WO1999051261A1

WO1999051261A1 - Use of urocortin and like polypeptides in therapy

Info

Publication number: WO1999051261A1
Application number: PCT/GB1999/001056
Authority: WO
Inventors: Anastasis Stephanou; Bhawan Brar; Richard Knight; David Latchman
Original assignee: University College London
Current assignee: University College London
Priority date: 1998-04-07
Filing date: 1999-04-07
Publication date: 1999-10-14
Anticipated expiration: 2000-10-07
Also published as: AU3340399A; GB9807504D0

Abstract

Urocortin (Ucn) protects primary cultures of rat cardiac myocytes from lethal ischaemic injury. Ucn expression is increased in cardiac myocytes following an ischaemic insult. Ucn or a cardioprotective equivalent thereof is used in the manufacture of a medicament for use in the treatment of cardiac ischaemia.

Description

- 1 -

USE OF UROCORTIN AND LIKE POLYPEPTIDES IN THERAPY

Field of the Invention The present invention relates to the use of urocortin, a 40 amino acid peptide, and derivatives thereof .

Background to the Invention Urocortin (Ucn) , has been cloned from both rat midbrain and human placental cDNA libraries (Vaughan, J., Donaldson, C, Bittencourt J. et al . Urocortin, a mammalian neuropeptide related to fish urotensin 1 and to corticotropin-releasing factor (CRH) . Nature. 1995: 378: 287-292; Donaldson, C.J., Sutton, S. ., Perrin, M.H. et al . Cloning and characterisation, of human urocortin. Endocrinology. 1996: 137: 2167-2170). Ucn is sometimes called corticotensin.

The human Ucn nucleotide sequence is predicted to encode a 124 amino acid protein comprising a 80 amino acid precursor and a 40 amino acid mature peptide (Donaldson, C.J., Sutton, S.W., Perrin, M. et al . Cloning and characterisation of human urocortin - Erratum. Endocrinology. 1996: 137: 3896). The deduced mature rat and human peptides share 95% identity at the amino acid level, and rat Ucn shares 45% sequence identity with rat CRH. The term "Urocortin" and the abbreviation "Ucn" are always used herein to refer to the mature peptide. Two distinct receptor genes for the CRH family of polypeptides, including Ucn, have been identified. Both encode seven transmembrane domain proteins and are functionally coupled to adenylate cyclase. The first, CRH-R1, was cloned from pituitary and brain (Chen, R., Lewis, K.A., Perrin, M.H., Vale, .W. Expression cloning -2 - of human corticotropm-releasmg factor receptor. Proc. Natl. Acad. Sci. USA. 1993: 90: 8967-8971; Chang, C, Pearse, R., O'Connell, S., Rosenfeld, M.G. Identification of a seven transmembrane helix receptor for corticotropm releasing factor and sauvagme m mammalian brain. Neuron. 1993: 11: 1187-1195).

Two splice variants of a second receptor, CRH-R2 and β, which differ m their N-termmal domains, have also been isolated. CRH-R2α has been isolated from brain (Lovenberg, T.W., Liaw, C. ., Grigoriadis, D.E. et al . Cloning and characterisation of a functionally distinct corticotropm-releasmg factor receptor subtype from rat brain. Proc. Natl. Acad. Sci. USA. 1995: 92: 836-840) CRH-R2β has been isolated from heart, brain and lung (Lovenberg et al , 1995; Kishimoto, T., Pearse, R.V., Lm, C.R., Rosenfeld, M.G. A sauvagme/corticotropm- releas g factor receptor expressed heart and skeletal muscle. Proc. Natl. Acad. Sci. USA. 1995: 92: 1108- 1112) . CRH and Ucn bind to and stimulate adenylate cyclase activity of the different receptor subtypes to different degrees with CRH-R2β having roughly lOx higher affinity for Ucn.

In the rat brain, Ucn mRNA and immunoreactivity are largely confined to the midbra Ed ger- estphal nucleus and the lateral superior olive, though weaker signals are found elsewhere. Ucn mRNA is also present m the pituitary and Ucn peptide can stimulate the secretion of Adrenocorticotropm (ACTH) by anterior pituitary corticoctroph cells. Ucn mRNA and peptide have also been found human placental and decidual cells throughout gestation.

The biological functions of Ucn have been unclear. Since Ucn, like CRH, binds avidly to the CRH-bmd g protein (CRH-BP) , its bioavailability will be determined by the proportion associated with CRH-BP. From its - 3- distribution in the brain, it has been proposed that Ucn may act as a neurotransmitter involved in the innervation of the ciliary ganglion and in the processing of auditory signals. Ucn also causes more pronounced and sustained falls in mean arterial blood pressure than CRH in vi vo, consistent with the higher affinity of the CRH-R2β receptor found in the heart for Ucn. It is also a more potent inhibitor than CRH of the oedema following thermal injury to the rat paw, again consistent with Ucn being an endogenous ligand for CRH-R2 receptors.

Summary of the Invention

We have now shown that Ucn is released from cultures of primary cardiac myocytes in response to ischaemic injury and that antagonists to the receptors of the CRH peptide family inhibit the protective effect of ischaemic conditioned media. Further, we have detected endogenous Ucn expression within the rat heart. We have shown that Ucn mRNA levels change following thermal injury to cardiac myocytes and that Ucn protects cardiac myocytes from cell death induced by hypoxia. Additionally, we have shown that Ucn protects isolated perfused hearts ex vivo .

