WO2012013597A1

WO2012013597A1 - Substances and the use thereof for influencing natriuretic peptide receptors

Info

Publication number: WO2012013597A1
Application number: PCT/EP2011/062655
Authority: WO
Inventors: Thomas Walther; Anja Schwiebs
Original assignee: Justus-Liebig-Universität Giessen
Priority date: 2010-07-28
Filing date: 2011-07-22
Publication date: 2012-02-02

Abstract

The invention comprises peptide or non-peptide receptor agonists of the natriuretic system, preferably NPRA and NPRB receptor agonists for the prevention and/or therapy of disease conditions which are accompanied by increased extracellular liquid volume and in which the second messenger cGMP develops protective properties and in which an anti-hypertrophic (antifibrotic) effect is desired. The invention in particular relates to peptide or non-peptide receptor agonists of the natriuretic system for treating congestive heart disease, coronary heart disease, organ and functional vascular disorders, nephrotic syndrome, high blood pressure, myocardial infarction, acute pulmonary damage, erectile dysfunction, primary or secondary fibrosis, cirrhosis of the liver, pulmonary fibrosis or for improving blood circulation of the arteries, veins and capillaries.

Description

Patent application

Name of the invention

Substances and their use for influencing natriuretic

peptide receptors

The present invention relates to peptidic and non-peptidic receptor agonists of the natriuretic system, preferably NPRA and NPRB receptor agonists for the prevention and / or treatment of disease states associated with increased extracellular fluid volume, in particular for the prevention and / or treatment of congestive heart disease coronary artery disease, organic and functional vascular disorders, nephrotic syndrome, high blood pressure disorder or to improve arterial, venous and capillary blood flow.

Likewise, the present invention relates to peptidic and non-peptidic receptor agonists of the natriuretic system, preferably NPRA and NPRB receptor agonists for the prevention and / or treatment of disease states in which the second messenger cGMP develops protective properties, in particular for the prevention and / or treatment of hypertrophic Cardiomyopathy, myocardial infarction, acute lung injury or erectile dysfunction.

Likewise, the present invention relates to peptidic and non-peptidic receptor agonists of the natriuretic system, preferably NPRA and NPRB receptor agonists for the prevention and / or treatment of disease states in which an antihypertrophic (antifibrotic) effect is desired, in particular for the prevention and / or therapy of a primary or secondary fibrosis, liver cirrhosis or pulmonary fibrosis.

Abbreviations and definitions

ACNP: natriuretic peptide of the invention, also referred to as ANP / CNP; Chimera of ANP and CNP

ANP: Atrial Natriuretic Peptide, Atrial Natriuretic Peptide; Also referred to as Atrial Natriuretic Factor, Atriopeptin, Natriuretic Peptide Type A, Cardionatrine, Cardiodilatin, Atrial Natriuretic Factor, Urodilatin

BNP: Brain Natriuretic Peptide; also referred to as B-type Natriuretic Peptides

BNP / CNP: chimera of BNP and CNP

cGMP: cyclic guanosine mono phosphates, cyclic guanosine monophosphate

Chimera: Combination of two or more pieces of peptide to a new peptide that does not occur in the wild type. Chimera also means those nucleic acid sequences which code for the chimeric peptides

CNP: C-type Natriuretic Peptide, C-type Natriuretic Peptide

Diuresis, diuretic: excretion of water with the urine

DNA: deoxyribonucleic acid, deoxyribonucleic acid

HEK cells: human embryonic kidney cells, human embryonic kidney cells; human cell line

Lusitropy, lusitrop: ability of the heart muscles for fast and complete relaxation

Natriurese, natriuretic: excretion of sodium ions with the urine

Non-peptidic receptor hormone: receptor agonist consisting of a non-peptidic substance, for example a low-molecular substance

NP: Natriuretic peptide containing the body's own natriuretic peptides ANP, BNP and CNP, as well as parts thereof, variants thereof or chimeras thereof NPRA: A-type Natriuretic Receptor, Natriuretic Peptide Receptor A

