WO2002083137A1

WO2002083137A1 - Use of npy y2 receptor antagonists

Info

Publication number: WO2002083137A1
Application number: PCT/SE2002/000729
Authority: WO
Inventors: Christina Abrahamsson; Margareta Nordlander
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2001-04-12
Filing date: 2002-04-12
Publication date: 2002-10-24
Anticipated expiration: 2003-10-12
Also published as: SE0101328D0

Abstract

The use of NPY Y2 receptor antagonists for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarctionpatients, patients with heart failure and patients with disturbed electrical stability of the heart.

Description

USE OF NPY Y₂ RECEPTOR ANTAGONISTS

This invention concerns the use of NPY Y₂ receptor antagonists for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

BACKGROUND ART

Experimental and clinical studies have revealed a significant relationship between a reduced cardiac vagal activity to the heart and cardiovascular mortality, including sudden death. (1, 2)

Reduced vagal activity and enhanced sympathetic activity, i.e. autonomic dysbalance can be measured clinically by heart rate variability (HRV) (2). Vagal activity is also determined clinically by several other means, including by measuring baroreflex sensitivity (BRS), i.e. bradycardia in response to a spontaneous or induced blood pressure elevation (3) and heart rate turbulence (4).

In patients who have experienced an acute myocardial infarction (AMI), vagal activity to the heart is reduced as measured by several means. Reduced BRS, HRV and heart rate turbulence have been identified as independent, prognostic markers for cardiac mortality in post- AMI patients (2,3), regardless of β-blocker treatment (4).

In patients with congestive heart failure (CHF) vagal activity is also reduced, as judged by measurements of HRV, and several studies illustrate an association between reduced HRV and sudden death. A relationship between a reduced vagal activity and electrical instability of the heart has been documented in animal experiments (5). This relationship was recently illustrated also clinically in a study in patients with CHF or ischemic heart disease and in need of implantable cardioverter defibrillators because of high risk for ventricular fibrillation (6). In these patients the number of episodes of ventricular fibrillation or ventricular tachycardias was associated with a reduced BRS, i.e. the lower the vagal activity the more frequent the need for defibrillation (6). It is well recognised that sympathetic activity is enhanced in patients with congestive heart failure (CHF) and that the therapeutic regimen of this disease and that of post AMI frequently includes betablocker treatment to eliminate the increased sympathetic activity. However, in order to fully restore the cardiac autonomic dysbalance in CHF and post-AMI patients, enhancement of vagal activity to the heart would also be required.

NPY FUNCTION AND NPY RECEPTORS

Four different subtypes of NPY receptors, Y,, Y₂, Y₄ and Y₅ have been identified. NPY receptors share a very low degree of homology (between 30 to 45%) amongst subtypes. However, individual subtypes are almost identical between different species (more than 90% identity). All subtypes belong to the superfamily of G protein coupled receptors and are negatively coupled through Gαi to adenylate cyclase, thereby decreasing the level of cAMP in cells. At least the Yi receptor is known to potentiate the Gαq mediated increase of iCa2⁺ in response to other neurotransmitters like noradrenalin.

The wide distribution of NPY in catecholamine containing neurones, especially around blood vessels led to investigation of the vasoactive properties of this hormone. Briefly, it was demonstrated that NPY causes (i) vasoconstriction of vascular smooth cells by direct action, (ii) potentiates noradrenalin-evoked vasoconstriction postjunctionally, and (iii) inhibits transmitter release prejunctionally. Subsequently, it was demonstrated that postjunctional activities require intact NPY whereas NPY]₃.₃₆ can also mimic the prejunctional effects of the intact hormone. These original observations led to the concept of pre and postjunctional receptors of NPY and provided the first evidence for the occurrence of NPY receptor subtypes. Postjunctional receptors which interacted only with intact NPY or PYY were referred to as Y], while the prejunctional receptors which interacted with NPY, PYY as well as C-terminal fragments such as NPY_l3-36 were described as Y₂. Subsequently, postjunctional Y₂ receptors were also found on vascular smooth muscle cells. NPY Y2 RECEPTORS

