DE19917990A1 - Medicament containing inhibitors of cell volume regulated human kinase h-sgk - Google Patents

Abstract

The invention relates to medicaments which contain inhibitors or activators of cell-volume regulated human kinase h-sgk. Medicaments of this type are suitable for treating conditions, in which an increased or reduced expression of h-sgk is identified.

Description

The present invention relates to medicaments containing inhibitors or activators of cell volume regulated human kinase h-sgk. Such drugs are used to treat Disease conditions in which an increased or decreased expression of the h-sgk is found suitable. The h-sgk and processes for their production have already been developed in the EP-0 861 896 described, the content of which is expressly part of the present Description should be.

Definitions

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)
TGFβ ₁

: tumor growth factor β ₁

,
NKCC: Na ⁺

-, K ⁺

-, 2Cl ^-

-Cotransporter
HEPES: [4- (2-hydroxyethyl) piperazino] ethanesulfonic acid
SEM: standard error of mean
Transdominant inhibitory kinase: mutation-altered h-sgk: lysine at position 127 was replaced by arginine (K127R); the mutation lies in the catalytic region and prevents the catalytic function of the kinase.

Increased expression of h-sgk is increasingly found in diabetes mellitus, arteriosclerosis, Alzheimer's disease, liver cirrhosis, Crohn's disease, fibrosing pancreatitis, pulmonary fibrosis and chronic bronchitis. The increased formation of h-sgk can be explained by stimulation of expression by TGFβ ₁ ( Fig. 1). Fibrosive diseases are caused by increased formation and reduced breakdown of matrix proteins. Both are effects of TGFβ ₁ . In fibroblasts, the increased expression of the matrix proteins can be prevented by inhibiting the NKCC with furosemide ( Fig. 2). So far it was unclear whether the increased expression of h-sgk is only the consequence or cause of the disease.

Surprising results now show that h-sgk activates Na ⁺ , K ⁺ , 2Cl ^- cotransport ( Fig. 3). From this it can be concluded that stimulation of the NKCC is triggered by h-sgk fibrosis. In addition to the Na ⁺ , K ⁺ , 2Cl ^- cotransport, the ENaC ( Fig. 4 and 5) and the MDEG are activated by the h-sgk.

The stimulating effect of h-sgk on the ENaC can be prevented 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 the ENaC can be suppressed, for example, by transdominant inhibitory kinase ( Fig. 5). Inhibitors of h-sgk, such as staurosporine, chelerythrine or other kinase inhibitors, could therefore be used in the therapy of the abovementioned diseases. In general, all known kinase inhibitors can be used. Kinase inhibitors are also commercially available in many cases, for example from Calbiochem-Novablochem GmbH, Lisztweg 1, D-65812 Bad Soden (see "1998 General Catalog"). Additional kinase inhibitors are available from other commercial and non-commercial sources known to those skilled in the art.

The h-sgk is increasingly expressed in epileptic seizures. The functional data we found show that the effects are suitable for reducing the excitability of neurons, since the activation of the NKCC leads to a decrease in the extracellular K ⁺ concentration, which results in hyperpolarization and thus inhibition of the activity of neurons . In addition, inhibition of MDEG should inhibit neuronal excitability. Accordingly, activators of the kinase that cross the blood-brain barrier could be used successfully in epileptic seizures. Conversely, inhibition of the kinase with pharmaceuticals that cross the blood-brain barrier could increase attention and the ability to learn. Activators of kinases have also long been known to the person skilled in the art, of which the protein kinase C activators are of particular interest (see, for example, Calbiochem-Novablochem 1998 General Catalog, loc. Cit.). Additional kinase activators are available from other commercial and non-commercial sources known to those skilled in the art.

Since the Na ⁺ , K ⁺ , 2Cl ^- 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 lead to hypertension and reduced expression of the kinase to hypotension.

