WO2001056590A2

WO2001056590A2 - Utilization of protein kinase inhibitor alpha

Info

Publication number: WO2001056590A2
Application number: PCT/EP2001/001002
Authority: WO
Inventors: Ralph KNÖLL
Original assignee: Schering Ag
Priority date: 2000-02-03
Filing date: 2001-01-31
Publication date: 2001-08-09
Also published as: DE10004858A1; AU2001233723A1; JP2003531114A; EP1253931A2; WO2001056590A3

Abstract

The invention concerns the utilization of PKI alpha and/or the derivatives thereof to lower blood pressure, as beta antagonists and for the production of a medicament for the prevention and/or treatment of diseases in which a beta antagonist can be used. The invention also concerns the utilization of an inhibitor of PKI alpha or a derivative thereof to lower blood pressure, as beta agonist and for the production of a medicament for the treatment and/or prevention of diseases in which a beta agonist can be used.

Description

Use of protein kinase inhibitor alpha

description

The present invention relates to the use of protein kinase inhibitor-alpha (PKI-alpha), the use of inhibitors of protein kinase inhibitor-alpha and methods for screening for beta-agonists and beta-antagonists.

The phenomenon of "myocardial stunning" was first described in 1975. This refers to the property of the myocardium to show long-lasting, reversible myocardial dysfunction after short-term ischemia as a result of coronary occlusions, ie the heart muscle does not show the expected contractility. This reversible state lasts for several hours and contradicts the prevailing view among experts that an ischemic state of more than 45 minutes is irrevocably associated with myocyte death.

The formation of oxygen radicals, calcium overload, dysfunction of the sarcoplasmic reticulum and troponin proteolysis have been discussed as pathogenicity mechanisms for the "myocardial stunning" (Bolli; Mechanism of myocardial "stunning". Circulation 1990: 723-38; and Wijns, Vatner, Camici. Hibernating myocardium, New England Journal of medicine, 1998, 173-81). However, each of these mechanisms is not suitable for fully describing the phenomenon (Duncker et al .; Myocardial stunning remainig questions. Cardiovasc Res 1998; 38: 549-558).

The protein kinase A present in the heart (henceforth also referred to as PKA), which regulates myocardial contractility by means of phospholamban and phosphorylation of troponin I, is controlled by receptors which are coupled to the adenylate cyclase, which in turn is regulated by catecholamines. The enzyme activity is both by protein kinase inhibitors (hereinafter referred to as PKI) and regulated by cAMP. There are two forms of protein kinase inhibitors in the heart: the alpha and beta forms (PKI-alpha and PKI-beta). Both act as competitive inhibitors by blocking the Cα and Cß subunits of active protein kinase A. The α-form is six times more effective than the β-form.

A protein kinase inhibitor known in the art is the protein kinase inhibitor alpha (also referred to herein as PKI-alpha). PKI-alpha has a length of 76 amino acids, whereby the part of the primary sequence that is essential for the inhibitory effect seems to be limited to the range of amino acids 14 to 22. This sequence is 100% identical in humans, pigs, chickens, rats and mice. At the level of the mRNA, the 5 'end of the total porcine mRNA shows a sequence homology of about 90% compared to that of the human, mouse, rat and chicken genome. A recombinant PKI-alpha comprising only amino acids 14 to 22 of the complete PKI-alpha amino acid sequence is cell permeable and available from Calbiochem (product number # 476485).

The porcine PKI-alpha coding cDNA has at its 5 'end a second open reading frame which codes for a 9 amino acid polypeptide and several different poly (A) signal sequences at its 3' end (GenBank # bankit256111 AF132737)

The present invention has for its object to provide new uses for PKI-alpha and for an inhibitor of PKI-alpha.

According to the invention, the object is achieved by using PKI-alpha and / or its derivatives for lowering blood pressure.

