UA79066C2 - Transdominant inhibitory kinase h-sgk in which lysine is substituted in the 127 position by arginine, and its use for preparation the remedy - 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

Description of the invention

The present invention relates to medicinal products containing inhibitors or activators of human p-5oK kinase 2, which regulates cell size. Such drugs are suitable for the treatment of disease states in which increased or decreased expression of y-zdkK is observed. Н-8оК, as well as the method of its preparation, (described in the European patent application EP-0 861 896), the detailed content of which is a component of the proposed description.

Explanation of concepts: p-voK: human serine and glucocorticoid-dependent kinase (Serine/Threonine-Kinase)

Emas: epithelial Ma" channel (sodium channel)

MOEO: rebirth in a mammal (U/aidtapp, K., I agatspeki, M. (1998) Sigtepi Oriop ip Meigorioiodu 8 : 418-424); synonym of the term "VMS" (Ma channel of the brain)

LLC: tumor growth factor B

Mkos: Ma", K-, 2SI- co-carrier

HERE5: (4-(2-hydroxyethyl)-piperazine|-ethanesulfonic acid

EARTH: standard error

Transdominant nominal by mutation p-5dK: lysine at position 127 is replaced by arginine (K 127 K); the mutation occurs in the inhibitory kinase: catalytic region and paralyzes the catalytic function of the kinase.

Increased expression of p-zdkK is clearly expressed in such diseases as: diabetes, arteriosclerosis, Alzheimer's disease, cirrhosis of the liver, Crohn's disease, fibrotic pancreatitis, pulmonary fibrosis and chronic bronchitis.

The increased formation of y-z9k can be explained by the stimulation of expression through the TOREZ factor (Fig. 1). The cause of fibrotic diseases is a high degree of formation and a weak degree of cleavage of matrix proteins. Both SM phenomena are the result of the action of the TOEV factor. In fibroblasts, it is possible to stop the increased expression of matrix o proteins by suppressing MKOS with furosemide (Fig. 2).

Until now, it was not clear whether the increased expression of p-z9K is only a consequence or a cause of the disease.

The data of the analysis confirm that y-zd9k activates Ma", K", 2CI" co-transport (Fig. 3). From this, we can conclude that the stimulation of the MKS with the help of p-zd9k causes fibrosis. Together with Ma 7", K", 2CI " by co-transfer with the help of p-zdK, EMas (Figs. 4 and 5) and MOEb are also activated. Gee)

The stimulatory effect of y-z9K on EMaS can be stopped with the help of a kinase inhibitor, such as staurosporine (Bidta, U-8204 Yueiveppoiep) or chelerythrine (Zidta, Ios.sii) (Fig. 4). In addition, the action of y-zudk on o

EMasS can be stopped, for example, with the help of a transdominant inhibitory kinase (Fig. 5). Therefore, Fu and zoK inhibitors, such as staurosporine, chelerythrine or other kinase inhibitors, could be used in the treatment of the above-mentioned diseases. Basically, all known kinase inhibitors are suitable for these purposes. Kinase inhibitors in many cases can be purchased from a commercial network, for example, the company SaIriospet-Momariospet Str. known to a specialist.

During an epileptic attack, p-z9K is repeatedly expressed. The data we found show that C 70 has beneficial effects that reduce the excitability of neurons, because the activation of MKSS leads to a decrease in the extracellular concentration of K" and, as a result, hyperpolarization and the associated inhibition of neuronal activity. :z" In addition this inhibition of the MOES should restrain the excitability of neurons. Therefore, kinase activators that cross the blood-brain barrier could be successfully used in epileptic seizures. On the contrary, inhibition of the kinase could, in combination with drugs that cross the blood-brain barrier - 15 , increase attentiveness and ability to learn. Kinase activators have long been known to those skilled in the art, of particular interest are C-activators of protein kinase (see SaIbiospet - Momariospet 1998 Sepega! Saiaisod, (Se) Ios.sii.). Other kinase activators can be purchased from other sources known to the specialist commercial and non-commercial sources. o Since Ma", K", 2СИ" co-transport and Ma" channel play an important role in renal Ma" resorption, and 5p puppy renal Ma" - resorption occurs with hypertension, it is worth assuming that the increase in the expression of the kinase

Me leads to hypertension, and a decrease in kinase expression leads to hypotension.