Many of the terminal fibres of the sympathetic and parasympathetic nervous systems lie in close proximity to each other in the heart, an arrangement, which provides the opportunity for both pre- and postjunctional interactivity. During high frequency sympathetic stimulation, NPY is co-released with noradrenalin (NA) from peripheral sympathetic nerve terminals and will stimulate presynaptic Y₂ receptors on neighbouring vagal nerve terminals. Activation of these Y₂ receptors inhibits the release of acetylcholine and will thereby attenuate the vagal influence on the heart. A means of enhancing cardiac vagal activity during high sympathetic activity would thus be to block the action of NPY on the prejunctional vagal Y₂ receptors.

Strong evidence that NPY released from sympathetic nerve endings attenuates vagal activation to the heart has been provided from animal experiments. In anaesthetised dogs and rats, Potter and co-workers have illustrated prolonged attenuation of cardiac vagal action following stimulation of the sympathetic nerve to the heart (7, 8). This attenuation of vagal bradycardia is still evident after α and β-adrenoceptor blockade and can be mimicked by injection of NPY, NPY_3-36 and other Y₂ receptor selective NPY analogues. This suggests that NPY released from sympathetic nerves may act on prejunctional vagal Y₂ receptors to inhibit release of acetylcholine.

In rats with decompensated heart failure due to volume overload by aortic-caval fistula the concentration of m-RNA for Y₂ receptors are upregulated (12). It is therefore likely that NPY acting on Y₂ receptors may be of pathophysiological importance in the cardiac autonomic dysbalance, present in post-AMI patients and in patients with CHF.

The cDNA encoding the human NPY Y₂ receptor as well as the human NPY Y₂ receptor gene have been cloned and characterised (14,15,16) WO 95/21245.

DE 19816889, DE 19816903, DE 19816915, DE 19816929 and DE 19816932 from Boehringer Ingelheim describe a number of closely related, non-peptidic, compounds claimed to be antagonists of the NPY Y₂ receptor. BRIEF DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that NPY Y₂ receptor antagonists can maintain normal vagal nerve activity during high sympathetic activity by inhibiting the NPY mediated attenuation of the vagal activity. The use of NPY Y₂ receptor antagonists has been identified as a new therapeutic concept for the treatment of patients at risk of sudden death due to cardiac arrhythmias, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

According to one aspect of the present invention there is provided the use of an NPY Y receptor antagonist for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

According to another aspect of the present invention there is provided the use of an NPY Y₂ receptor antagonist in the manufacture of a medicament for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

According to yet another aspect of the present invention there is provided a method of identifying a compound potentially useful for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart , which comprises assaying the compound for its ability to antagonise the activity of a NPY Y₂ receptor wherein the assay comprises measurement of NPY Y₂ receptor activity using cells which express a NPY Y₂ receptor or membranes or isolated receptors prepared from such cells.

According to another aspect of the present invention there is provided a method of preparing a pharmaceutical composition which comprises: i) identifying a compound as useful for treatment of patients at risk of sudden death due to cardiac arrhythmias, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart according to a method as described herein; and ii) mixing the compound or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient or diluent.

According to yet another aspect of the present invention there is provided a pharmaceutical composition which comprises: i) a compound identified as useful for treatment of patients at risk of sudden death due to cardiac arrhythmias, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart, according to a method as described herein; and ii) a pharmaceutically acceptable excipient or diluent.

LEGENDS TO FIGURES

Figure 1 : Effects of increasing doses of a selective Y₂ antagonist (Boehringer Ingelheim substance BIIE0246 and vehicle on inhibition of vagal bradycardia by PYY in anaesthetised guinea pigs. Vagal bradycardia in response to stimulation of the right cervical n. vagus is attenuated by i.v. injection of PYY, and infusion of the Y₂ antagonist attenuates this inhibition, thereby reinforcing vagal nerve activity. 100 % equals extent of inhibition of vagal bradycardia caused by PYY injection.

