Note: Descriptions are shown in the official language in which they were submitted.
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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 PKCa, (3~, (3Z (~31 and X32 are
alternately
spliced forms of the same gene) and 'y; 2) novel PKCB, E, r~, and 8; 3)
atypical
PKC~, ~., r1 and v ; and 4) PKC~. The a, ~3" (32 and y isoforms are Ca2+,
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 Ca2+ (House et al. , i n , 2~$, 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).
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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 PKCB 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 PKCB in said mammal.
Preferably, PKCB 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 PKCB surfaces.
Figure 2 illustrates the inhibition of eosinophil superoxide production by
the PKCB-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
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angioedema associated with eosinophilia, ulcerative colitis, inflammatory
bowel
disease, vernal conjunctivitis, giant papillary conjunctivitis, and allergic
conjunctivitis.
According to the invention, PCKB 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, PCKB activity can be modulated by
administering an effective amount of a chemical agent that acts upon PKCB
(e.g.,
an inhibitor), such as the specific inhibitor of the PKCB 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 PKCB 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 PKCB expression
(Shanmugam et al., Mol. Cell. Endocrinoloav, 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 PKCB can
also be employed, such as gene therapy. The complete nucleotide coding
sequence of PKCB is available (see GenBank Accession No.: 5453969).
Furthermore, human recombinant PKCB protein is also commercially available
(BioMol Cat. No. SE-147). DNA encoding PKCB can be used to alter the
amount of PKCB in a cell. Alternatively, DNA encoding any protein which
interacts with PKCB, so as to modulate its activity (e.g., inhibit or increase
its
activity), is also envisioned.
DNA encoding PKCB, or a PKCB 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
PKCB, a PKCB modulating protein, or comprising DNA complementary to DNA
encoding PKCB or a PKCB modulating protein. This can be done by any
procedure useful for the introduction into a particular cell (e.g., physical
or
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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 Laboratorv 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
PKCB in a mammal, for example, by administering to the mammal an effective
amount of "antisense" mRNA transcripts or antisense oligonucleotides that
encode PKCB which, when expressed from an expression cassette in a host cell,
can alter PKCB 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 PKCB. The introduction of PKCB 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 PKCB. Thus,
the
introduced nucleic acid may be useful to modulate the expression of PKCB in
mammals with an eosinophil-associated indication. For example, the
administration of an expression vector encoding PKCB peptide may increase the
PKCB activity and thus be efficacious for diseases which are characterized by
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decreased levels of PKCB. Likewise, the administration of an expression vector
comprising antisense PKCB sequences may be useful to prevent or treat a
disorder associated with increased PKCB expression.
In addition, the administration of a dominant-negative mutant of
5 PKCB may also specifically inhibit PKCS expression. (lain et al., J. Biol.
Chem., 274, 24392-24400 (1999)). Modulation of PKCB activity by the
administration of antibodies which specifically react with PKCB is also
contemplated. An anti-PKCB antibody is commercially available from BioMol
(Cat. No. SA-148).
The invention will now be illustrated by the following non-
limiting examples.
Example 1
The following procedure was carned out to assess the ability of
lidocaine to bind PKCB.
Materials and Methods. Sensor chip CMS (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 NaCI) 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. PKCB 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,
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followed by injection of PKCB, followed by blocking of remaining activated
carboxyl groups by 1 M ethanolamine, pH 8.5. The flow rate throughout was
pl/min. Low-density and high-density surfaces were prepared by controlled
PKCB injections (6176.0 R.U.s and 15968.0 R.U.s respectively). A control
5 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
~g/ml, 10 ~g/ml, 25 ~g/ml and 40 ~g/ml. These solutions were then injected
10 over prepared surfaces at a flow rate of 20 ~1/min for two minutes (Figure
1).
Results
PKCB, coupled to the sensor chip surface, remained active to the
binding of lidocaine following the immobilization procedure. Two different
surface densities of PKCB were employed to show that binding of lidocaine to
PKCB was specific. Comparison to the control surface also discriminates
between bulk refractive index changes and specific binding interactions. This
data demonstrates that lidocaine binds PKCB.
Example 2
In the following assay, Rottlerin, a selective blocker of PKCB,
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
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production curve (usually 20 to 50 minutes following IL-S 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 PKCB-selective blocker, rottlerin, inhibited the rate of
superoxide production and magnitude of degranulation in a concentration-
dependent manner, with ICSO values (0.7 pM and 0.6 pM respectively) for
rottlerin consistent with inhibition of PKCs catalytic activity (3-6 pM).
Thus,
PKCB 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 PKCB 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.