Accordingly, the present invention provides: - use of urocortin, or a cardioprotective derivative thereof, in the manufacture of a medicament for use in the treatment of cardiac ischaemia; a method for the treatment of a patient who has suffered a cardiac ischaemia, which method comprises the step of administering to the patient a therapeutically effective amount of urocortin, or a cardioprotective derivative thereof; and an agent for treating cardiac ischaemia comprising urocortin or a cardioprotective derivative thereof. Description of the Figures

Figure 1 shows the densitometric analysis of Ucn expression relative to that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in H9c2 cells at various times after exposure to 42°C heat shock for 20 min;

Figure 2 shows the protective effects of 10^"8M Ucn on 6 hours hypoxic exposure of primary cardiac myocytes. Cell death was assessed by trypan blue uptake (left hand figure) and by lactic dehydrogenase (LDH) release (right hand figure) ;

Figure 3a: Cultures of neonatal rat ventricular myocytes were treated with purified recombinant human CRH-BP (known to bind CRH and CRH-like peptides including urocortin) 24 hours before exposure to 6 hours of lethal ischaemia. Myocyte cell death, measured by LDH released activity is signficantly increased (**p<0.01, n=6 +_. S.E.M.) in the CRH-BP treated cells from the untreated cells exposed to lethal ischaemia and those exposed to control normoxic conditions. Figure 3b: Administration of 0, 0.1, 10 and 10000 nM helical CRH results in a dose-dependent increase in both LDH released activity and cell death (Figure 3c) as measured by trypan blue exclusion (n=8 +_. S.E.M. for each dose). The untreated cells released 0.41 ± 0.01 released activity and 16.19% of these cells were permeable to trypan blue. The experiments have been repeated three times to confirm the data.

Figure 4: Conditioned media from myocytes exposed to a two hour ischaemic insult protect fresh cardiac myocytes from lethal ischaemia in comparison to untreated cells as assessed by both LDH release (*p<0.05) in comparison to untreated cells exposed to ischaemia.

Figure 5: Addition of CRH, Ucn and urotensin reduce lethal ischaemic induced death of cardiac myocytes, with -5- a rank order of potency: Ucn > urotensin > CRH where cell survival is measured using both trypan blue exclusion and (Figure 5b) LDH released activity (The data are representative of 4 experiments, n=16, S.E.M.). Figure 6: A 2 hour ischaemic insult of cardiac myocytes transfected with the Ucn promoter containing the C/EBP (CCAAT/enhancer-binding protein) consensus site ligated upstream of a luciferase reporter results in a 2- fold increase in reporter activity. Ischaemic cardiac myocytes also show significant increases in expression of the C/EBP transcription factors C/EBPβ (NF-IL6; approximately 4-fold) and C/EBPδ (NF-IL6β; approximately 10-fold) , consistent with these enhancers being at least partially responsible for the increased expression of Ucn in ischaemic cardiac myocytes.

Detailed description of the invention

The invention is concerned with the use of Ucn or a cardioprotective derivative thereof to treat cardiac ischaemia. The Ucn is typically human Ucn, in particular the 40 amino acid mature human peptide. The full human Ucn amino acid sequence including the precursor sequence is shown in SEQ ID NOS: 1 and 2 (Genbank accession number U43177) . The mature peptide is Asp-83 to Val-122 inclusive.

A cardioprotective derivative is a derivative which protects cardiac myocytes from cell death. It can thus reduce cell death induced by ischaemia in cardiac myocytes. This may be determined in vi tro by the following procedure: cardiac myocytes are incubated with a test polypeptide (test cardiac myocytes) ; ischaemia is induced by thermal stress, hypoxia or the use of a specific ischaemic buffer in the test cardiac myocytes and in control cardiac myocytes that - 6- have not been incubated with the test polypeptide; the respective proportions of the test cardiac myocytes that die as a result of the ischaemic challenge and of the control cardiac myocytes that die as a result of the ischaemic challenge are determined; and the ability of the test polypeptide to reduce cell death attributable to the challenge is thus assessed.

Such a procedure will be well known to those skilled in the art. Detailed descriptions of the procedure are set out in the following Examples. Optionally the proportions of the test and control cardiac myocytes that die by apoptosis and necrosis as a result of the ischaemic challenge may be determined. A polypeptide is cardioprotective if it is able to reduce death of the test cardiac myocytes by 30% or more, for example 40% or more or 50% or more, with respect to the control cardiac myocytes. For example, there is a 50% reduction in cell death if 80% of the control cardiac myocytes and 40% of the test cardiac myocytes die as a result of the ischaemic challenge. Preferably, a cardioprotective derivative reduces cell death by 60% or more or 70% or more. A cardioprotective derivative may bind to CRH-BP. A cardioprotective derivative typically possesses an amino acid sequence that is substantially homologous to that of human Ucn. The term derivative is taken to encompass allelic variants and species homologues . An allelic variant is a variant which occurs naturally in a human and which will function in a substantially similar manner to human Ucn. In particular, it has cardioprotective activity. Similarly, a species homologue is a functionally equivalent polypeptide which occurs naturally in another species. Such a homologue may occur in animals such as mammals (e.g. rats or -7- rabbits), especially primates, pigs, sheep, cows or goats. Within any one species, a homologue may exist as several allelic variants.