In a further aspect of the invention, the object is achieved according to the invention by using PKI-alpha and / or its derivatives as a beta-antagonist. In a still further aspect of the invention, the object is achieved by the use of PKI-alpha and / or its derivatives for the production of a medicament for the prevention and / or treatment of diseases in which a beta-antagonist can be used.

In the use according to the invention it can be provided that the PKI-alpha is one of SEQ ID No. 2 amino acid sequence shown comprises.

In one embodiment of the uses according to the invention, the PKI-alpha is encoded by a nucleic acid which corresponds to the coding sequence of the sequence shown in SEQ ID No. 1 or a sequence resulting therefrom as a result of the degeneracy of the genetic code or a nucleic acid hybridizing thereon.

In the uses according to the invention it can also be provided that the PKI-alpha is used in a shortened form.

In a preferred embodiment of the uses according to the invention it can be provided that the PKI-alpha contains the amino acids 14 to 22 of the PKI-alpha sequence, in particular the sequence of PKI-alpha according to SEQ ID No.1 or SEQ ID No. 2 includes.

In particularly preferred embodiments of the uses according to the invention it is provided that the PKI-alpha or its derivative is used for the treatment and / or prevention of diseases which are selected from the group consisting of cardiac arrhythmias, hyperkinetic cardiac syndrome, angina pectoris, myocardial infarction, acute myocardial infarction , Heart failure, arterial hypertension, essential and renal hypertension, portal hypertension, bleeding from esophageal varices, pheochromocytoma, overdosage of beta-sympathomimetics and choline receptor blockers, glaucoma simplex, hyperthyroidism, thyroid toxicity regimen, migraines, and migraines, migraines, migraines, erectile dysentery, migraines, migraines, and migraines, migraines, migraines, migraines, migraines, and migraines. In further particularly preferred embodiments of the uses according to the invention, it is provided that the PKI-alpha or its derivative is used for post-infarct medication, prophylaxis of the reinfarcts and / or as a sedative.

According to the invention, the object is also achieved by using an inhibitor of PKI-alpha or a derivative thereof for increasing blood pressure.

In a further aspect, the object is achieved by using an inhibitor of PKI-alpha or a derivative thereof as a beta agonist.

In a still further aspect, the object is achieved by using an inhibitor of the PKI-alpha or a derivative thereof for the manufacture of a medicament for the treatment and / or prevention of diseases in which a beta-agonist can be used.

In a preferred embodiment of the uses according to the invention, it is provided that the inhibitor influences the transcription of the PKI-alpha.

In the use according to the invention it can further be provided that the PKI-alpha is a PKI-alpha as described herein.

In a further embodiment of the uses according to the invention it is provided that the inhibitor of the PKI-alpha comprises an amino acid sequence which is selected from the group consisting of the sequences with the SEQ ID No. 3 and SEQ ID No. 4 includes.

In particularly preferred embodiments of the invention

Uses the PKI-alpha inhibitor is intended to be used to treat and / or prevent diseases selected from the group consisting of heart failure, heart attack, chronic and acute heart failure and hypotension. In a further aspect, the object is achieved by a method according to the invention for screening for agents for lowering blood pressure, beta-antagonists and / or agents for the treatment and / or prevention of

Diseases where a beta-antagonist can be used, it is contemplated that

a mixture of a beta receptor, a beta agonist of the beta receptor and a candidate beta antagonist is provided, and

the extent to which the binding behavior of the beta agonist to the beta receptor changes as a result of the candidate beta antagonist.

In one embodiment of the method according to the invention it is provided that the change in the binding behavior of the beta-agonist to the beta-receptor observed under the influence of the candidate beta-antagonist is compared with that under the influence of the beta-antagonist as described herein , observed change in the binding behavior of the beta agonist to the beta receptor.

In a preferred embodiment of the method according to the invention it is provided that the beta receptor is selected from the group comprising betal and beta2 receptors.