Figure 2: Change in RR interval (heart rate) in response to stimulation of the right cervical n. vagus in anaesthetised dogs (n=6). In the control sequence vagal bradycardia is inhibited 50 % subsequent to sympathetic stimulation (SS; 20 Hz). Administration of the Y₂ antagonist in a dose resulting in a plasma concentration of 60 nmol/1, totally inhibited this attenuation

Figure 3. Map of the BPV-based expression vector pAM194. mMT-1, the murine metallthionine-1 enhancer and promoter element. rB-globin, genomic fragment containing exon II, intron II, exon III, and downstream elements of the rabbit β-globin gene. PML2d, a pBR322 derivative for propagation of the vector in E. coli.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention there is provided use of an NPY Y₂ receptor antagonist for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

The compound BIIE0246 described in DE 19816929 is a potent NPY Y₂ receptor antagonist that has proved effective in the assays described herein and which is potentially suitable for use for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

New NPY Y₂ receptor antagonists to be used in the treatment according to the present invention can be identified by the methods described and claimed herein by the present inventors.

According to another aspect of the present invention there is provided use of an NPY Y₂ receptor antagonist in preparation of a medicament for the treatment of patients at risk of sudden death due to cardiac arrhythmias, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

In yet another aspect the present invention provides a method of treating mammals at risk of sudden death due to cardiac arrhythmias, for example post myocardial infarction mammals, mammals with heart failure and mammals with disturbed electrical stability of the heart comprising administering a therapeutically effective amount of an NPY Y₂ receptor antagonist to a mammal in need thereof. In a further aspect the present invention provides a method of preventing sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart comprising administering a therapeutically effective amount of an NPY Y₂ receptor antagonist to a patient in need thereof.

In a still further aspect the present invention provides a pharmaceutical composition for use in the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart comprising an NPY Y₂ receptor antagonist and a pharmaceutically acceptable carrier.

According to yet another aspect of the present invention there is provided a method of identifying a compound potentially useful for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart which comprises; i) providing a plurality of compounds to be tested; and ii) assaying the compounds for their ability to antagonise the activity of an NPY Y₂ receptor where the assay comprises measurement of NPY Y₂ receptor activity using cells which express an NPY Y₂ receptor or membranes prepared from such cells; and iii) selecting a number of candidate compounds which show NPY Y₂ receptor antagonist activity; and iv) determining the pA2 of the candidate compounds and identifying compounds with pA2 > 5 as useful for treatment of patients at risk of sudden death due to cardiac arrhythmias.

Alternatively a compound may initially be identified as potentially useful in this invention by having an IC50 of less than 10 micromolar in a Y2 receptor binding assay.

The compounds to be tested can be selected from e.g. random chemical libraries, natural product libraries, directed compound libraries prepared by combinatorial chemistry or conventional chemical synthesis. Such libraries include random peptide libraries (17,18), libraries made of D- and/or L- configuration amino acids, or phosphopeptides libraries (19).

The assay used to determine the effect of a compound on the activity of a NPY Y₂ receptor can be based on measurement of binding of a ligand to the NPY receptor, such as NPY , NPY₃.₃₆ and other Y₂ receptor selective NPY analogues, in the presence compared to in the absence of the compound. To facilitate the detection of binding of the ligand to the NPY Y₂ receptor the ligand can preferable be labelled, e.g. a radioisotope like ¹²⁵I or ³H.

The assay can also be a functional assay where a functional response of the cell expressing the NPY Y₂ receptor following addition of a NPY Y₂ receptor ligand can be measured. Examples of such assays are measurement of cAMP production or metabolic changes in the cells (microphysiometry).

The cells used in the assay can be cells naturally expressing a NPY Y₂ receptor or transfected cells expressing a recombinant NPY Y₂ receptor. Preferably the NPY Y₂ receptor is the human recombinant NPY Y₂ receptor.