Allelic variants and species homologues can be obtained using standard procedures. In particular, a human Ucn nucleotide sequence can be used to probe libraries made from mammalian cells to obtain clones encoding the allelic or species variants. The clones can be manipulated by conventional techniques to identify a polypeptide of the invention which can then be produced by recombinant or synthetic techniques known per se . Preferred species homologues include primate, procine, ovine, bovine or caprine species homologues.

Further, the sequence of human Ucn or of allelic variants and species homologues can be modified to provide a suitable polypeptide. One or more amino acid substitutions may be made, for example 1, 2, 3 or 4 or more, for example up to 10 substitutions may be made provided that the modified polypeptide retains cardioprotective activity. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

ALIPHATIC Non-polar G P

I L V

Polar - uncharged C S T M

N Q

Polar - charged D E

K R

AROMATIC H F W Y

Alternative modifications include one or more amino acid deletion, rearrangement or extension. An extension at the N-terminal and/or C-terminal may be provided. The length of each extension may be relatively short, for example from 1 to 10 or from 3 to 6 amino acid residues. Alternatively the length of each extension may be much longer, for example from 10 to 200 or from 30 to 100 amino acid residues. Any suitable extension may be provided, provided the resulting polypeptide is non- toxic, or at least suitable for use in animals of humans, and is cardioprotective. The length of each extension may thus serve as a carrier sequence. A fusion protein may thus be provided.

Generally, a modified polypeptide is at least 70% homologous to the amino acid sequence of human Ucn, for example to the mature human Ucn amino acid sequence shown in SEQ ID NOS: 1 and 2 from Asp-83 to Val-122. There may be at least 80%, at least 90% or at least 95% homology. Methods of measuring protein homology are well known in the art and it will be well understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology") . Generally homology is calculated over a region of at least 20, preferably at least 30, for example 35 or more contiguous amino acids. The cardioprotective derivative of Ucn may be a cardioprotective fragment of human Ucn, an allelic variant or species homologue thereof, or of a cardioprotective modified version of human Ucn or an allelic variant or species homologue thereof. The fragment may be from 6, for example from 8 or from 10, to 15, for example to 20 or to 25, amino acids long.

A cardioprotective fragment may be identified using standard procedures. These may involve fragmentation of a polypeptide such as human Ucn using proteolytic enzymes or chemical agents and then determining the ability of - 9- each fragment to protect cardiac myocytes from cell death induced by ischaemia. Alternatively, the DNA encoding a polypeptide such as human Ucn may be fragmented by restriction enzyme digestion or other well-known techniques and then introduced into an expression system to produce fragments (optionally fused to a carrier polypeptide) . Again, the cardioprotective ability of the resulting fragments can be assessed.

Another approach is to chemically synthesise short peptide fragments, for example from 6 to 20 amino acids long) which cover the entire sequence of a full-length polypeptide such as human Ucn with each peptide overlapping the adjacent peptide. This overlap can be from 1 to 10 amino acids. Each peptide is then assessed for its cardioprotective ability. Finally, sequence homologies may be studied to identify a candidate motif that may be associated with cardioprotection. Such a prediction may then be tested by producing an appropriate polypeptide and assessing its ability to protect against cell death induced by ischaemia in cardiac myocytes.

The cardioprotective fragment may also be provided with the N-terminal and/or C-terminal extension. The length of each extension may be relatively short, for example from 1 to 10 or from 3 to 6 amino acid residues. Alternatively the length of each extension may be much longer, for example from 10 to 200 or from 30 to 100 amino acid residues. Any suitable extension may be provided, provided the resulting polypeptide is non- toxic, or is at least suitable for use in animals and humans, and is cardioprotective. The extension may thus serve as a carrier and/or targetting sequence. The fragment may thus be presented as a fusion protein. A polypeptide for use in the invention may be prepared by chemical synthesis. A polypeptide may be built up from single amino acids and/or preformed - 1 0- peptides of two or more amino acids in the order of the sequence of the desired polypeptide. Solid-phase or solution methods may be employed. The resultant polypeptide may be converted into a pharmaceutically acceptable salt if desired.

In solid-phase synthesis, the amino acid sequence of the desired polypeptide is built up sequentially from the C-terminal amino acid which is bound to an insoluble resin. When the desired peptide has been produced, it is cleaved from the resin. When solution-phase synthesis is employed, the desired polypeptide may again be built up from the C-terminal amino acid. The carboxy group of this acid remains blocked throughout by a suitable protecting group, which is removed at the end of the synthesis.

Whichever technique, solid-phase or solution-phase, is employed each amino acid added to the reaction system typically has a protected amino group and an activated carboxy group. Functional side-chain groups are protected too. After each step in the synthesis, the amino protecting group is removed. Side-chain functional groups are generally removed at the end of the synthesis. The resultant polypeptide may then be converted into a pharmaceutically acceptable salt. It may be converted into an acid addition salt with an organic or inorganic acid. Suitable acids include acetic, succinic and hydrochloric acid. Alternatively, the polypeptide may be converted into a carboxylic acid salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.