NPRB: B-type Natriuretic Receptor, Natriuretic Peptide Receptor B

Nucleic acid sequence: Sequence of the nucleotides of any nucleic acid, for example DNA or RNA

Peptidic receptor agonist: Receptor agonist consisting of a protein or peptide or a compound of a protein or peptide with a non-peptide Receptor: substance with the ability to activate a receptor and thus trigger a reaction of the receptor

RNA: ribonucleic acid, ribonucleic acid

Vasodilatation, vasodilatory: dilation of blood vessels, thereby lowering blood pressure

Description of the general field of the invention

Currently, three distinct families of endogenous natriuretic peptides are known, namely the Atrial Natriuretic Peptide (ANP), the Brain Natriuretic Peptide (BNP) and the C-type Natriuretic Peptide (CNP). The natriuretic peptides are structurally related endogenous molecules which have essentially natriuretic, diuretic and vasodilatory activities and thus play an important role in the homeostasis of the fluid balance as well as the control of the blood pressure. Likewise, the natriuretic peptides affect the release of cGMP and thus have a direct cardioprotective effect. Likewise, the natriuretic peptides have an antihypertrophic (antifibrotic) effect. The individual naturally occurring natriuretic peptides bind to different receptors (natriuretic peptide receptors) and can therefore develop different effects.

ANP selectively binds to the receptor NPRA and thereby unfolds a direct natriuretic effect and an increase in diuresis and by the vasodilatory effect a reduction in blood pressure. Similarly, ANP reduces the release of renin and aldosterone, thereby inhibiting the renin-angiotensin-aldosterone system.

BNP binds to the receptor NPRA and thus unfolds a direct natriuretic effect and an increase in diuresis and by the vasodilatory effect a reduction in blood pressure. BNP also has a renin inhibitor and positive lusitropic effect. BNP and the N-terminal fragment of proBNP can also be used to diagnose and predict heart disease by measuring the plasma level of BNP or the N-terminal fragment of proBNP in patients. The naturally occurring BNP consists of 32 amino acids and is therefore also referred to as BNP 1 -32.

CNP binds to the receptor NPRB and acts as a vasodilatory and growth-inhibiting peptide, but has no natriuretic activity. CNP also promotes bone growth.

ANP, BNP and CNP each have an amino acid loop formed by an intramolecular disulfide bridge between two cysteine amino acids, with ANP, BNP and CNP each having different lengths of C- and N-terminal ends.

The receptor NPRA and the receptor NPRB are stimulated by different natriuretic peptides, with the stimulation of both receptors simultaneously showing a therapeutic effect in a large number of diseases and thus being extremely positive.

State of the art

The amino acid sequences of the naturally occurring natriuretic peptides and the nucleic acid sequences coding therefor are known.

Likewise, the use of natriuretic peptides for the treatment of a variety of diseases is known. BNP and the N-terminal fragment of proBNP are used to diagnose cardiac diseases.

BNP 1 -32 under the trade name Natrecor ^® (Neseritide) Available in USA. The art describes the production of natriuretic and vasodilatory peptides which correspond to the amino acid structure of the BNP and can be used to treat disease states associated with an elevated extracellular fluid state. Thus, a recombinantly produced human BNP and its use as a medicament are described (US 5,1 14,923, US 5,674,710).

Nucleic acid sequences are known which code for peptides having natriuretic activity (for example for cardiodilatin), as well as the corresponding amino acid sequences (EP 0224676 A1). The use of natriuretic peptides for the diagnosis or treatment of diseases which are associated with an elevated extracellular fluid state is known (US 5,114,923, US 5,674,710, EP 0224676 A1, US 0,170,756 A1).

The preparation of peptides having a desired amino acid sequence, for example by means of genetic engineering or biochemical methods is known. These preparation methods are established as standard methods and known to the person skilled in the art (US Pat. Nos. 4,851,349, EP 0224676 A1).