In yet another embodiment of the method according to the invention it is provided that the beta agonist is a sympathomimetic, in particular one which is selected from the group comprising noradrenaline, adrenaline, dopamine and dobutamine.

Finally, it can be provided in the method according to the invention that the beta-agonist is a beta-agonist as described herein. In a still further aspect, the object is achieved by a method of screening for agents for lowering blood pressure, beta-agonists and / or agents for the treatment and / or prevention of diseases in which a beta-agonist can be used, wherein

a transcription system for a beta-antagonist is provided,

a candidate beta agonist is added to the transcription system and

To what extent the transcription for the beta-antagonist changes due to the beta-agonist.

In a preferred embodiment of the method according to the invention it is provided that the change in the transcription of the beta-antagonist observed under the influence of the candidate beta-agonist is compared with the change in the observed under the influence of the beta-agonist as described herein Transcription of the beta antagonist.

In a further embodiment of the method it is provided that the beta-antagonist is one as described herein.

In yet another embodiment of the method according to the invention it is provided that the beta-antagonist can be obtained by one of the methods according to the invention.

The present invention is based on the surprising finding that PKI-alpha has a negative inotropic, that is to say an effect which worsens the myocardial function. This effect comes about through an inhibition of protein kinase A, which is responsible for the phosphorylation of Phospholamban and troponin is responsible (so-called beta-adrenergic signal transduction). As a result of the negative inotropic effect of PKI-alpha, inhibition of PKI-alpha in turn improves myocardial function, particularly in the case of heart failure.

The uses according to the invention are all based on the knowledge described above that PKI-alpha has a concentration-dependent, negative inotropic effect and is involved in beta-adrenergic signal transduction. In one aspect, this means that PKI-alpha can be used to lower blood pressure or to produce a medicament. In another aspect, it follows that PKI-alpha can be used as a beta antagonist. The latter is due to the fact that the effect of the beta-receptors binding the catecholamines on the contractility-regulating protein kinase A can be counteracted by the protein kinase inhibitors, which thus show a beta-antagonistic effect.

As a result of the beta-antagonistic effect of the PKI-alpha, it is also suitable for the manufacture of medicaments for the treatment of diseases in which a beta-antagonist is generally used. In other words: due to the beta-adrenergic effect of PKI-alpha, this or derivatives thereof can be used in medication in place of or in addition to the beta-antagonist (s) previously used. Basically, there are no restrictions, so that PKI-alpha or a medicament containing it now provides a replacement for any medicament containing a beta-antagonist as pharmaceutical active ingredient.

Since the PKI-alpha is not only expressed in muscle tissue, but also in nerve tissue, in particular in the brain, neurophysiological fields of indication for PKI-alpha and inhibitors thereof are also within the scope of the present invention. As an example, reference is made here to migraines, for the therapy of which beta-antagonists are used are, which can be replaced by PKI-alpha according to the invention. It is apparent to the person skilled in the art that any tissue which expresses PKI-alpha and develops pathological or changed conditions can be treated therapeutically or preventively according to the invention with PKI-alpha and inhibitors of the same.

There are a number of advantages with the uses according to the invention. Thus, with the use of the PKI-alpha and an inhibitor thereof, the group of pharmaceutically active compounds is expanded considerably, particularly to the extent that a completely new class of compounds is introduced into this therapeutically very important indication area. In addition, the use of PKI-alpha and inhibitors thereof is also advantageous in that they start very early in the cascade of beta-adrenergic signal transduction and are therefore not subject to the undesired down-regulation of the beta-receptors which is associated with various disadvantages. These advantages will be further explained in the following using heart failure.