A polypeptide for use in the invention may also be prepared by recombinant DNA methodologies. Thus, a DNA sequence encoding the polypeptide is provided. An expression vector is prepared which incorporates the DNA sequence and which is capable of expressing the - 1 1 - polypeptide when provided in a suitable host. The DNA sequence is located between translation start and stop signals in the vector. Appropriate transcriptional control elements are also provided, in particular a promoter for the DNA sequence and a transcriptional termination site. The DNA sequence is provided in the correct frame such as to enable expression of the peptide to occur in a host compatible with the vector.

Any appropriate host-vector system may be employed. The vector may be a plasmid. In that event, a bacterial or yeast host may be used. The resulting polypeptide that is expressed can then be isolated and purified using standard techniques.

A polypeptide of the invention is used to treat cardiac ischaemia. The polypeptides protect against necrotic and/or apoptotic cell death. They can therefore be used to treat myocardial ischaemia. Myocardial ischaemia occurs when the heart muscle does not receive an adequate blood supply and is thus deprived of necessary levels of oxygen and nutrients. The most common cause of myocardial ischaemia is atherosclerosis, causing blockages in the blood vessels (coronary arteries) that provide blood flow to the heart muscle. The ischaemia may be attributable to low cardiac output, angina, clot formation or an arterial spasm. A therapeutically effective amount of the polypeptide is administered to a patient, typically to a human patient, in need of such treatment. The polypeptide can thus alleviate the ischaemia. The polypeptide is typically administered by injection. The polypeptide may be administered by intravenous or intramuscular injection, or orally provided it can be protected from the acid environment in the stomach. In emergencies it may be injected directly into the heart or into blood about to flow into the - 12 - heart. The timing of administration of the polypeptide should be determined by medical personnel, but generally should occur as soon as possible after an ischaemic episode has been detected or is suspected of having taken place, for example within four hours of such an episode. The amount of polypeptide that is given to a patient will depend upon a variety of factors including the nature of the patient under treatment and the severity of the condition under treatment. Typically, however, from 5 to 1000 mg of polypeptide may be given, for example from 10 to 500 mg. The exact dose will be determined by the medical personnel attending to the patient.

A polypeptide is formulated for administration with a pharmaceutically acceptable carrier or diluent. Any carrier or diluent suitable for a pharmaceutical composition and suitable for the selected route of administration may be employed. Water for injection or physiological saline, for example phosphate-buffered saline, may be used for a formulation intended for injection.

Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate and the like. Capsules, tablets and pills may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate or hydroxypropylmethyl cellulose .

The following Examples illustrate the invention.

Example 1

1. MATERIALS AND METHODS

Cells

The H9c2 rat embryonic cardiac myoblast cell line was obtained from the ATCC (Rockville, Maryland, US) and was maintained in continuous culture in Dulbecco's - 13 - odified Eagles medium containing 10% foetal calf serum. Primary Sprague Dawley rat myocyte cultures were obtained from pups less than 2 days old and were cultured as described previously (Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure and chronotropic response to isoprotenol. Circ. Res. 1982: 50: 101-116). Brains were obtained from normal adult Sprague Dawley rats.

Cell death was assessed using Trypan blue uptake and by the release of lactic dehydrogenase using a commercially available kit (Sigma, Poole, GB) . H9c2 cells were thermally shocked by exposing them to 42°C for 20 minutes, after which they were returned to a 37°C incubator. Cell pellets were recovered at the times indicated in the Results, and stored at 80°C for RNA extraction. Primary myocyte cultures were rendered hypoxic by exposure to an atmosphere of 5% C0₂ and 95% argon for 6 hours in a tissue culture chamber. RNA extraction and Reverse Transcription

Total cellular RNA was extracted by the method of Chomczynski and Sacchi (Chomczynski P, Sacchi N. Single- step method of RNA isolation by acid guanidiniu thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987: 62: 123-127). 1 μg RNA was reverse transcribed for 15 minutes at 42°C with 15 units of AMV reverse transcriptase, oligo(dT)₁₅ primer (0.5 μg) , dNTP mixture (1 mM each) and rRNasin ribonuclease inhibitor (20 units) .

Polymerase Chain Reaction (PCR) cDNAa were amplified in a reaction volume of 50 μl containing 200 uM final concentrations of mixed dNTPs, 2 mM MgCl₂, 0.5 μM primers and 1.5 units of Taq polymerase (all reagents from Promega, Southampton, GB) . Primers - 14 - for Ucn and the housekeeping gene, glyceraldehyde-3- phosphate dehydrogenase (G3PDH) were designed using the OLIGO 4.0 programme. Amplification was performed on a Perkin Elmer 4800 thermal cycler for 35 cycles on settings of 94°C for 45 seconds, 64°C for 1 minute 30 seconds (58°C for G3PDH) and 72°C for 1 minute 30 seconds .

The following sets of primers were used (all primers were from Cruachem, Glasgow, GB) . G3PDH: AAG GTC GGA GTC AAC GGA TTT and AAG GTG GAG GAG TGG GTG TCG Ucn: (forward) GGC TGC GGC GGC GAA TGT GGT;

(reverse) CAG CAG GTG GAA GGT GAG GTC (UcnRl) and AGG TGG GGG AAA GGG TCA AGG (UcnR2) . The predicted sizes of the amplification products are 872 bp (G3PDH) , 196 bp (UcnRl) and 329 bp (UcnR2). Purification and characterisation of amplification products

Amplification products were electrophoresed through 1.5% agarose gels containing ethidium bromide and visualised under an ultra violet transilluminator (Genetic Research Instrumentation, Dunmow, Essex, GB) . Bands of the predicted sizes were excised and the DNA purified using the Geneclean II kit (BIO 101 Inc, Vista, California, US) . Restriction digestion was performed for 18 hours at 37°C using Bam HI and Pst 1 (Promega, Southampton, GB) , and the products were separated on a 2% ethidium bromide-stained agarose gel. Sequencing was performed using the dye terminator cycle sequencing method and the two reverse Ucn PCR primers.