The present invention thus also relates to the use of h-sgk inhibitors for Manufacture of medicines for the treatment of diabetes mellitus, arteriosclerosis, M. Alzheimer's, cirrhosis of the liver, Crohn's disease, fibrosing pancreatitis, pulmonary fibrosis, chronic Bronchitis, radiation fibrosis, scleroderma, cystic fibrosis and other fibrosis Diseases and therapy for arterial hypertension. Furthermore, you can Medicines containing inhibitors or activators of h-sgk for the regulation of the neuronal Excitability can be used. The use of the inhibitors is particularly advantageous Staurosporine or chelerythrine and their analogues.

RESULTS Diabetic kidney

The h-sgk is expressed only sparingly in the normal kidney. Single cells in glomerulum, late proximal and distal tubules show clear h-sgk expression. In contrast in addition there are clusters of cells with massive h-sgk- in the diabetic kidney Expression.  

arteriosclerosis

In the vascular walls of arteriosclerotic vessels, massive amounts of h-sgk- expressing cells.

M. Alzheimer

There are only a few cells in the normal brain that express h-sgk. These cells are probably oligodentroglia cells. In brains with Alzheimer's disease, the number h-sgk- expressing cells significantly increased.

Cirrhosis of the liver

In the normal liver, only copper cells express h-sgk. This is the case with cirrhosis of the liver Tissue, however, is strewn with h-sgk-expressing cells.

Crohn's disease

In normal intestinal tissue, the h-sgk is only expressed in the enterocytes. At However, Crohn's disease, the kinase is also found in the connective tissue.

Fibrous pancreatitis

In the normal pancreas, the h-sgk is found in azinar cells and also in duct cells. There are isolated h-sgk-expressing mononuclear cells around the pancreatic ducts. At fibrosing pancreatitis, the expression of the kinase is significantly increased.

Pulmonary fibrosis and chronic bronchitis

Massive expression of h-sgk is seen in pulmonary fibrosis and chronic bronchitis observed.

Stimulation of h-sgk expression by TGFβ ₁

The expression of h-sgk is stimulated by TGFβ ₁ ( Fig. 1). Since TGFβ _{1 is formed} in fibrosis-inflamed tissue, this finding explains the increased expression of h-sgk in inflamed tissue.

TGFβ ₁ stimulates the expression of the matrix protein Biglycan, an effect that is prevented by the NKCC inhibitor furosemide.

TGFβ ₁ stimulates the expression of biglycan. In the presence of the NKCC inhibitor furosemide, the effect of TGFβ ₁ on biglycan expression is completely prevented. So the fibrosing effect of TGFβ ₁ requires activation of the NKCC ( Fig. 2).

Stimulation of the NKCC by h-sgk

The increased expression of the kinase in fibrosing tissue could have various meanings, which is not causally related to the fibrosis. However, experiments with the two-electrode voltage clamp show that the activity of the NKCC is massively stimulated by the h sgk ( Fig. 3). In view of the furosemide sensitivity of the biglycan synthesis, this finding clearly proves a causal role of h-sgk in the fibrosis.

Stimulation of the ENaC by h-sgk

This effect can be prevented by the kinase inhibitors staurosporine and chelerythrine. As Fig. 4 shows, the current through ENaC increases massively through co-expression with the h-sgk. The kinase therefore stimulates the ENaC. The activation of the ENaC by the h-sgk can be completely prevented by the kinase inhibitors staurosporine and chelerythrine.

The stimulation of the epithelial ENaC by the h-sgk can be reversed by coexpression of the transdominant inhibitory kinase h-sgk:
As Fig. 5 shows, the stimulating effect of h-sgk coexpression on the ENaC-mediated Na ⁺ current can be prevented by coexpression of a transdominant inhibitory kinase. This transdominant inhibitory kinase (compare with "definitions") is so modified on the catalytic unit that it can no longer function. However, since it attaches to the substrate, it displaces the active kinase and thus suppresses its effect. The transdominant inhibitory kinase not only suppresses the increase in ENaC activity by exogenous h-sgk, but also apparently suppresses stimulation by the endogenous h-sgk.