Heart failure is one of the most important diseases in western industrialized countries due to its high prevalence (1-1.5%) and morbidity of the adult population as well as its correspondingly high costs (Sharpe N. et al .; Lancet 352 (suppi I), 3 - 7 (1998)). A number of medications are used to treat them, but only a few of them are really effective, ie they will continue to be effective for many years to come. These mainly include ß-blocking substances and ACE inhibitors (Cleland, J; Lancet 352 (suppi I), 1 - 2 (1998)). In contrast, the calcium antagonists (e.g. adalat-nifedipine) that are still in use have fallen into disrepute (Prescrire Int 7 (35), 90-91 (1998); and Ishibashi, Y. et al .; Clin Exp Pharmacol Physiol 26 (5-6 ), 404-10 (1999)). In particular, their acute effects, which seem to increase the mortality of the treated patients, call for caution when using these drugs. In contrast, ß-blockers seem to have considerable advantages, particularly in terms of mortality, for the affected patients. Unfortunately, these drugs work via the ßl- and ß2- Receptors that are subject to the so-called down regulation and can therefore miss their place of action (Massie, B .; Lancet 352 (suppi I), 29-33 (1998). For this reason, substance classes are increasingly sought which have their place of action further above the ß- As a result of the inhibition of protein kinase A, especially by intracoronary administration, the PKI-alpha, in particular amino acids 14-22 thereof, appears to belong in the desired substance class.

In this context, an inhibitor of PKI-alpha appears to be particularly interesting, in particular the inhibitor of PKI-alpha (PKI-alpha-1) encoded by the second open reading frame of PKI-alpha. Thus, as desired, both the beta-antagonistic and the beta-agonistic effects can be brought to bear by using PKI-alpha or an inhibitor thereof.

The amino acid sequence of PKI-alpha is shown in SEQ ID No.1 together with the nucleic acid sequence encoding it and as an amino acid sequence as such in SEQ ID No. 2. It turns out for the person skilled in the art that the PKI-alpha is not restricted to the PKI-alpha which contains the sequences SEQ ID No. 1 and SEQ ID No. 2 sequences shown. Rather, a PKI-alpha is understood here to mean any protein kinase inhibitor which influences the activity of protein kinase A. To this extent, the term protein kinase inhibitor is based on a functional definition.

As a result of the functional definition of the term protein kinase inhibitor used herein, the nucleic acid sequence shown in SEQ ID No.1 is also only an example of a sequence coding for a protein kinase inhibitor. Other sequences result from the degeneracy of the genetic code.

The protein kinase inhibitor-alpha has a length of 76 amino acids in its native form. In fact, that extends to the inhibitory Effect-relevant region of the primary sequence of amino acids 14 to 22, so that shortened ("truncated") forms of protein kinase inhibitors, in particular PKI-alpha, can also be used according to the invention. The use of PKI-alpha, whose Sequence is limited to amino acids 14 to 22 of the complete amino acid sequence This shortened PKI-alpha is membrane-permeable or membrane-permeable, ie it can enter the intracellular space via a cytoplasmic membrane and there the intracellular compartments, in particular the sarcoplasmic reticulum and the protein kinase A, PKI-alpha and in particular a shortened form and especially amino acids 14 to 22 of the complete primary sequence of the PKI-alpha, as shown in SEQ ID No. 1 or SEQ ID No.2, can thus achieve an intracoronary, intraperitoneal form or given intravenously.

It is within the capabilities of the person skilled in the art that PKI-alpha and its inhibitor can be present in modified form. Such a modification can take place, for example, in connection with the requirements of galenics. Other modifications can affect the pharmacological and toxicological properties.

Formulations comprising PKI-alpha or an inhibitor thereof may include the usual pharmaceutical excipients and ingredients such as pharmaceutically acceptable carriers and buffers.