Densitometric comparison of expression of Ucn and G3PDH amplification products was performed on a PDI DeskTop densitometer with Quantity One software (PDI, Huntington, New York, US) . - 15-

2 . RESULTS

Expression of Ucn in cardiac myocytes

Electrophoresis through 1.5% agarose gels revealed that PCR products of the expected 196 and 329 bp had been amplified from mRNA prepared frpm primary cardiac myocyte cultures and whole rat brain. Identical amplification products were also obtained from the H9c2 cell line. No Ucn product was amplified under the same or similar conditions from a bronchial epithelial and a neuroblastoma cell line.

Characterisation of the amplification product

The Ucn amplification products each contained a restriction site for Bam HI at position 168 and for Pst 1 at position 279 as determined from an electrophorogram of restriction digests. Thus, with the product generated from H9c2 cells using UroR2 as reverse primer, Bam HI generated a 304 bp product and Pst 1 generated products of 137 and 192 bp . A predicted 25 bp Bam HI product was not visible on the gel. Similar digestion fragments were obtained with the 329 bp product from rat brain and primary cardiac myocytes.

Direct sequencing of the products generated from brain and from both cardiac myocytes with the UroR2 reverse primer showed complete identity with the published Ucn sequence from nucleotides 144-433 apart from untypeable nucleotides at positions 407 and 423 (data not shown) . Modulation of Ucn expression by heat shock

Expression of the housekeeping gene, G3PDH, remains unchanged after heat shock. Increases in Ucn expression were apparent immediately after heat shock, were sustained for the following 18 hours and returned to control values after 24 hours. Mean densito etric values for Ucn normalised to G3PDH from three experiments are shown in Fig. 1. Eighteen hours after thermal shock, - 1 6- mean Ucn mRNA expression was 1.8 x the control value. Protective effects of Ucn against ischaemic injury

Primary cardiac myocytes, preincubated with 10^"8 M Ucn were protected from the effects of lethal (6 hours) ischaemia. Both the proportion of trypan blue positive cells and the release of LDH into the medium, were significantly (p<0.01, Students t test) reduced in the Ucn treated cells (Fig. 2) . A similar, but less pronounced, protective effect of Ucn was seen with thermal injury (data not shown) .

3. DISCUSSION

The data reported here show that rat cardiac myocytes express Ucn mRNA, that the level of expression is increased by thermal shock and that Ucn protects cardiac myocytes from cell death induced by hypoxia. The increased expression of Ucn following thermal injury, and the protective effects of exogenous Ucn against cell death induced by hypoxia, suggest that Ucn can function as an endogenous cardioprotective agent. Hypoxia induces both necrotic and apoptotic forms of cell death in cardiac myocytes, although hypoxia is associated more with necrosis and reperfusion than with apoptosis. The Ucn-mediated reduction in LDH release observed here suggests that Ucn is protecting against necrotic cell death.

CRH is particularly associated with the hypothalamic response to physical and psychological stress. The data reported here suggests that the related peptide, Ucn, can fulfil a similar role in mediating the response of cardiac myocytes to cellular stress. The only CRH receptor found in the heart is CRH-R2β which has at least lOx higher affinity for Ucn than CRH, both in terms of binding affinity and in the induction of cAMP . This suggests that endogenous Ucn may be the preferred ligand -17- for cardiac CRH receptor.

Example 2

1. MATERIALS AND METHODS Cell culture

Ventricular myocytes isolated from the hearts of neonatal rats (Sprague Dawley) that were less than 2 days old were cultured as described previously (Simpson, P., Savion S., 1982) with the following modifications. After collagenase digestion the cells were pre-planted m medium consisting of DMEM (1000 mg glucose/L GIBCO), 1 mMol/L L-glutamme, 100 U/ml penicillm/streptomycm (GIBCO BRL) supplemented with 15% (v/v) fetal calf serum on 10cm tissue culture dishes. Pre-platmg of the cell suspension for 30 minutes allows contaminating fibroblasts to attach and the myocytes remain free within the culture media.

Subsequent to this incubation, the cardiac myocyte cell suspension was transferred onto six-well (3cm) gelatin coated plates (Falcon) at a density of 10⁵ cells per well. This plating system yields cell cultures that are more than 95% myocytes as determined by indirect staining with a monoclonal mouse antibody to Desmm (Wu, C.F., Bishopric, N.H., Pratt, R.E. Atrial natπuretic peptide induces apoptosis in neonatal rat cardiac myocytes. J. Biol. Chem. 1997; 272 (23): 14860-6). After 24 hours, the cell media was replenished with the above media containing reduced FCS at 1% (v/v) for an additional 24 hours before experimentation (hereafter referred to as growth medium) . Within 3 days a confluent monolayer of spontaneously beating myocytes was formed. Preconditioning of cardiac myocytes using CRH and CRH- like peptides

Cardiac myocytes preconditioned by incubating CRH, Ucn and Urontensm at concentrations of 10^~6, 10^"8, 10^-10 M - 1 8 - in 1ml of growth media for 24 hours in a humidified atmosphere of 21% 0₂, 5% C0₂ at 37°C. Preconditioned media was obtained from the cells by incubating 1 ml of the following control buffer; 137mM CaCl₂, 3.8 mM KC1, 0.49 mM MgCl₂, 0.9 mM CaCl₂.2H20, 4 mM HEPES (pH 7.4) with the cells with the cells at 37°C in the ischaemic chamber for 2 hours.