The NPY Y₂ receptors may be expressed in a variety of hosts such as bacteria, plant cells, insect cells, fungal cells and human and animal cells. Eukaryotic recombinant host cells are especially preferred. Examples include yeast, mammalian cells including cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be used and which are commercially available include, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The expression vector comprising a nucleic acid encoding a NPY Y₂ receptor may be introduced into host cells to express a polypeptide of the present invention via any one of a number of techniques including calcium phosphate transformation, DEAE-dextran transformation, cationic lipid mediated lipofection, electroporation or infection

The transfected host cells are propagated and cloned, for example by limiting dilution, and analysed to determine the expression level of recombinant NPY Y₂ receptor. Identification of transformed host cells which express the NPY Y₂ receptor may be achieved by several means including immunological reactivity with antibodies and/or the detection of biological activity using the assays described herein.

According to another aspect of the present invention there is provided a method of preparing a pharmaceutical composition which comprises: i) identifying a compound as useful for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart according to a method as described herein; and ii) mixing the compound or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient or diluent.

The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).

The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents. Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal track, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.

Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame). Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.

The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.

Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.

Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedure well known in the art.

Compositions for administration by insufflation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30μ or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.

Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.

For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990 which is incorporated herein by reference. The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient.

For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990 which is incorporated herein by reference.

The size of the dose for therapeutic or prophylactic purposes of a compound will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.

In using a compound for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. .Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred.

EXAMPLES

Example 1. Attenuation of vagal inhibition by NPY Yτ_antagonist

We have used similar approaches as Potter, to document that administration of a Y₂ antagonist may block the attenuation of vagal bradycardia caused by administration of a NPY analogue with actions on Y₂ receptors. For this reason, a method to evaluate the potency of Y₂ antagonists in anaesthetised guinea pigs has been developed (9). The vagal nerve is repeatedly stimulated and the extent of bradycardia recorded. Administration of PYY (polypeptide YY, an analogue to NPY) inhibits the vagal bradycardia by activating prejunctional vagal Y₂ receptors so that acetylcholine release (from the cardial vagal nerve) is attenuated. The potency of Y₂ antagonists to attenuate this inhibition is then determined. The effective dose of the Boehringer Ingelheim Y antagonist BIIE0246 (described in DE 19816929) has been determined to be 21 nmol/kg/30 min in this model, when given as i.v. infusion ( see fig 1 below)

In anaesthetised dogs we have illustrated that Y₂ receptor blockade inhibits the prolonged NPY driven attenuation of vagal bradycardia obtained after sympathetic stimulation of the heart (see figure 2 below).

We have thus discovered that inhibition of prejunctional NPY Y₂ receptors will maintain a high vagal activity on the heart during intense sympathetic activation. This observation is novel and can be used as a new principle to treat patients at risk of sudden death from cardiac arrhythmia.

Example 2. In vitro assays

2.1 Preparation of cells expressing the recombinant human NPY Y?_receptor

The 1.18kb cDNA encoding the human NPY Y2 receptor (EMBL HS362691) (15) was cloned from a human brain cDNA library (Clontech) by PCR according to the manufacturer's recommendations (Perkin Elmer). The following primers containing the indicated engineered restriction enzyme sites were used: forward primer (Hind III): 5 ' CG A AGCTTCGGC AGCCC A AC ATGGGTCC A AT AGGTGC AG AG 3 ' , reverse primer (Sal I): 5' CGGTCGACTTAGACATTGGTAGCCTCTGT 3'. The resulting PCR fragment was cloned into a TA-vector according to the protocol supplied by the manufacturer (Invitrogen). The NPY Y2 receptor cDNA was excised from the TA-vector by Hind III / Sal I digestion and subcloned into an expression vector containing a mMT-1 promoter and a rB-globin polyadenylation sequence (20). The accuracy of the sequence of the cloned NPY Y2 receptor cDNA was verified by DNA sequencing on an ABI Prism 377 DNA sequencer (Perkin Elmer) and the corresponding plasmid was designated pAM194.