Beta blockers, i.e. Beta-antagonists, and thus PKI-alpha and their derivatives according to the invention, are used, for example, as or for

1. Antiarrhythmic drugs (irregular heartbeat)

2. Hyperkinetic heart syndrome

3. Anti-anginal drugs (angina pectoris) 4. Improvement of the prognosis in patients with myocardial infarction (post-infarct medication), acute myocardial infarction and prophylaxis of the pure infarct

5. Therapy for heart failure

6. Antihypertensives (arterial hypertension, essential and renal hypertension)

7. Portal vein hypertension, bleeding from esophageal varices

8. pheochromocytoma (only applicable after previous alpha blockade)

9. Overdose of ß-sympathomimetics and m cholinoceptor blockers

10. Glaucoma on the eye (Glaucoma simplex)

11. Hyperthyroidism and thyrotoxicosis

12. Therapy of central nervous diseases such as migraines and as a sedative (central damping effect) (essential tremor,

Arousal tremor, alcohol withdrawal syndrome)

The use according to the invention of an inhibitor of PKI-alpha (hereinafter also referred to as PKI-alpha-1) is based in turn on the surprising finding that PKI-alpha has a concentration-dependent, inotropic effect and is involved in beta-adrenergic signal transduction. In the light of these properties of PKI-alpha, an inhibitor of PKI-alpha can modify and reverse the effects of PKI-alpha. As a result, an inhibitor of PKI-alpha can be used to increase blood pressure or a suitable medication. After the inhibitor of PKI-alpha has been inhibited by an inhibitor of the beta adrenergic signal transmission, it acts as a beta agonist and can be used accordingly. This use generally includes the use in the manufacture of a medicament for the treatment and prevention of diseases in which beta-agonists can be used: thus an inhibitor of PKI-alpha can be used in medication in addition to the beta-agonist (s) previously used become. Basically, there are no restrictions, so that the PKI-alpha inhibitor now provides a replacement for any medicament containing a beta-agonist as active pharmaceutical ingredient.

The term inhibitor of PKI-alpha is based on a functional definition. An inhibitor of PKI-alpha is thus any compound which inhibits the action of PKI-alpha, irrelevant to what extent the inhibition of PKI-alpha takes place, to which chemical compound class the inhibitor of PKI-alpha can be assigned and on based on what mechanism the PKI-alpha inhibitor works.

A mechanism that can be the basis of the action of an inhibitor of PKI-alpha is the inhibition of the transcription of PKI-alpha. The gene product of the second open reading frame of the PKI-alpha gene, for example, acts as an inhibitor in this sense. It appears from Wang X. et al .; that the gene product can be the active agent. Mol Pharm 54, 514-524 (1998). The amino acid sequence of this human gene product was

Met Trp He Phe Gly Ser Asn Asp

and is as SEQ ID No. 3, together with the nucleic acid sequence encoding it, was incorporated herein.

The corresponding pork sequence is Met Trp lle Phe Val Ser Asn Asp

and is as SEQ ID No. 4, together with the nucleic acid sequence encoding it, was incorporated herein.

The difference between the two types of humans and pigs in the form of an amino acid exchange at position 5 of the amino acid sequence is based on an exchange at the nucleic acid level, with position 2 of the fifth codon being changed. In the case of the sequence in humans, glycine is found as the fifth amino acid, whereas in the case of the pig sequence it is valine. The corresponding base exchange is a change from G (human) to T (pig).

It is obvious to the person skilled in the art that deviations from those in SEQ ID No. 3 and SEQ ID No. 4 disclosed sequences are possible as long as the derivative derived from the sequences still has an inhibitory effect on PKI-alpha, in particular its transcription.

Beta agonists and thus inhibitors of PKI-alpha, as disclosed herein, can be used for

1. Increase in contractility of the myocardium (desirable for severe heart failure or in heart attack patients (chronic heart failure and acute heart failure)

2. Hypotension

It is obvious to the person skilled in the art that the uses according to the invention are therapeutic and preventive Treatments can be used, especially on people who need such treatment.

The methods according to the invention are methods by means of which compounds are determined or provided which are known as PKI-alpha, i.e. can be used as inhibitors of protein kinase A or as inhibitors thereof, in particular in connection with one or more of the diseases or indication fields disclosed herein. The methods can also be directed to the fact that one or more of a number of compounds should be determined with a corresponding property (“screening”). A compound which may have a corresponding property is also referred to herein as "Candidate" connection referred to.