The media was removed, centrifuged at 1000 rpm for 10 minutes and the supernatant incubated with fresh cells, in the presence and absence of 10^~8 M a helical

CRH(9-41). CRH-binding protein (25 μg/ml) and α helical CRH(9-41) (0, 1, 10, 10000 nM) was added to untreated cells for 24 hours prior to exposure to lethal ischaemia. Lethal simulated ischaemia and determination of cardiocvte viability

Following "preconditioning" of the cardiocytes, the cells were subjected to a more severe stress comprising of "lethal" simulated ischaemia. The normal growth media of the cardiocyte cultures was replaced with 1 ml of ischaemic buffer composed of 137mM CaCl₂, 12 mM KC1, 0.49 mM MgCl₂, 0.9 mM CaCl₂.2H₂0, 4 mM HEPES, 20 mM Na lactate (Sigma) (pH6.2) (Esumi, K., Nishida, M., Shaw, D., Smith, T.W., Marsh, T.W., Marsh, J.D. NADH measurements in adult rat myocytes during simulated ischaemia. Am. J. Physiol. 1991:260 (6 Pt 2): 1743:52). The cultures were incubated at 37°C in the ischaemic chamber for 6 hours.

The ischaemic buffer was then removed, centrifuged at 1000 rpm for 10 minutes and the supernatant snap frozen under liquid N₂ until assay for lactate dehydrogenase (LDH) activity. LDH activity released from the cardiocytes was assessed using a spectrophotometric assay kit (Sigma) . For trypan blue exclusion, the cells were washed with phosphate buffered saline (PBS) , trypsinised for 2 minutes in 0.25 mg/ml trypsin in versene (Gibco-BRL) and then neutralised by the addition - 1 9- of new-born calf serum. Cells were centrifuged, the supernatant aspriated and the cardiocytes resuspended in 100 μl of PBS. Following addition of an equal volume of 0.8% trypan blue in PBS, 250 cells were scored 3 times per plate of cells using a haemocytometer .

Processing of the Ucn precursor in ischaemic myocytes

Ucn is synthesised as a precursor which is subsequently cleaved to yield the 40 amino acid mature peptide. To assess precursor cleavage following ischaemia, whole cell extracts were prepared in SDS-PAGE sample buffer and aliquots containing equal protein concentrations were separated on gradient polyacrylamide gels. Separated proteins were Western blotted onto nitrocellulose membranes and probed with a Ucn-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA) . Membranes were washed in PBS/0.05% Tween and incubated with a peroxidase conjugated anti-goat antibody (Santa Cruz) followed by detection using the enhanced chemiluminescence kit (ECL, Amersham) .

Activation of the Ucn promoter in ischaemia

The region of the published Ucn promoter sequence containing a C/EBP consensus binding site (Zhao, L., Donaldson, C.J., Smith, G.W., Vale, W.W. The structures of the mouse and human Urocortin genes (Ucn and UCN) . Genomi cs 1998, 50, 23-33) was amplified by PCR and the identity of the sequence established by restriction digestion. The fragment was ligated upstream of a luciferase reporter and the construct transfected into primary cultures of cardiac myocytes by calcium phosphate precipitation as described previously (Stephanou, A., Okosi, A., Knight, R.A., Chowdrey, H.S., Latchman, D.S. C/EBP activates human corticotropin-releasing hormone gene promoter. Mol . Cell . Endocrinol . 1997, 134, 41-50). -20-

Cells were cultured with normal growth medium for 24 hours and then with Esumi control buffer (137mM CaCl₂, 3.8mM Kcl, 0.49mM MgCl₂, 0.9mM CaCl₂.2H₂0, 4mM HEPES pH7.4; Esumi et al . , 1991) for 4 hours at 37°C in an atmosphere of 5% C0₂, 0% 0₂, 95% argon in a hypoxia chamber.

Subsequent to this period, the cells were returned to a normoxic environment of 21% 0₂, 5% C0₂ at 37°C for 24 hours before harvesting for luciferase activity. The cells were harvested with 1 mM EDTA and lysed with three cycles of freeze-thawing. Assays for luciferase activity (with assay times from 2-4h, depending upon the level of activity detected) were performed using samples of equal protein concentration, previously determined by the method of Bradford (Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Bi ochem 1976; 72: 248-254). To control for differences in transfection efficiency, results of luciferase activity were corrected for β-galactosidase expression in the same transfection.