Ф This invention also relates to the use of y-z9K inhibitors for the preparation of drugs for the treatment of diabetes, arteriosclerosis, Alzheimer's disease, cirrhosis of the liver, Crohn's disease, fibrotic pancreatitis, pulmonary fibrosis, chronic bronchitis, radiation fibrosis, scleroderma, cystic fibrosis fibrosis and other fibrous diseases, as well as for the treatment of arterial hypertension. In addition, drugs containing inhibitors or activators of p-39OK can be used to regulate neuronal (F, excitability. The best use as inhibitors is staurosporine or chelerythrine, as well as their analogues. ka Results

Diabetic kidney: 60 p-z9K is only weakly expressed in a normal kidney. Clear expression of p-z3OK is found in individual cells of the kidney glomerulus, late proximal and distal tubules. In contrast, clusters of cells with massive p-5OK expression are found in the diabetic kidney.

Arteriosclerosis:

In the walls of arteriosclerotic vessels, an increase in the array of p-5akK expressing cells is found. 65 Alzheimer's disease:

Only individual cells expressing p-z9K were found in the normal brain. These cells are obviously -D-

oligodendroglial cells. In Alzheimer's disease, there is a significant increase in the number of cells expressing p-5OK.

Cirrhosis:

In a normal liver, y-zadak is expressed only in Kupffer cells. In cirrhosis of the liver, tissues are dotted with cells expressing p-5acK.

Crohn's disease:

In normal intestinal tissue, y-z9K is expressed exclusively in enterocytes. In Crohn's disease, the kinase is also detected in the connective tissue. 70 Fibrous pancreatitis:

In the normal pancreas, p-39K is detected in acinar cells and migrating cells. Single groups of y-z9K - expressing mononuclear cells are found around the entrance to the pancreas. In fibrotic pancreatitis, the expression of kinase is clearly increased.

Pulmonary fibrosis and chronic bronchitis:

Massive expression of p-zoK is observed in pulmonary fibrosis and chronic bronchitis.

Stimulation of p-zac expression using the TOEV factor:

The expression of p-59K is stimulated with the help of the TORErp factor (Fig. 1). Since TOEB 5 are formed in fibrous inflamed tissues, this explains the increased expression of p-5dK in inflamed tissue.

TOEV.u stimulates the expression of the matrix protein biglycan, a process that is suspended under the influence of the MKOS inhibitor furosemide.

Ltd. stimulates the expression of biglycan. In the presence of the MKSS inhibitor furosemide, the effect of the TOEV 4 factor on the expression of biglycan is completely paralyzed. Thus, the fibrotic action of the TOEV factor in assumes activation

ISS (Fig. 2).

Stimulation of MKOS with p-5aK: sch

Elevated kinase expression in fibrotic tissues may have a different meaning that is not causally related to fibrosis. However, experiments with a two-electrode voltage clamp show that the activity of MKSS is massed and) stimulated by Pp-5OoK (Fig. 3). These research data, taking into account the sensitivity of furosemide in the synthesis of biglycan, clearly prove the causal role of p-zaK in fibrosis.

Stimulation of EMas by means of p-5ak: Hezo This process can be stopped by means of kinase inhibitors staurosporine and chelerythrine. As shown in

Fig. 4, the flux through EMas is significantly increased due to co-expression with p-59K. The kinase thus stimulates EMas. Under the influence of staurosporine and chelerythrine kinase inhibitors, it is possible to completely stop the activation of EMasS with the help of p-5OK.

Stimulation of epithelial EMasS with the help of p-z9K can change directly in the opposite direction due to me) co-expression of the transdominant inhibitory p-zOOK kinase:

As shown in Fig. 5, the stimulatory effect of y-zdk-co-expression on the Mo" flow caused by EMasS can be stopped by co-expression of a transdominant inhibitory kinase. This transdominant-inhibitory kinase (see the section "Explanation of concepts") during catalysis changes as follows, that it can no longer develop its " function. But since it is layered on the substrate, it displaces the active kinase and thus inhibits its action. The transdominant-inhibitory kinase inhibits not only the growth of EMaC activity with the help of exogenous i) c B-5OK, but and, as can be seen, suppresses stimulation by endogenous p-509kC. MOES is completely excluded due to co-expression with p-5ak: "" As shown in Fig. b, the expression of MOES induces in oocytes a strong flow of Mo ", which is activated by reducing the extracellular pH value. The channel is completely blocked due to co-expression with y-zoK. This leads to the conclusion that y-z9K suppresses neuronal excitability. -|

