Note: Descriptions are shown in the official language in which they were submitted.
CA 02369078 2001-10-19
as originally filed
Medicaments comprising inhibitors of the cell volume-regulated human
kinase h-sgk
The present invention relates to medicaments comprising inhibitors or
activators of
the cell volume-regulated human kinase h-sgk. Such pharmaceuticals are
suitable
for the therapy of pathological states in which an increased or reduced
expression
of h-sgk is found. EP-0 861 896 has already described h-sgk and processes for
its
preparation, and the contents thereof are expressly intended also to form part
of the
present description.
Definitions of terms:
h-sgk: human serum and glucocorticoid dependent kinase (serine/threonine
kinase)
ENaC: epithelial Na+ channel
MDEG: mammalian degenerin (Waldmann, R., Lazdunski, M. (1998) Current
Opinion in Neurobiology 8: 418-424); a synonymous term is "BNC"
(brain Na+ channel)
TGFI3i: tumor growth factor (31
NKCC: Na+, K+, 2C1- cotransporter
HEPES: [4-(2-hydroxyethyl)piperazino]ethanesulfonic acid
SEM: standard error of mean
Trans-
dominant
inhibitory
kinase: h-sgk modified by mutation: lysine in position 127 has been replaced
by arginine (K127R); the mutation is located in the catalytic region and
suppresses the catalytic function of the kinase.
An increased expression of h-sgk is often found in diabetes mellitus,
arteriosclerosis, Alzheimer's disease, cirrhosis of the liver, Crohn's
disease,
fibrosing pancreatitis, pulmonary fibrosis and chronic bronchitis. The
increased
production of h-sgk can be explained by stimulation of expression by TGF13~
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(fig. 1 ). Fibrotic disorders are caused by increased formation and reduced
breakdown of matrix proteins. Both are effects of TGFl3l. Increased expression
of
the matrix proteins in fibroblasts can be suppressed by inhibiting the NKCC
with
furosemide (fig. 2). It has to date been unclear whether the increased
expression of
h-sgk is only a consequence or is the cause of the disorder.
Surprising findings now prove h-sgk activates Na+, K+, 2C1- cotransport (fig.
3). It
can be concluded from this that stimulation of NKCC by h-sgk induces fibrosis.
Besides Na+, K+, 2Cl- cotransport, h-sgk also activates ENaC (figs. 4 and 5)
and
MDEG.
The stimulating effect of h-sgk on ENaC can be suppressed by kinase inhibitors
such as, for example, staurosporine (Sigma, D-82041 Deisenhofen) or
chelerythrine (Sigma, loc. cit.) (fig. 4). In addition, the effect of h-sgk on
ENaC can
be suppressed, for example, by traps-dominant inhibitory kinase (fig. 5).
Inhibitors
of h-sgk such as staurosporine, chelerythrine or other kinase inhibitors might
therefore be employed in the therapy of the abovementioned disorders.
Generally
suitable for this purpose are all known kinase inhibitors. Kinase inhibitors
are also
commercially available in many cases, for example from Calbiochem-
Novabiochem GmbH, Listweg l, D-65812 Bad Soden (see "1998 General
Catalog"). Further kinase inhibitors can be obtained from other commercial and
noncommercial sources known to the skilled worker.
Expression of h-sgk is increased in an epileptic seizure. The functional data
we
have found show that the effects are suitable for reducing the excitability of
neurons because activation of NKCC leads to a reduction in the extracellular
K+
concentration, which is followed by hyperpolarization and thus inhibition of
the
activity of neurons. In addition, the inhibition of MDI?G ought to inhibit
neuronal
excitability. Accordingly, kinase activators which cross the blood-brain
barrier
might be employed successfully for epileptic seizures. Conversely, kinase
inhibition with drugs crossing the blood-brain barrier might increase
attentiveness
and learning ability. Kinase activators have moreover been known to the
skilled
worker for a lengthy period, among which the protein kinase C activators are
particularly of interest (see, for example, Calbiochem-Novabiochem 1998
General
Catalog, loc. cit.). Further kinase activators can be obtained from other
commercial
and noncommercial sources known to the skilled worker.