The prolongation of the half-life of a natriuretic peptide in the blood of a patient by the combination of the natriuretic peptide with an active group which can form a compound with blood components is known (WO 2004/01 1498 A2).

The preparation of peptide chimeras from two or more different parent peptides is known, even if the parent peptides are natriuretic peptides (US 2009 / 0,170,756 A1, US 5,583,108).

Disadvantages of the Prior Art To date, only natriuretic peptides or peptide chimeras have been described which bind only to one of the known natriuretic peptide receptors. Thus, when using these known natriuretic peptides or peptide chimeras, there is a risk that the known natriuretic peptides or peptide chimeras lose their therapeutic effectiveness as soon as the corresponding natriuretic peptide receptor is down-regulated. This usually occurs in particular with the natriuretic peptide receptor A (NPRA, A-type Natriuretic Receptor), which is downregulated in various disease states. As a result, a natriuretic peptide used as a drug loses its effectiveness because the corresponding receptor via which it exerts its effect is no longer available in sufficient quantity or quality.

The known natriuretic peptides stimulate only one of the two receptors for natriuretic peptides (NPRA or NPRB). This leads to the fact that the use of one of the known native receptor agonists leads to a loss of therapeutic efficacy in the regularly occurring down-regulation of the corresponding receptor. It is also advantageous for the treatment of a variety of diseases when both natriuretic peptides receptors (NPRA and NPRB) are simultaneously stimulated to achieve enhanced efficacy at the same dosage level

Since none of the known receptor agonists simultaneously stimulate both receptors (NPRA and NPRB), the known natriuretic peptides provide only limited therapeutic benefit.

task

The object of the invention is therefore the provision of substances which bind to both the natriuretic peptide receptor A and B and thereby trigger the desired effects, without a down-regulation of one of the two receptors reduces the effect of the substances of the invention, and their use as medicines.

The object of the invention is therefore to provide substances which can act more efficiently on the NPRA than the native BNP. Furthermore, it is an object of the invention to provide substances which simultaneously bind to the two natriuretic peptide receptors A and B and thus allow the desired effects of such binding in an improved form.

It is likewise an object of the invention to use the substances according to the invention as medicaments in a large number of diseases.

solution

The objects are achieved according to the claims.

By providing a novel natriuretic peptide (ACNP, ANP / CNP) according to SEQ ID NO: 1 and its derivatives homologues and analogues, a receptor agonist of the natriuretic peptide receptors is available which offers a solution to the disadvantages in the prior art.

The natriuretic peptide ACNP according to the invention binds to both the natriuretic peptide receptor A and the natriuretic peptide receptor B on the basis of its amino acid sequence. By providing the novel natriuretic peptides hBNP 1 -30, mBNP 1 -30 and pBNP 1 -30 and their derivatives, homologues and analogs, receptor agonists of the natriuretic peptide receptors are available which are active both on the natriuretic peptide receptor A and on the natriuretic peptide receptor B and thus allow simultaneous stimulation of both natriuretic peptide receptors. In addition, the peptides hBNP 1-30, mBNP 1-30 and pBNP 1-30 according to the invention stimulate the natriuretic peptide receptor A significantly more potent than the naturally occurring BNP 1-32, so that the use of the receptor agonists hBNP 1-30, mBNP 1 - 30 and pBNP 1 -30 leads to a greater blood pressure reduction and relief of the heart at the same dosage. It has been shown that hBNP 1 -30 is more potent than BNP 1 -32 in in vitro and in vivo experiments and results in greater blood pressure reduction and cardiac relief at the same dose.

By providing the novel natriuretic peptides hBNP 1 -29 and mBNP 1 -29 and their derivatives, homologs and analogues, receptor agonists of the natriuretic peptide receptors are available which bind to both the natriuretic peptide receptor A and the natriuretic peptide receptor B and thus to a Stimulation of both natriuretic peptide receptors simultaneously.