Mouse C127 fibroblasts (ATCC CRL1616) were co-transfected with pAM194 and an expression plasmid (pS86) encoding the G418 selectable marker using the calcium- phosphate method (21) Non-transfected cells were selected against using 800μg/ml G-418 (Gibco BRL) in the cell culture medium Dulbecco's MEM/Nut mix F12 (1 :1) supplemented with 2mM L-glutamine and 10% foetal bovine serum (GIBCO BRL). Twenty-four positive clones were manually picked, expanded and analysed for human ■ NPY Y2 receptor production using radioligand binding. One high producing clone, UME4, was selected and these C127-hY2 cells were used in the assays described below.

2.2 Binding assays 2.2.1 Filtration assay

To establish the in vitro affinity of compounds for the Y₂ receptor a receptor binding assay is used. Prior to the binding experiments C127-hY2 cells are harvested and frozen in Krebs-Ringer buffer (KR; 137 mM NaCl, 2.7 mM KC1, 2.1 mM MgC12, 1.8 mM CaC12, 20 mM Hepes; pH 7.4) complemented with 0.2 % (w/v) bovine serum albumin (BSA) and 0.025 % (w/v) bacitracin. The radioligands used are either ¹²⁵I-PYY or ³H-NPY (Amersham Pharmacia). The non specific binding is determined in the presence of 1 μM PYY or NPY. The cells, radioligand, and agonists (PYY or NPY for non specific binding) is diluted in KR buffer supplemented with 0.2 % (w/v) BSA. Test compounds are dissolved and diluted in dimethyl sulphoxid (DMSO) at concentrations resulting in a final DMSO concentration of 1.5 % (v/v). The cells are incubated together with radioligand and test compounds in 96 well plates for 60 minutes at 30°C. The incubation is stopped by rapid filtration through a polyethyleneimine pretreated GF/C filtermat using a Skatron cell harvester. The filtermats are washed with ice-cold Krebs Ringer buffer without BSA, dried, and scintillant (Meltilex, Wallac OY) is melted through the filter. The radioactivity is counted using a Trilux counter (Wallac).

The specific ¹²⁵I-PYY binding is calculated as the difference between binding in the absence and presence of 1 μM PYY. The effect of the compounds are expressed as a percentage of the specific binding in each concentration tested. The results are analyzed using the Xlfit program, using the Levenburg Marquardt algorithm for the curve fitting. For a compound to be considered a hit in the binding assay the IC₅o value needs to be 10 μM or lower.

BIIE0246 was used as a reference compound for in vivo studies, and then showed an in vitro affinity of 2.5 nM on recombinant human Y₂ receptors using the filtration assay and ³H-NPY as the radioligand.

2.2.2 SPA assay

An alternative binding assay is the homogenous Scintillation Proximity assay (SPA), where no separation step is necessary. This assay can be run in either 96 or 384 well format using the C127-hY2 cells and ¹²⁵I-PYY as the radioligand. As above, the cells, radioligand, and agonist are diluted in KR buffer supplemented with 0.2 % (w/v) BSA. Test compounds are dissolved and diluted in dimethyl sulphoxid (DMSO) at concentrations resulting in a final DMSO concentration up to 2 % (v/v).

The cells are preincubated with WGA coated PVT SPA beads (Amersham Pharmacia) for 2 hours at +4°C during constant slow shaking. The beads plus cells are then mixed with radioligand, 1 μM PYY for non specific binding, and test compounds. The plates are allowed a 5 to 10 hours incubation before the radioactivity is counted using a Trilux counter (Wallac).

2.3 Functional assays

2.3.1 Microphysiometer assay

This assay uses a Cytosensor microphysiometer (Molecular Devices). In short, the extracellular acidification rate is used as a measure of the metabolic activity in the cells upon agonist activation.

C127-hY2 cells are seeded in 12 well cups (Molecular Devices Capsule kits) at a density of 300 000 cells per well 24 hours prior to the experiment. On the day of the experiment the media surrounding the cells is changed to running media, i.e. DMEM/Nut Mix without sodium bicarbonate (i.e. non buffered) supplemented with 0.1 % (w/v) BSA, and the cell cups are transferred into the Cytosensor flow chambers. The cells are allowed an one hour stabilisation period in the flow chambers before any additions are made, to obtain a stable baseline in the acidification rate. All dilutions of agonists and compounds are performed in running media, and all additions are made with the lowest concentration first. The additions are made during the first 40 seconds of a two minute pump cycle. The acidification rate is measured over 30 seconds in the end of each pump cycle. Additions of agonists and/or test compounds are repeated every half-hour to allow the cells to recover in between.