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Since the Na+, K+, 2C1- cotransport and the Na+ channel are crucial for renal
Na+
absorption and an increased renal Na+ absorption is associated with
hypertension, it
must be assumed that increased expression of the kinase leads to hypertension
and
reduced expression of the kinase leads to hypotension.
The present invention thus also relates to the use of inhibitors of h-sgk for
producing medicaments for the treatment of diabetes mellitus,
arteriosclerosis,
Alzheimer's disease, cirrhosis of the liver, Crohn's disease, fibrosing
pancreatitis,
pulmonary fibrosis, chronic bronchitis, radiation fibrosis, scleroderma,
cystic
fibrosis and other fibrosing disorders, and for the therapy of essential
hypertension.
Medicaments comprising inhibitors or activators of h-sgk can additionally be
employed to regulate neuronal excitability. It is particularly advantageous to
use
the inhibitors staurosporine or chelerythrine and their analogs.
RESULTS
Diabetic kidney:
Expression of h-sgk in the normal kidney is only low. A few cells in the
glomerulus, late proximal and distal tubule show distinct h-sgk expression. In
contrast to this, cells with massive h-sgk expression accumulate in the
diabetic
kidney.
Arteriosclerosis:
Cells massively expressing h-sgk are frequently found in the walls of
arteriosclerotic vessels.
Alzheimer's disease:
Only a few cells expressing h-sgk are found in the normal brain. These cells
are
probably oligodendroglial cells. The number of h-sgk-expressing cells is
significantly increased in brains with Alzheimer's disease.
Cirrhosis of the liver:
Only copper cells express h-sgk in the normal liver. However, in cirrhosis of
the
liver the tissue is dotted with h-sgk-expressing cells.
Crohn's disease:
In normal intestinal tissue, h-sgk is expressed exclusively in the
enterocytes.
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However, in Crohn's disease, the kinase is also found in connective tissue.
Fibrosing pancreatitis:
In the normal pancreas, h-sgk is found in acinar cells and in duct cells. A
few
h-sgk-expressing mononuclear cells are found around the pancreatic ducts.
There is
a marked increase in kinase expression in fibrosing pancreatitis.
Pulmonary fibrosis and chronic bronchitis:
Massive expression of h-sgk is observed in pulmonary fibrosis and chronic
bronchitis.
Stimulation of h-sgk expression by TGF131:
The expression of h-sgk is stimulated by TGF13, (fig. 1). Since TGFl31 is
produced
in fibrotic/inflamed tissue, this finding explains the increased expression of
h-sgk
in inflamed tissue.
TGFl3~ stimulates the expression of the matrix protein biglycan, an effect
which is
suppressed by the NKCC inhibitor furosemide:
TGFI31 stimulates the expression of biglycan. In the presence of the NKCC
inhibitor furosemide, the effect of TGF131 on biglycan expression is
completely
suppressed. Thus activation of NKCC is a precondition for the fibrotic effect
of
TGFf3 ~ . (fig. 2).
Stimulation of NKCC by h-sgk:
The significance of the increased expression of the kinase in fibrotic tissue
might
be manifold and not causally connected with the fibrosis. However, experiments
with the two-electrode voltage clamp have shown that the activity of NKCC is
massively stimulated by h-sgk (fig. 3). In view of the furosemide sensitivity
of
biglycan synthesis, this finding unambiguously demonstrates a causal role of h-
sgk
in fibrosis.
Stimulation of ENaC by h-sgk:
This effect can be suppressed by the kinase inhibitors staurosporine and
chelerythrine. As fig. 4 shows, there is a massive increase in the current
with ENaC
through coexpression with h-sgk. The kinase therefore stimulates ENaC. The
kinase inhibitors staurosporine and chelerythrine are able completely to
suppress
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the activation of ENaC by h-sgk.
