fulbright et al., J. Immunol.. 160. 5546-5553 (1998)). 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). Results The PKCδ-selective blocker, rottlerin, inhibited the rate of superoxide production and magnitude of degranulation in a concentration- dependent manner, with IC 50 values (0.7 μM and 0.6 μM respectively) for rottlerin consistent with inhibition of PKC δ catalytic activity (3-6 μM). Thus, PKCδ likely plays a central role in the regulation of NADPH oxidase activity and degranulation in eosinophils. Accordingly, Applicant has discovered that agents that modulate the activity of PKCδ are useful to treat diseases wherein activation of eosinophils is implicated (e.g., hypersensitivity diseases such as asthma). All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims: WHAT IS CLAIMED IS: 1. The use of a chemical agent that modifies PKCδ activity to prepare a medicament for treating a hypersensitivity disease or condition in a mammal, provided the agent is not lidocaine or another topical anesthetic. 2. The use of claim 1, wherein the agent inhibits PKCδ activity. 3. The use of claim 1, wherein the agent is rottlerin, antisense mRNA, an antisense oligonucleotide, or an antibody. 4. The use of claim 1, wherein the agent is DNA. 5. The use of claim 1 , wherein the agent is not rottlerin. 6. The use of claim 1, wherein the agent is rottlerin. 7. The use of claim 1, wherein the agent is antisense mRNA. 8. The use of claim 1, wherein the agent is an antisense oligonucleotide. 9. The use of claim 1, wherein the agent is an antibody. 10. The use of claim 1 , wherein the hypersensitivity disease or condition is asthma. 11. A therapeutic method for treating a hypersensitivity disease or condition in a mammal comprising modulating PKCδ activity in the mammal; wherein PKCδ activity is not modulated by the administration of lidocaine or another topical anesthetic. 12. The method of claim 11 , wherein the PKCδ activity is modulated by administration of an effective amount of a chemical agent. 13. The method of claim 12, wherein the PKCδ activity is inhibited. 14. The method of claim 12, wherein the chemical agent is rottlerin. 15. The method of claim 11 or 12, wherein the chemical agent is not rottlerin. 16. The method of claim 11 wherein the PKCδ activity is modulated by gene therapy. 17. The method of claim 11 , wherein the PKCδ activity is modulated by the administration of antisense mRNA, or a fragment thereof, which specifically binds PKCδ. 18. The method of claim 11, wherein the PKCδ activity is modulated by the administration of antisense oligonucleotide, or fragment thereof, which specifically binds PKCδ. 19. The method of claim 11 , wherein the PKCδ activity is modulated by the administration of antibodies which specifically react with PKCδ. 20. The method of any one of claims 11-19 wherein the hypersensitivity disease or condition is asthma.

Inventors: GLEICH GERALD J, GLEICH, GERALD, J.
Applicants: GLEICH GERALD J, MAYO FOUNDATION, GLEICH, GERALD, J., MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH
Application year: 2000
Main classification: A61K31
IPC codes: A61K31, A61K39, A61K45, A61K48, A61P1, A61P11, A61P17, A61P27, A61P37, A61P43