To establish the antagonistic properties of compounds concentration response curves of the agonist is run in the absence and presence of different concentrations of the test compound. Schild regression analysis, plotting the dose ratio against the antagonist concentration, results in the pA2 value (-log(Ki)). pA2 values of 5 and higher, without any effect of the maximum agonist activation, indicates Y2 antagonism. 2.3.2 cAMP assay

Since the Y₂ receptor in negatively coupled to the adenylate cyclase, forskolin is used to induce cAMP production. NPY or PYY is then used to inhibit the forskolin induced cAMP production, and a test compound used to inhibit the agonist induced inhibition of the forskolin response.

Cells are seeded in sterile cell culture treated 96 well plates the day before the experiment. On the day of the experiment the media is removed by inverting the plate, and the cells are washed with assay buffer (Hepes 2.5 mM, Tris 2.5 mM, NaCl 140 mM, KCl 5 mM, CaCl₂ 1.8 mM, bovine serum albumin 0.2 % (w/v), glucose 4.5 g/1, IMX 0.1 mM; pH 7.5).

The cells are preincubated with the assay buffer for 15-20 minutes at 37°C, then test compound, agonist, and forskolin is added in that order. 20 minutes after the forskolin addition the incubation is stopped by decanting the buffer and addition of lysis reagent. The measurement of intracellular cAMP is performed using the cAMP SPA direct screening assay system from Amersham Pharmacia (RPA 559). In short, following lysis of the cells the main part of the lysis reagent is transferred to new (SPA) plates, and reagents, SPA beads and tracer is added to the wells. The plate is incubated in room temperature for 15 to 20 hours and the radioactivity is counted in a Microbeta counter. A cAMP standard curve is run on each plate, and the cAMP production in each well is determined from the standard curve.

REFERENCES

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Claims

1. The use of an NPY Y₂ receptor antagonist for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment including post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

2. The use of an NPY Y₂ receptor antagonist in the manufacture of a medicament for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment.

3. The use acording to claim 1 or 2 wherein the patients are post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart.

4. A method of treating mammals at risk of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction mammals, mammals with heart failure and mammals with disturbed electrical stability of the heart comprising administering a therapeutically effective amount of an NPY Y₂ receptor antagonist to a mammal in need thereof.

5. A method of preventing sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart comprising administering a therapeutically effective amount of an NPY Y₂ receptor antagonist to a patient in need thereof.

6. A pharmaceutical composition for use in the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart comprising an NPY Y₂ receptor antagonist and a pharmaceutically acceptable carrier.

7. A method of identifying a compound potentially useful for the prevention of sudden death due to cardiac arrhythmias in patients in need of such treatment, for example post myocardial infarction patients, patients with heart failure and patients with disturbed electrical stability of the heart which comprises; i) providing a plurality of compounds to be tested; and ii) assaying the compounds for their ability to antagonise the activity of an NPY Y₂ receptor where the assay comprises measurement of NPY Y₂ receptor activity using cells which express a NPY Y receptor or membranes prepared from such cells; and iii) selecting a number of candidate compounds which show NPY Y₂ receptor antagonist activity; and iv) determining the pA2 of the candidate compounds and identifying compounds with pA2 > 5 as useful for treatment of patients at risk of sudden death due to cardiac arrhythmias.

8. The use according to any of claims 1 to 3 or the method according to claim 4 or claim 5 or the composition according to claim 6 wherein the NPY Y2 antagonist is BIIE0246 described in DE 19816929.

9. The use according to any of claims 1 to 3 or the method according to claim 4 or claim 5 or the composition according to claim 6 wherein the NPY Y2 antagonist has been identified by the method of claim 7.