Stimulation of epithelial ENaC by h-sgk can be reversed by coexpression of the
traps-dominant inhibitory kinase h-sgk:
As fig. 5 shows, the stimulating effect of h-sgk coexpression on the ENaC-
mediated Na+ current can be suppressed by coexpression of a traps-dominant
inhibitory kinase. This traps-dominant inhibitory kinase (compare with
"definitions
of terms") is modified on the catalytic unit in such a way that it can no
longer
display its function. However, since it binds to the substrate it displaces
the active
kinase and thus suppresses its effects. The traps-dominant inhibitory kinase
not
only suppresses the increase in ENaC activity due to exogenous h-sgk but
evidently
also suppresses the stimulation by endogenous h-sgk.
MDEG is completely blocked by coexpression with h-sgk:
As fig. 6 shows, expression of MDEG in oocytes induces a strong Na+ current
which is activated by lowering the extracellular pH. The channel is completely
blocked by coexpression with h-sgk. It must be concluded from this that h-sgk
inhibits neuronal excitability. Examples:
Example 1: In situ hybridization
Tissue from normal pancreas, liver, vessels, brain, lung, kidney and
intestine, and
tissue with diabetic nephropathy, arteriosclerosis, Alzheimer's disease,
cirrhosis of
the liver, Crohn's disease, fibrosing pancreatitis and pulmonary fibrosis was
embedded in paraffin in 4% paraformaldehyde/0.1 M sodium phosphate buffer
(pH 7.2) for 4 hours. Tissue sections were dewaxed and hybridized as described
previously (Kandolf, R., D. Ameis, P. Kirschner, A. Canu, P.H. Hofschneider,
Proc. Natl. Acad. Sci. USA 84: 6272-6276, 1987; Hohenadl, C., K. Klingel, J.
Mertsching, P. H. Hofschneider, R. Kandolf., Mol. Cell. Probes 5: 11-20, 1991;
Klingel, K., C. Hohenadl, A. Canu, M. Albrecht, M. Seemann, G. Mall, R.
Kandolf, Proc. Natl. Acad. Sci. USA, 89: 314-318, 1992).
The hybridization mixture contained either 35S-labeled sense RNA coding for h-
sgk or 35S-labeled antisense RNA complementary to the latter RNA (500 ng/ml of
each) in 10 mM Tris-HC1, pH 7.4; 50% (vol/vol) deionized formamide; 600 mM
NaCI; 1 mM EDTA; 0.2% polyvinylpyrrolidone; 0.02% Ficoll; 0.05% calf serum
albumin; 10% dextran sulfate; 10 mM dithiothreitol; 200 ~g/ml denatured
CA 02369078 2001-10-19
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sonicated salmon sperm DNA and 100 p,g/ml rabbit liver tRNA.
Hybridization with RNA probes was carried out at 42°C for 18 hours. The
slides
were washed as described (Hohenadl et al., 1991; Klingel et al., 1992), and
then
incubated in 2x standard sodium citrate at 55°C for 1 hour.
Unhybridized single-
stranded RNA probes were digested by RNase A (20 pg/ml) in 10 mM Tris-HCI,
pH 8.0/0.5 M NaCI at 37°C for 30 min. Tissue samples were then
autoradiographed for three weeks (Klingel et al., 1992) and stained with
hematoxylin/eosin.
Example 2: Transcriptional regulation of biglycan and h-sgk
Cells were cultivated in RPMI/5% C02/10 mM glucose at 37°C, pH
7.4,
supplemented with 10% (vol/vol) fetal calf serum (FCS). The cells were grown
to
90% confluence and then homogenized in TRIZOL (GIBCO/BRL) (about 0.4x 106
per sample). Total RNA was prepared in accordance with the manufacturer's
instructions. Northern blots were fractionated by electrophoresis through 10
g/1
agarose gels with 15 or 20 ~g of total RNA with separate control in the
presence of
2.4 mol/1 formaldehyde. RNA was transferred by vacuum (Appligene Oncor Trans
2o- DNA Express Vacuum Blotter, Appligine, Heidelberg, Germany) to positively
charged nylon membranes (Boehringer Mannheim, Germany) and crosslinked
under ultraviolet light (UV Stratalinker 2400, Stratagene, Heidelberg,
Germany).