In the method according to the invention for determining or screening a compound having a beta-antagonistic effect in the broadest sense, the receptor provided can be present as an isolated molecule, for example in solution or immobilized. However, it is also within the scope of the present invention that the receptor is present in a biological system. Such a biological can be an in vitro system or an in vivo system. A typical in vitro system is a translation or transcription system. A typical in vivo system is a cellular system, for example a cell, in particular a myocardial or nerve cell, which contains the genetic information for PKI-alpha. However, a cellular system can also be a cell into which, preferably by means of genetic engineering methods, a nucleic acid coding for PKI-alpha or a part thereof or its or its expression controlling nucleic acid is introduced. When using the in vivo and in vitro systems, use can be made of the stress inducibility of PKI-alpha, which is also disclosed here. In the method according to the invention it can further be provided that the effectiveness of a candidate antagonist is assessed on the basis of a comparison of the activity of PKI-alpha and its derivatives, as described herein. As a result, the effectiveness of the tested compounds can be quantified at a very early stage in the screening or development process of novel beta-antagonists and beta-agonists. The method according to the invention can also provide that a beta agonist as described herein is used.

In the method according to the invention for determining or screening a compound having a beta-agonistic effect in the broadest sense, the beta-agonist provided can be present as an isolated molecule, for example in solution or immobilized. However, it is also within the scope of the present invention that the candidate agonist is present in a biological system. Such a biological system can be an in vitro system or an in vivo system. A typical in vitro system is typically a cell-free translation or transcription system. A typical in vivo system is a cellular system, for example a cell, in particular a myocardial or nerve cell, which contains the genetic information for the candidate beta-agonist. However, a cellular system can also be a cell into which, preferably by means of genetic engineering methods, the nucleic acid coding for the candidate beta-agonist or a part thereof or its or its expression controlling nucleic acid is introduced.

The transcription system for a beta-antagonist used in the process according to the invention can be an in vivo system or an in vitro system. A typical in vitro system is a translation or transcription system. A typical in vivo system is a cellular system, for example a cell, in particular a myocardial or nerve cell, which contains the genetic information for PKI-alpha. On However, the cellular system can also be a cell into which, preferably by means of genetic engineering methods, the nucleic acid coding for PKI-alpha or a part thereof or its or its expression-controlling nucleic acid is introduced. When using the in vivo and in vitro systems, use can be made of the stress inducibility of PKI-alpha, which is also disclosed here.

It is also within the scope of the method according to the invention that the transcription system for a beta-antagonist also includes the candidate beta-agonist or the nucleic acid coding for it or controlling its expression. Furthermore, it is within the scope of the methods according to the invention that beta-antagonists and beta-agonists as disclosed and described herein or that can be determined or determined as such by one of the methods disclosed herein are used therein.

In the method according to the invention it can further be provided that the effectiveness of a candidate beta agonist is assessed on the basis of a comparison of the change in the transcription of the beta antagonist observed under the influence of the candidate beta agonist with that under the influence of a beta -Agonists, as described herein, observed changes in beta antagonist transcription. As a result, the effectiveness of the tested compounds can be quantified at a very early stage in the screening or development process of novel beta-agonists.

The invention will be further illustrated by the following figures, examples and the sequence listing, from which further features and advantages of the invention may result. It shows:

Fig. 1 shows the function of protein kinase A and PKI-alpha in beta-adrenergic signal transduction: More precise

Signal transduction cascade of the ßl and ß2 receptors (ßl and ß2 AR) shown that activate the protein kinase A via a, ß and y-G protein subunits as well as the adenylate cyclase (AC) and cAMP increases. The protein kinase A can be inhibited by the PKI-alpha, whereby the effects of the protein kinase A can be antagonized. Protein kinase A phosphorylates in particular phospholamban, which explains the positive inotropic effect.