MDEG is completely switched off by coexpression with h-sgk:
As Fig. 6 shows, expression of MDEG induces a strong Na ⁺ current in oocytes, 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 the h-sgk inhibits neuronal excitability.

Examples example 1 In situ hybridization

Tissues from normal pancreas, liver, vessels, brain, lungs, kidney and intestine, as well Tissue with diabetic nephropathy, arteriosclerosis, Alzheimer's disease, cirrhosis of the liver, M. Crohn's, fibrosing pancreatitis and pulmonary fibrosis were in 4% paraformaldehyde / 0.1 M Sodium phosphate buffer (pH 7.2) embedded in praffin for 4 hours. Tissue sections were dewaxed and hybridized as previously described (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. Mali, R. Kandolf, Proc. Natl. Acad. Sci. USA, 89: 314-318, 1992).

The hybridization mixture contained either ³⁵ S-labeled sense RNA coding for h-sgk or ³⁵ S-labeled antisense RNA (500 ng / ml in each case) complementary to the latter in 10 mM Tris-HCl, pH 7.4; 50% (vol / vol) deionized formamide; 600 mM NaCl; 1mM EDTA; 0.02% polyvinyl pyrrolidone; 0.02% Ficoll; 0.05% calf serum albumin; 10% dextran sulfate; 10 mM dithiothreitol; 200 µg / ml denatured sonicated salmon sperm DNA and 100 µg / ml rabbit liver tRNA.

Hybridization with RNA samples 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 for 1 Incubated for 1 hour at 55 ° C in 2 × standard sodium citrate. Single-stranded RNA not hybridized Samples were analyzed by RNase A (20 µg / ml) in 10 mM Tris-HCl, pH 8.0 / 0.5 M NaCl for 30 min digested at 37 ° C. Tissue samples were then autoradiographed for three weeks (Klingel et al., 1992) and stained with hematoxylin / eosin.

Example 2 Transcriptional regulation of Biglykan and the h-sgk

Cells were cultured in RPMI / 5% CO _2/10 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) (approx. 0.4 × 10 ⁶ per sample). Total RNA was prepared according to the manufacturer's instructions. Northern blots were electrophoresed with 15 or 20 µg total RNA with separate control in the presence of 2.4 mol / l formaldehyde through 10 g / l agarose gels. Using a vacuum (Appligene Oncor Trans DNA Express Vacuum Blotter, Appligene, Heidelberg, Germany), RNA was transferred to positively charged nylon membranes (Boehringer Mannheim, Germany) and crosslinked under ultraviolet light (UV Stratalinker 2400, Stratagene, Heidelberg, Germany) . Overnight was hybridized with DIG-Easy-Hyb (Boehringer Mannheim) at a sample concentration of 25 µg / l at 50 ° C. The digoxigenin (DIG) labeled samples were generated by PCR as previously described in detail (Waldegger et al. (1997) PNAS 94: 4440-4445). For autoradiography, the filters were on average 5 min. exposed to an X-ray film (Kodak).

Example 3 Two-electrode voltage clamp and tracer flow experiments

The dissection of Xenopus laevis, the extraction and treatment of the oocytes have been described in detail earlier (Busch et al. 1992). The oocytes were injected with 1 ng cRNA from NKCC, ENaC or MDEG with or without simultaneous injection of the h-sgk. Two-electrode voltage and current clamp experiments could be performed 2-8 days after injection. Furosemide-inhibitable Na ⁺ influx by the NKCC was determined by ²² Na ⁺ uptake in the oocytes, which was determined using a scintillation counter. Na ⁺ currents (ENaC) were filtered at 10 Hz and recorded with a recorder. The experiments were usually carried out on the 2nd day after cRNA injection. The bath solution contained: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , and 5 mM HEPES at pH 7.5 and the holding potential was -50 mV. In all experiments, the pH was adjusted by titration with HCl or NaOH. The flow rate of the bath liquid was set to 20 ml / min, which ensured a complete change of solution in the measuring chamber within 10-15 s. All data are given in the form of arithmetic mean ± SEM.