Exemplary Embodiments The exemplary embodiments emerge from the attached sequence listing SEQ ID NO: 1 -6.

Embodiment 1:

The natriuretic peptide ACNP, ANP / CNP with the amino acid sequence SLRRS SCFGL KLDRI GSMSG LGCNS FRY and a disulphide bridge between the two cysteine amino acids at position 7 and 23 as well as its derivatives, homologues and analogues. Embodiment 2:

The natriuretic peptide hBNP 1 -30 with the amino acid sequence SPKMV QGSGC FGRKM DRISS SSGLG CKVLR and a disulphide bridge between the two cysteine amino acids at positions 10 and 26 as well as its derivatives, homologues and analogues.

Embodiment 3:

The natriuretic peptide mBNP 1 -30 with the amino acid sequence NSKVT HISSC FGHKI DRIGS VSRLG CNALK and a disulphide bridge between the two cysteine amino acids at positions 10 and 26 as well as its derivatives, homologues and analogues.

Embodiment 4:

The natriuretic peptide pBNP 1 -30 with the amino acid sequence SPKTM RDSGC FGRRL DRIGS LSG LG CNVLR and a disulfide bridge between the two cysteine amino acids at positions 10 and 26 as well as its derivatives, homologues and analogues. Embodiment 5:

The natriuretic peptide hBNP 1 -29 with the amino acid sequence SPKMV QGSGC FGRKM DRISS SSGLG CKVL and a disulphide bridge between the two cysteine amino acids at positions 10 and 26 as well as its derivatives, homologues and analogues.

Embodiment 6:

The natriuretic peptide mBNP 1 -29 with the amino acid sequence NSKVT HISSC FGHKI DRIGS VSRLG CNAL and a disulphide bridge between the two cysteine amino acids at positions 10 and 26 as well as its derivatives, homologues and analogues. Explanations to the figures

FIG. 1 shows the amino acid sequence of a peptidic receptor agonist ACNP according to the invention (ANP / CNP) according to SEQ ID NO: 1 with a disulphide bridge between the two cysteine amino acids at positions 7 and 23.

FIG. 2 shows the generation of the second messenger cGMP in HEK cells transfected with the natriuretic peptide receptor A (NPRA) or the natriuretic peptide receptor B (NPRB) after stimulation with various natriuretic peptides. The upper diagram shows that ACNP stimulates the NPRA in the same way as the native receptor agonists ANP and BNP. The lower diagram shows that ACNP stimulates the natriuretic peptide receptor B in the same way as the native receptor agonist CNP. FIG. 3 shows the generation of the second messenger cGMP in HEK cells transfected with the natriuretic peptide receptor A (upper diagram) or the natriuretic peptide receptor B (lower diagram) after stimulation with different natriuretic peptides. The figure shows that shortening the C- or N-terminal ends of the BNP leads to a significant reduction in the stimulability of the NPRA and to an increase in the stimulability of the NPRB.

FIG. 4 shows the result of the blood pressure measurements from 3 groups of mice to which saline, BNP 1 -32 or BNP 1 -30 were injected in each case. The y-axis shows the change in blood pressure in mmHg. The graph shows that the blood pressure at the injection of BNP 1 -32 and BNP 1 -30 decreases compared to the control group that received only saline. BNP 1 -30 reduces blood pressure to a higher degree than BNP 1 -32, which is statistically significant. As a control, the cGMP values in the plasma of these animals were measured, which is shown in the second graph. cGMP arises from the stimulation of natriuretic petid receptors by natriuretic peptides and is significantly involved in vascular relaxation by natriuretic peptides. Thus, the generation of cGMP is a measure of the biological activity of the peptides. The y-axis shows the concentration of cGMP in pmol / ml. By stimulation with BNP 1 -32 and BNP 1 -30 are increased cGMP Values measurable compared to the saline group. Stimulation by BNP 1-30 leads to a significantly increased formation of cGMP over BNP 1-32.