Polyacrylamide gel electrophoresis and Western blotting: analysis the C/EBP family C/EBPβ (NF-IL6) , C/EBPδ (NF- IL6B) and NF-kB

The cardiac myocytyes were cultured with CRH at a concentration of 10^"8 M in growth media for 24 hours. Approximately 1x10" cells were harvested in lOOμl of 2x concentrated SDS PAGE sample buffer for analysis of C/EBP family C/EBPβ (NF-IL6) , C/EBPδ (NF-IL6β) and NF-kB protein expression. Proteins separated by SDS-PAGE were subsequently transferred onto nitrocellulose membranes (Hybond C, Amersham, GB) and were then probed using antibodies to C/EBPβ (NF-IL6) , C/EBPδ (NF-IL6β) and NF-kB (all from Santa Cruz Biotechnology, Santa Cruz, CA) . The - 2 1 - membranes were subsequently washed in PBS/0.05% Tween and incubated with a peroxidase conjugated rabbit anti-mouse IgG antibody (DAKO) at a dilution of 1/2000 followed by detection using an enhanced Chemiluminescence kit (ECL, Amersham) . The relative protein levels were determined using densitometry (Bio Rad) normalising to the actin band on a duplicate Coomassie Brilliant Blue R250 (BDH) stained gel.

Assessment of apoptosis; TUNEL assay and flow cytometry The number of apoptotic nuclei were assessed by using terminal transferase from calf thymus (Boehringer Mannhemim) and Fluorescein-12-2 ' -deoxy-uridine-5 ' - triphosphate. Terminal transferase catalyses a template independent addition of deoxyribonucleotide triphosphate to the 3' -OH ends of double- and/or single-stranded DNA and has been used for assessment of apoptotic nuclei as described previously. Briefly cardiac myocytes were exposed to an ischaemic insult in conditioned media, in the presence and absence of 10^~6 M Ucn and helical CRH

(9-41) within the ischaemic chamber. The cells were then returned to a normoxic environment for 2h and fixed in 4% paraformaldehyde for 30 min at 25°C and washed with PBS three times. Terminal deoxynucleotidyl transferase was added to the cells for 2h in a 37°C humidified incubator. After washing with PBS the cells were then imaged with fluorescent microscopy. The percentage of apoptotic nuclei is expressed as a percentage of total nuclei. For flow cytometry the cells were fixed in acetone-methanol (4:1), pelleted and treated with RNase for 15 minutes at 37°C. They where then stained with propidium iodide for 20 minutes at 37°C in the dark and analysed on a FACScan (Becton Dickinson, Mountain View, California, US) .

RNA extraction and RT-PCR -22-

Primary cultures of cardiac myocytes were exposed to 0 and 2 hours of lethal ischaemia and the cells pelleted immediately after these timepoints and 24 hours later. RNA extraction, reverse transcription and PCR were performed as described in Example 1. Amplification in respect of Ucn was performed under the same conditions as for G3PDH. The following sets of Ucn primers were used (from Cruachem, Glasgow, Scotland):

Ucn (forward) GGC TGC GGC GGC GAA TGT GGT reverse 1 CAG CAG GTG GAA GGT GAG GTC (Ucn RI) reverse 2 AGG TGG GGG AAA GGG TCA AGG (Ucn R2 )

The predicted sizes of the amplification products are 872 base pairs (G3PDH) , 196 base pairs (UcnRl) and 329 base pairs (UcnR2) .

Statistical analysis

Data are expressed as means +_. S.E.M. Differences among means were compared among the treatment groups using the Students t-Test. The experiments were repeated at least three times with a minimum of n=4 for each experiment .

2. RESULTS

Inhibition of the cardioprotective effects of ischaemic preconditioned media bv CRH-BP and helical CRH (9-41) Cultures of neonatal rat ventricular myocytes were treated with purified recombinant human CRH binding protin (CRH-BP; Woods, R.J., Grossman, A., Saphier, P., Kennedy, K., Ur. E., Behan, D., Potter, E., Vale, W., Lowry, P.J. Association of human corticotropin-releasing hormone to its binding protein in blood may trigger clearance of the complex. J Clin Endocrinol Metab, 1994; 78(1) : 73-6) which has been shown to bind Ucn and with -23- the CRH receptor antagonist, ot helical CRH(9-41), before exposure to 6 hours of ischaemia. Myocyte cell death, measured by lactate dehydrogenase release (LDH) (Figure 3a) is significantly increased the CRH-BP and α helical CRH (9- 1) treated cells (Figure 3b, c) . The addition of 25 μg/ml of CRH-BP to the extracellular media of cardiac myocytes cultured m a normoxic environment produced no effect on myocyte cell death as measured by both trypan blue exclusion and LDH release at time points of 24and 48 hours (data not shown) .

Conditioned media from myocytes exposed to a two hour ischaemic msult protect fresh cardiac myocytes from lethal ischaemia. This cardioprotective effect on LDH release (Figure 4) and apoptosis assessed by DNA end- labelling by the terminal deoxynucleotidyl transferase (TdT) assay (TUNEL) (Table 1 below) and flow cytometry (data not shown) was abrogated in the presence of a. helical CRH (9-41) (Figure 4 and Table 1) and by CRH-BP (data not shown) . These results suggest that endogenous Ucn is involved m cardiac preconditioning.