Hybridization was carried out over night with DIG-Easy-Hyb (Boehringer
Mannheim) at a probe concentration of 25 pg/1 at 50°C. The digoxigenin
(DIG)-
labeled probes were produced by PCR as described in detail earlier (Waldegger
et
al. (1997) PNAS 94: 4440-4445). For the autoradiography, the filters were
exposed
to an X-ray film (Kodak) for an average of S min.
Example 3: Two-electrode voltage clamp and tracer flux experiments
Dissection of Xenopus laevis, and the obtaining and treatment of the oocytes
has
been described in detail earlier (Busch et al. 1992). The oocytes were each
injected
with 1 ng of cRNA of NKCC, ENaC or MDEG with or without simultaneous
injection of h-sgk. It was possible to carry out two-electrode voltage and
current
clamp experiments 2-8 days after the injection. Na+ influx which could be
inhibited
by furosemide through the NKCC was measured by the 22Na+ uptake, which was
determined with a scintillation counter, into the oocytes. Na+ currents (ENaC)
were
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filtered at 10 Hz and recorded with a pen recorder. The experiments were
normally
carried out on the second day after cRNA injection. The bath solution
contained:
96 mM NaCI, 2 mM KCI, 1.8 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES at
pH 7.5 and the holding potential was -50 mV. The pH was adjusted by titration
with HCl or NaOH in all the experiments. The flow rate of the bath liquid was
set
at 20 ml/min, which ensured a complete change of solution in the measurement
chamber within 10-15 s. All the data are stated in the form of arithmetic
means ~
SEM.
Figure legends:
Fig. 1: Stimulation of h-sgk expression by TGF13~:
The expression of h-sgk is stimulated by TGF13~. The effect of TGF131 after
0.5 to 6 h is shown (top). The phorbol ester PDD (4-alpha-phorbol
12,13-didecanoate; stimulates protein kinase C) and the Cap ionophore
ionomycin (Sigma, loc. cit; increases the intracellular Ca++ concentration)
likewise stimulate h-sgk expression (below).
Fig. 2: Stimulation of biglycan expression by TGFI3,:
The expression of biglycan (B) is stimulated by osmotic swelling of cells
(hypo = h, top left) and by TGFf31 (top right). The effect of TGF131 on
biglycan expression is almost completely suppressed in the presence of the
NKCC inhibitor bumetanide (b) (control = c).
Fig. 3: Stimulation of the NKCC by h-sgk:
The uptake which can be inhibited by furosemide of 22Na+ in oocytes
[uptake (nmo1/20 min/oocyte) = u] which express the NKCC is massively
stimulated by h-sgk. NKCC-injected oocytes do not show a higher Na+
influx than uninfected oocytes (n.i.). This Na+ influx is not inhibited by the
NKCC inhibitor furosemide (= F) (top). Expression of h-sgk alone does not
lead to stimulation of the Na+ influx. Coexpression of h-sgk with NKCC
leads to a large increase in the Na+ influx, and this increase is completely
suppressed by furosemide (below).
Fig. 4: Stimulation of the ENaC by h-sgk:
The current through the ENaC (I) increases massively through coexpression
with h-sgk. Treatment of the oocytes with the kinase inhibitors
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staurosporine (S) or chelerythrine (C) suppresses the activation of the Na+
channel by h-sgk.
Fig. 5: The stimulation of the ENaC by h-sgk can be reversed by coexpression
of
the trans-dominant inhibitory kinase:
oocytes expressing ENaC and h-sgk simultaneously show very much larger
currents (I) than do oocytes expressing only the ENaC. Coexpression of the
trans-dominant inhibitory kinase suppresses the stimulation of the ENaC by
h-sgk.
Fig. 6: Inhibition of the MDEG by h-sgk:
The current through the MDEG (I) increases with the duration of the
incubation [day (T) 1-4]. The current is completely suppressed by
coexpression with h-sgk (peak = p; plateau = pl).