Figure legends

Fig.

1
Stimulation of h-sgk expression by TGFβ ₁

:
Expression of h-sgk is expressed by TGFβ ₁

stimulates. The effect of TGFβ _{1 is shown}

after 0.5 to 6 h (top). Phorbol ester PDD (4-alpha-phorbol-12,13-didecanoate; stimulates protein kinase C) and Ca ⁺⁺

Ionophore ionomycin (Sigma, loc. Cit .; increases intracellular Ca ⁺⁺

Concentration) also stimulate h-sgk expression (bottom).

Fig.

2nd
Stimulation of biglycan expression by TGFβ ₁

:
Biglycan expression becomes osmotic cell swelling (hypo, top left) and by TGFβ ₁

(top right) stimulated. In the presence of the NKCC inhibitor bumetanide (bum), the effect of TGFβ is ₁

on the Biglycan expression almost completely prevented.

Fig.

3rd
Stimulation of the NKCC by h-sgk:
The furosemide-inhibitable uptake of ²²

Well ⁺

in oocytes that express the NKCC is massively stimulated by the h-sgk. NKCC injected oocytes show no higher Na ⁺

Inflow as non-injected oocytes (ni). This Na ⁺

Influx is not inhibited by the NKCC inhibitor furosemide (top). Expression of the h-sgk alone does not stimulate the Na ⁺

- inflow. Co-expression of h-sgk with NKCC leads to a strong increase in Na ⁺

Inflow, which is completely prevented by furosemide (below).

Fig.

4th
Stimulation of the ENaC by h-sgk:
The current through the ENaC (I) increases massively through co-expression with h-sgk. Treatment of the oocytes with the kinase inhibitors staurosporine or chelerythrine prevents the activation of Na ⁺

-Channels through h-sgk.

Fig.

5
The stimulation of the ENaC by h-sgk can be reversed by coexpression of the transdominant-inhibitory kinase:
Oocytes that express ENaC and h-sgk simultaneously have much larger currents (I) than oocytes that only express ENaC. Co-expression of the transdominant inhibitory kinase prevents stimulation of the ENaC by the h-sgk.

Fig.

6
Inhibition of MDEG by h-sgk:
The current through the MDEG (I) increases with the duration of the incubation (days 1-4). The current is completely prevented by co-expression with h-sgk.

Claims (9)

1. Medicament containing an inhibitor of cell volume regulated human kinase h sgk. 2. Medicament according to claim 1, wherein the inhibitor staurosporine or chelerythrine is. 3. Medicament containing an activator of the cell volume regulated human kinase h sgk. 4. Medicament according to claim 3, wherein the activator cross the blood-brain barrier can. 5. Use of an inhibitor of the cell volume regulated human kinase h-sgk, in particular staurosporine or chelerythrine, for the manufacture of a medicament for Treatment of fibrosis diseases. 6. Use according to claim 5, which is one or more of the following Diseases are: atherosclerosis, Alzheimer's disease, cirrhosis of the liver, Crohn's disease, fibrosing pancreatitis, pulmonary fibrosis, chronic bronchitis, radiation fibrosis, Scleroderma or cystic fibrosis. 7. Use of an inhibitor of the cell volume regulated human kinase h-sgk, in particular staurosporine or chelerythrine, for the manufacture of a medicament for Treatment of diabetes mellitus or arterial hypertension. 8. Use of an activator of the cell volume regulated human kinase h-sgk for Manufacture of a medicament to treat epilepsy. 9. Use according to claim 8, wherein the activator crosses the blood-brain barrier can.