Fig. 2 shows the decrease in the function of the left cardiac ventricle

Administration of PKI-alpha expressed as dp / dt and external cardiac work (EHW).

EXAMPLES

Example ^ Investigations of the effect of PKI-alpha on a working rat heart

The hearts of a total of six Sprague Dawley rats weighing approximately 300 g were isolated and, as in Schulze et al. (Schulze et al .; Circulation 1990: 959-69). Hemodynamic parameters including contractility, external cardiac work (EHW), coronary artery and aortic flow were recorded approximately 15 minutes after antegrade perfusion. Cell permeable, recombinant PKI-alpha (amino acids 14 to 22, Calbiochem # 476485) was then administered. The compound was infused directly into the atrium cannula using a motor pump. The infusion rate was continually adjusted to the ejection volume (the sum of aortic and coronary flow) and increased at five-minute intervals in order to reach the final PKI-alpha concentrations in the perfusate of 0.5, 1.0 and 2.0 μmol / l. The results relating to the external heart volume and the course of dp / dt _max under these test conditions are shown in FIG. 2. The infusion of the shortened -PKI-alpha caused a pronounced, reversible, concentration-dependent (up to 2 μmol) depression of the myocardial function, which was comparable to the dysfunction observed after short coronary occlusion. The measured reduction in external cardiac work and dp / dt-max was significant in the paired t-test for all concentrations tested. Perfusion with the shortened PKI-alpha led to a reduction in external cardiac work by 43% and a decrease in left ventricular function, expressed as dp / dt-max, by 53%.

It follows from these studies that the shortened form of the PKI-alpha is suitable for lowering blood pressure and in general for use as a new form or class of substance of a beta-antagonist.

Example 2: Investigations on cell cultures

The protein kinase inhibitor-alpha, truncated to amino acids 14 to 22, was added to cell cultures of myocytes (e.g. C9H2 cells, available from the American Type Culture Collection) and the activity of protein kinase A was determined. A dose-dependent decrease in activity was found.

The 9 amino acid long PKI-alpha inhibitor (the gene product of the second open reading frame of the PKI gene in humans and in pigs) was added to cell cultures of myocytes and the expression of the PKI-alpha and the activity of the protein kinase A were determined. An increase in the activity of the PKA depending on the dose of the PKI-alpha inhibitor was found.

Cell cultures of myocytes were transfected with an expression plasmid with the sequence of PKI-alpha, as described, for example, in Olsen (Olsen et al .; Endocrinology 5; 1246 - 1256 (1991)) and the expression of the PKI- alpha determined. At the same time, the activity of protein kinase A, which is specifically inhibited by PKI-alpha, was determined. (Protein kinase A is contained in both striated and smooth muscle cells (Wang X. et al .; Mol Pharm 54, 514-524 (1998))). Various concentrations of the protein kinase inhibitor-alpha-inhibitor (PKI-alpha-1) were added to these cell cultures. The expression of the PKI-alpha was reduced depending on the concentration of the PKI-alpha inhibitor.

The disclosure of the various references cited herein is hereby fully incorporated by reference.

The features of the invention disclosed in the above description, the claims and the drawing can be essential both individually and in any combination for the implementation of the invention in its various embodiments.

Claims

Expectations

1. Use of PKI-alpha and / or its derivatives for lowering blood pressure.

2. Use of PKI-alpha and / or its derivatives as a beta antagonist.

3. Use of PKI-alpha and / or its derivatives for the manufacture of a medicament for the prevention and / or treatment of diseases in which a beta-antagonist can be used.

4. Use according to one of claims 1 to 3, characterized in that PKI-alpha one in SEQ ID No. 2 shown

Amino acid sequence includes.

5. Use according to one of claims 1 to 3, characterized in that the PKI-alpha is encoded by a nucleic acid which corresponds to the coding sequence of SEQ ID No. 1 shown sequence or one resulting from the

Degeneracy of the genetic code or a hybridizing nucleic acid.