Tissues of a normal pancreas, liver, blood vessels, brain, lungs, kidneys and intestines, as well as tissue (av) in diabetic nephropathy, arteriosclerosis, Alzheimer's disease, cirrhosis of the liver, Crohn's disease, fibrotic

FU 50 pancreatitis | pulmonary fibrosis in 495 paraformaldehyde/0.1M sodium phosphate buffer (pH7.2) is poured into paraffin for 4 hours. Paraffin is removed from tissue sections and hybridization is carried out as previously described 4) IKapao!i, K., O. Ateiiv, R. Kigespeg A., Sapy, R.N. Noivsppeideg, Rhos. May Asai. si. OBA 84: 6272-6276, 1987; Nopepadi, S, K.. Kiipde!i, 9U. Megizspipd, R.N. Noivsppeideg, K. Kapadoii, Moi. Sei. Rhobrev 5: 11-20, 1991; Kiipdei, K.., S. Nopepadi, A. Sapy, M. AIBBgespi, M. bZeetapp, b. MaiYi, K. Kapaoii Rgos. May Asai. beat 0 CYZA, 89: 314-318, 19921.

Gee! The hybridized mixture contains either p-zokK coding, C8-labeled sense RNA, or complementary to the last RNA,

C55-labeled antisense RNA (if necessary, 500 ng/ml) in 10 mM Tris-HCl, pH7.4; 50965 (volume/volume) of deionized formamide; BOOMM of the Masses; iM EOTA (ethylenediaminetetraacetic acid); 0.02905 polyvinylpyrrolidone; 0.0295 ficol; 0.0590 calf serum albumin; 1095 dextran sulfate; 10mm 60 dithiothreitol; 200mcg/ml of denatured sonicated (homogenate obtained under the influence of ultrasound) DNA of salmon sperm and 100mg/ml of rabbit liver RNA.

Hybridization with RNA samples is carried out at a temperature of 422C for 18 hours. Carriers of objects are washed, as described in INopepaFdi ei ai., 1991; Kiipday! ei and I., 19921), and then incubation is carried out for 1 hour at a temperature of 55°C in 2x standard sodium citrate. Unhybridized samples of single-stranded RNA are subjected to enzymatic degradation with RNase A (20 mg/ml) in 10 mM Tris-HCl, pHe8.0/0.5 M Mass for

REFERENCE at a temperature of 37. Tissue samples are autoradiographed for three weeks (Kiipdei, 1992) and stained with hematoxylin/eosin.

Example 2: Transcriptional regulation of biglycan and p-5okK.

Cells are cultured in KRMI/596 CO2/10 mM glucose at a temperature of 372C, pH7.4, adding up to 1095 (vol/vol) fetal calf serum (EC5). Cells are cultured to 9095 confluence and then homogenized in Trizol (VIVSO/VEI) (approximately 0.4x109U per sample). Total RNA is prepared according to the manufacturer's instructions.

Norton blots are separated by electrophoresis with 15 or 20 μg/ml total RNA with a separate control in the presence of 2.4 mol/l formaldehyde using 10 g/l agar gel. In a vacuum (Arriidepe Opsog Tgapv 70 OMA Ehrgezv Masiit Wioceg, Arriidepe, Heidelberg, Germany), RNA is transferred to positively charged nylon membranes (Woephipdeg Mannheim, Germany) and cross-linked under the influence of ultraviolet light (M

ZigayappKeg 2400, Zigayadape, Heidelberg, Germany). Hybridization is carried out during the night using ris-Eazu-Nuv (Voeigipdeg Mannheim) at a sample concentration of 25mg/l at a temperature of 502C. Digoxigenin (BIS)-labeled samples are obtained by polymerase chain reaction (PCR), as described in detail in |MaIdeddeg eyai!. (1997) RMAB5 94: 4440-4445). For autoradiography, filters are exposed to X-ray film (Kodak) for an average of 5 minutes.

Example 3: Experiments using a two-electrode voltage clamp and labeled atoms.