FIG. 5 shows the activity measurement of various natriuretic peptides against natriuretic peptide receptors in cells. The y-axis shows the concentration of cGMP in pmol / ml. cGMP is caused by stimulation of receptors by natriuretic peptides. Thus, the generation of cGMP is a measure of the biological activity of the peptides. For this experiment, human embryonic kidney (HEK293) cells were transfected with the NPRA or NPRB so that these cells overexpress the respective receptor. After stimulation with the respective peptide, the cGMP concentration was measured. In the first graph, it can be seen that ANP and BNP 1 -32, as described in the literature, stimulate the NPRA, leading to the formation of cGMP. The BNP metabolite BNP 1 -30 is also capable of this and shows a higher stimulation than BNP 1 -32. In the second graph, it can be seen that BNP 1 -32 can not stimulate the NPRB, as also known from the literature. BNP 1 -30 shows significant biological activity at the NPRB.

Claims

claims

1 . Receptor agonists, characterized in that they possess at least one activity selected from vasodilation, natriuresis, diuresis, renin-suppressing activity, antihypertrophic (antifibrotic) activity and influencing the second messenger cGMP.

2. Rezeptoragonisten according to claim 1, characterized in that they are peptidic or non-peptidic receptor agonists of the natriuretic system.

3. Rezeptoragonisten according to claim 1, characterized in that they are peptidic receptor agonists.

4. Rezeptoragonisten according to claim 3, characterized in that they have an amino acid sequence according to SEQ ID NO: 1 or are derivatives, homologs or analogues thereto.

5. Rezeptoragonisten according to claim 4, characterized in that the two cysteine amino acids are connected at position 7 and 23 by a disulfide bridge.

6. Rezeptoragonisten according to claim 3, characterized in that they have an amino acid sequence according to SEQ ID NO: 2-4 or derivatives, homologs or analogues thereto.

7. Rezeptoragonisten according to claim 6, characterized in that the two cysteine amino acids are connected at position 10 and 26 by a disulfide bridge.

8. Rezeptoragonisten according to claim 3, characterized in that they have an amino acid sequence according to SEQ ID NO: 5-6 or derivatives, homologs or analogues thereto.

9. receptor agonist according to claim 8, characterized in that the two cysteine amino acids are connected at position 10 and 26 by a disulfide bridge.

10. Nucleic acid sequences, characterized in that they encode for the receptor agonists described in claim 3.

1 1. A pharmaceutical preparation, characterized in that it contains at least one receptor agonist according to claim 1.

12. A pharmaceutical preparation according to claim 1 1, characterized in that it is used for the diagnosis, prevention and / or treatment of diseases that are associated with an increased extracellular fluid volume, or in which the second messenger cGMP develops protective properties or in which antihypertrophic (anti-fibrotic) effect is desired.

13. A pharmaceutical preparation according to claim 11, characterized in that it is used for the diagnosis, prevention and / or treatment of congestive heart disease, coronary heart disease, organic and functional vascular disorder, nephrotic syndrome, hypertensive disease, hypertrophic cardiomyopathy Myocardial infarction, acute lung injury, erectile dysfunction, primary or secondary fibrosis, cirrhosis of the liver, pulmonary fibrosis or to improve arterial, venous and capillary perfusion.

14. A pharmaceutical preparation according to claim 1 1, characterized in that it is used for the diagnosis, prevention and / or treatment of diseases according to claim 12 in mammals.

15. A pharmaceutical preparation according to claim 1 1, characterized in that it is used for the diagnosis, prevention and / or treatment of diseases according to claim 12 in humans.

16. The use of a pharmaceutical preparation according to claim 1 1, characterized in that it is used for the diagnosis, prevention and / or treatment of diseases according to claim 12 in mammals.

17. The use of a pharmaceutical preparation according to claim 1 1, characterized in that it is used for the diagnosis, prevention and / or treatment of diseases according to claim 12 in humans.