6. Use according to one of claims 1 to 5, characterized in that the PKI-alpha is used in a shortened form.

7. Use according to claim 6, characterized in that the PKI-alpha amino acids 14 to 22 of the PKI-alpha sequence, in particular the sequence of PKI-alpha according to SEQ ID No.1 or SEQ ID No. 2 includes.

8. Use according to one of claims 1 to 7, characterized in that the PKI-alpha for treatment and / or Disease prevention is used, which are selected from the group consisting of cardiac arrhythmias, hyperkinetic heart syndrome, angina pectoris, myocardial infarction, acute myocardial infarction, heart failure, arterial hypertension, essential and renal hypertension, portal vein hypertension, bleeding from oesophageal varices,

Pheochromocytoma, overdose of beta-sympathomimetics and choline receptor blockers, glaucoma simplex, hyperthyroidism, thyrotoxicosis, migraine, essential tremor, excitement tremor and alcohol withdrawal syndrome.

9. Use according to one of claims 1 to 8, characterized in that the PKI-alpha is used for post-infarct medication, prophylaxis of the reinfarcts and / or as a sedative.

10. Use of an inhibitor of PKI-alpha or a derivative thereof for increasing blood pressure.

11. Use of an inhibitor of PKI-alpha or a derivative thereof as a beta agonist.

12. Use of an inhibitor of PKI-alpha or a derivative thereof for the manufacture of a medicament for the treatment and / or prevention of diseases in which a beta agonist can be used.

13. Use according to one of claims 10 to 12, characterized in that the inhibitor influences the transcription of the PKI-alpha.

14. Use according to one of claims 10 to 13, characterized in that the PKI-alpha is a PKI-alpha according to one of claims 1 to 9.

15. Use according to one of claims 10 to 14, characterized in that the inhibitor of PKI-alpha comprises an amino acid sequence which is selected from the group comprising the sequences with SEQ ID No. 3 and SEQ ID No. 4 includes.

16. Use according to any one of claims 10 to 15, characterized in that the inhibitor of PKI-alpha is used for the treatment and / or prevention of diseases which are selected from the group consisting of heart failure, heart attack, chronic and acute heart failure and hypotension includes.

17. A method of screening for agents for lowering blood pressure, beta-antagonists and / or agents for the treatment and / or prevention of diseases in which a beta-antagonist can be used, characterized in that

18. The method according to claim 17, characterized in that the change in the binding behavior of the beta-agonsite to the beta-receptor observed under the influence of the candidate beta-antagonist is compared with that under the influence of the beta-antagonist according to one of claims 1 to 9 observed change in the binding behavior of the beta agonist to the beta receptor.

19. The method according to claim 17 or 18, characterized in that the beta receptor is selected from the group comprising betal and beta2 receptors.

0. The method according to any one of claims 17 to 19, characterized in that the beta agonist is a sympathomimetic, in particular one selected from the group comprising noradrenaline, adrenaline, dopamine and dobutamine.

21. The method according to any one of claims 17 to 20, characterized in that the beta agonist is a beta agonist according to one of claims 10 to 16.

22. A method of screening for agents for lowering blood pressure, beta-agonists and / or agents for the treatment and / or prevention of diseases in which a beta-agonist can be used, characterized in that

a transcription system for a beta-antagonist is provided,

a candidate beta agonist is added to the transcription system and

23. The method according to claim 22, characterized in that the change in the transcription of the beta-antagonist observed under the influence of the candidate beta-agonist is compared with that under the

Influence of the beta-agonist according to one of claims 11 to 16 observed change in the transcription of the beta-antagonist.

24. The method according to claim 22 or 23, characterized in that the beta-antagonist is one according to one of claims 1 to 9.

5. The method according to any one of claims 22 to 24, characterized in that the beta-antagonist is obtainable by a method according to one of claims 17 to 21.