Dissection of Heporiz Iaemiz, obtaining and processing of oocytes is described in detail in |Vyzsy eiya!|. 1992). Oocytes are injected with cRNA from MKSS, EMaS or MOBES with or without simultaneous injection of y-zodkK. Experiments with two-electrode terminals of voltage and current can be carried out 2-8 days after the injection. Furosemide inhibition of the penetration of Ma" using MKS is detected by the absorption of Ma" by oocytes, which is determined using a scintillation counter. The flow of Ma " (EmMasC) is filtered at a frequency of 10 Hz and recorded using a recorder. Experiments are usually carried out on the second day after the injection of TsRNA.

The solution in the bath contains: Ubmm of sodium chloride, 2mm of potassium chloride, 1.9mm of calcium chloride, 1mm of magnesium chloride and MM of NEREZ at a pH of 7.5 and a maintained potential of -50tM. In all experiments (9), the pH value is set by titration with hydrochloric acid or caustic soda. The flow rate through the bath is 2 Oml/min., thanks to which a complete change of the solution in the measuring chamber is ensured within 10-15 seconds. All data are given in arithmetic mean values -5EM.

Explanation to the drawings

Fig. 1 Stimulation of the expression of n-gdK with the help of the TOEV factor: (Se)

The expression of p-z9Kk is stimulated by the factor TOEV 3. The effect of the factor TOEV is shown. after 0.5-6 hours (above). The phorbol ester ROIU (4-alpha-phorbol-12,13-didecanoate; stimulates protein kinase C) and the ionophore Ca"" o ionomycin (Zidta, Ios.sia; increases the intracellular concentration of Ca"") simultaneously stimulate the expression of /F)

V-hOK (below). m

Fig. 2 Stimulation of biglycan expression using the TOEV factor:

The expression of biglycan (B) is stimulated by osmotic cell swelling (below, above, left) and the factor

Ltd. (right, above). In the presence of the MKSS-inhibitor bumetanide (B), the effect of the factor TOEV 5 on the expression of biglycan is almost completely stopped (control-c). " 20 Fig. 3 Stimulation of MKOSS using n-5OK: z

Absorption of 22Ma" by oocytes, restrained by furosemide (absorption (nmol/20 min./oocyte) -C|, expressing with MKSOS, massed with steam p-voK. o other' and MKS bi ,; ulted with the help of p-5d9K. Oocytes, other "injected" are not characterized by "high Ma penetration" compared to non-injected oocytes (p.i.). Ma penetration is not inhibited by the MKOSS inhibitor furosemide (-E) (above). The expression of y-z9K by itself does not leads to stimulation of penetration of Ma". Co-expression of p-89k with MKOSS leads to a strong increase of penetration of Ma", which is completely paralyzed by furosemide.

Fig.A4 Stimulation of EMas with the help of p-5aKk: i-o Flow through EMas (1) strongly increases due to co-expression with p-zdk. Treatment of oocytes with kinase inhibitors (c) staurosporine (5) or chelerythrine (C) paralyzes the activity of the Mo" channel due to H-5OK.

Fu 50 Fig5 Stimulation of EMa by means of p-z9K can appear in the opposite way due to the co-expression of a transdominant inhibitory kinase: 4) Oocytes simultaneously expressing EMasC and p-z9K show a much higher flux (1) compared to oocytes expressing only EMasS. Co-expression of a transdominant inhibitory kinase stops the stimulation of EMas by p-5OK.

Fig. b Suppression of MOES with P-5oK: o The flow caused by MOES (1) increases as the duration of incubation |day (T) 1-4) increases. As a result of co-expression with p-59K, the flow stops completely (peak-r; plateau:-ri). name) in

Claims (2)

The formula of the invention 1. Transdominantly inhibitory p-zoK kinase, in which lysine at position 127 is replaced by arginine. 2. Use of a transdominantly inhibitory y-zu9K kinase, in which lysine at position 127 is replaced by arginine, for the preparation of a medicinal product for the treatment of diseases accompanied by a decrease in the activity of the epithelial sodium channel and/or the Ma", K", 2CI co-transporter selected from groups: liver cirrhosis, fibrotic pancreatitis, pulmonary fibrosis, radiation fibrosis, scleroderma, cystic fibrosis, chronic bronchitis, arterial hypertension, Crohn's disease and Alzheimer's disease. s o (Se) "so "v) (22) h- " shi s z -I se) ((c) b 50 42) F) name) 60 b5