Apototic nuclei (%)

Untreated cells 26.29 ± 0.17 (21% 0_2/ 5% C0₂) n=4

Ischaemic cells 41.33 + 4.11 (0% 0_2, 5% C0₂) n=4

Ischaemic cells 26.03 + 1.85 (0% 0_2/ 5% C0₂) n=4 lO^'6 M CRH

Preconditioned media 28.05 + 3.21 treated cells (0% 0₂, 5% C0₂) n=7

-24 -

Preconditioned media 26.14 + 4.13 treated cells + 10^"8 M CRH

(0% 0₂, 5% C0₂) n=7

Preconditioned media 27.31 ± 4.18 treated cells + 10^~8 M Ucn

(0% 0₂, 5% C0₂) n=7

Preconditioned media 48.01 + 4.48 treated cells + 10^~8 M a CRH (9-41)

(0% 0₂, 5% C0₂) n=7

The cardioprotective effects of exogenouslv administered CRH, urotensin and Ucn against lethal ischaemic injury Exogenous CRH-like peptides also have cardioprotective effects against lethal simulated ischaemia. Addition of nanomolar concentrations of Ucn, urotensin and CRH protected cardiac myocytes from ischaemia-induced necrotic (Figs 5a and b) and apoptotic (Table 1) cell death. Both trypan blue uptake and LDH release revealed that Ucn is more potent than urotensin and CRH, with a 71, 52 and 31% reduction in LDH release with lOnM Ucn, urotensin and CRH respectively.

Transactivation of the Ucn promoter bv ischaemia

Cardiac myocytes, transfected with a promoter fragment containing an NF-IL6 consensus site ligated into a luciferase reporter vector showed increased reporter gene activity following ischaemia (Fig 6) . RT-PCR revealed that the expression of urocortin mRNA increased following a 2 hour ischaemic insult, being 10.6 fold greater than control values 24 hours after ischaemia (as assessed by BIO-RAD GS 670 Image densitometer) . -25-

Increased expression of C/EBPβ (NF-IL6) , C/EBPδ and NF-kB in ischaemia and regulation of the CRH promoter

In addition, cardiac myocytes showed increased expression of C/EBP transcription factors C/EBPβ (NF- IL6), C/EBPδ (NF-IL6β) and NF-kB after a two hour ischaemic insult.

3. DISCUSSION

We have shown here that both exogenous and endogenous Ucn protect primary cultures of rat cardiac myocytes from lethal ischaemic injury and that Ucn expression is increased in cardiac myocytes following an ischaemic insult. This suggests that Ucn produced by cardiac myocytes has a direct involvement in the cardiovascular response to stress.

In this study we have also shown that ischaemic injury increases the expression of specific transcription factors C/EBPβ (NF-IL6) , C/EBPδ (NF-IL6β) and NF-kB within the heart which may in turn activate the Ucn promoter. Autocrine/paracrine release of the Ucn confers protection from necrotic and apoptotic myocardial cell death.

The data show that, in response to ischaemic stress, cardiac myocytes increase transcription of the Ucn gene and process the precursor polypeptide. The results are consistent with the release of the mature Ucn peptide which, following its binding to the cardiac CRH-R2 receptor, protects myocytes from necrotic and apoptotic cell death induced by the ischaemia. Thus Ucn itself, and derivatives thereof, may be clinically useful therapeutic agents in the treatment of human myocardial ischaemia .

There is a relatively small, but significant release of CRH into the culture medium of cardiac myocytes following ischaemic insult. However, there is a greater -26- mduction of Ucn mRNA from ischaemic myocytes suggesting that Ucn, acting through its preferential CRH-R2β receptor, may be the more significant and important cardioprotective mediator. This is substantiated by the greater protective potency of Ucn compared to CRH.

Example 3

Cardioprotective effects of Urocortin on isolated hearts To confirm the cardioprotective effects of Ucn observed m vivo, protection experiments were also performed on isolated perfused hearts ex vivo . Hearts from adult male Wistar rats were perfused with oxygenated normothermic Krebs-Hemsleit beffer on a Lagendorf apparatus . After a 20 minute period of stabilization, the middle coronary artery was ligated to produce regional ischaemia for 35 minutes. The ligature was then released and the hearts reperfused for a further 120 minutes. Ucn (10-8M) was added to the perfusate either for 30 minutes prior to the ischaemia or added to the perfusate during the first 30 minutes of reperfusion. The risk zone (R) was defined using fluorescent microspheres and the mfarct size (I) using TTC staining and the results are expressed as the %I/R. The %I/R in untreated hearts was 45.67+/-6.36 (SD, n=8). When Ucn was added before the period of ischaemia, this was reduced to 15.06+/-11.12% (n=3, p<0.01) and when Ucn was included only during the first 30 minutes of reperfusion, to 29.89+/-7.92% (n=4, p<0.03) . The data therefore show that Ucn remains cardioprotective even when added after the ischaemic period. This suggests that Ucn and derivatives thereof may be useful in a clinical setting when administered after the onset of acute myocardial infarction.

Claims

-27-CLAIMS

1. Use of urocortin, or a cardioprotective derivative thereof, in the manufacture of a medicament for use in the treatment of cardiac ischaemia.

2. Use according to claim 1, wherein human urocortin or a cardioprotective derivative thereof is employed.

3. A method for the treatment of a patient who has suffered a cardiac ischaemia, which method comprises the step of administering to the patient a therapeutically effective amount of urocortin, or a cardioprotective derivative thereof.

4. Urocortin, or a cardioprotective derivative thereof, for use in a method of treatment of the human or animal body by therapy.

5. An agent for treating cardiac ischaemia comprising urocortin or a cardioprotective derivative thereof .