WO2010013012A9

WO2010013012A9 - Hypothermia inducing polypeptides and uses thereof

Info

Publication number: WO2010013012A9
Application number: PCT/GB2009/001885
Authority: WO
Inventors: Carin SJÖLUND; Björn Ulrik WALSE; Tadeusz Wieloch
Original assignee: Auril Ab
Priority date: 2008-08-01
Filing date: 2009-07-31
Publication date: 2010-08-12
Also published as: WO2010013012A2; WO2010013012A4; WO2010013012A3

Abstract

The present invention provides a polypeptide capable of binding to a receptor for glucagon-like peptide-1 (GLP-1) for use in inducing or maintaining hypothermia in a subject in need thereof, wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 1 or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the hypothermia-inducing activity of said amino acid sequence. The invention further provides pharmaceutical compositions comprising the polypeptides of the invention, together with methods and uses of the same.

Description

NOVEL POLYPEPTIDES AND USES THEREOF

Field of Invention

The present invention relates to polypeptide-based agents for use in the induction and/or maintenance of hypothermia in a subject in need thereof. In particular, the invention provides methods for treating or preventing neuronal damage in brain following an ischemic episode, such as a stroke or cardiac arrest.

Background

It is well known that hypothermia can postpone damage to tissues caused by inadequate blood flow and oxygen deprivation. The prompt induction of hypothermia can have a significant impact on the rate of survival for patients suffering from a variety of conditions including, but not limited to, ischemia due to cardiac arrest (e.g. myocardial infarction), stroke, haemorrhage, traumatic injury, spinal cord injury and asphyxia.

Systemic hypothermia has historically been accomplished by immersion of the patient's body in a cool bath. Today, there are several commercial systems available for the induction of systemic hypothermia via physical means. They typically consist of blankets or pads where cooled water is circulated through channels in the walls of the blanket or pad, and the patient's body is maintained in intimate contact.

However, there are several drawbacks to this approach: (a) it may take several hours to lower a patient's body to therapeutic temperatures, (b) hypothermia cannot be initiated until after the patient has been admitted to the hospital, (c) the entire patient's body is cooled in a slow and uniform manner, with protective levels of hypothermia in the brain not achieved until the whole body reaches protective levels of hypothermia. Moreover, when such physical methods are used to induce hypothermia, the patient's body naturally seeks to compensate for the drop in body temperature by increasing cellular processes to generate heat; in so doing, the body tries to work against the hypothermia treatment.

Thus, there is a need for new methods of inducing and maintaining hypothermia in the acute treatment of ischemic episodes, such as a stroke or cardiac arrest.

Summary of Invention

The first aspect of the invention provides a polypeptide capable of binding to a receptor for glucagon-like peptide-1 (GLP-1) for use in inducing or maintaining hypothermia in a subject in need thereof,

wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 1 :

AU-01: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO: 1]

or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the hypothermia-inducing activity of said amino acid sequence.

In a related embodiment, the invention provides polypeptides for use in inducing or maintaining hypothermia in a subject in need thereof, wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 1, or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the hypothermia-inducing activity of said amino acid sequence.

Thus, the invention provides polypeptide-based medicaments for lowering body temperature and, particularly, the temperature of the brain (i.e. cerebral hypothermia).

As indicated above, a disadvantage of the known physical means for lowering body temperature is that the patient's body naturally seeks to compensate by generating more heat internally. In contrast, the present invention comprises the induction or maintenance of hypothermia by chemical means. In effect, the polypeptides of the invention appear to act by lowering the body's internal thermostat, resulting in the reduction of body temperature. The advantage of such chemical induction or maintenance of hypothermia is that it does not provoke the body's compensatory heat generating mechanisms associated with physical hypothermia induction.

The amino acid sequence of SEQ ID NO: 1 corresponds to the known peptide, exendin- 4, originally isolated from Heloderma suspectum (GiIa monster); for example, see GenBank Accession Nos. AAB 22006 and AAB51130).

It will be appreciated by persons skilled in the art that the invention encompasses polypeptides comprising or consisting of SEQ ID NO: 1 , as well as fragments, variants, fusions and derivatives of this amino acid sequence which retain the hypothermia- inducing activity of exendin-4.

By "retains the hypothermia-inducing activity" we mean that the fragment, variant, derivative or fusion is able to induce hypothermia in a subject. Thus, by "retains" we include fragments, variants, derivatives and fusions which retain, at least in part, the ability of the polypeptide of SEQ ID NO: 1 to lower body temperature. Preferably, such polypeptides retain hypothermia-inducing activity in ischemic subjects.

The ability of fragments, variants, derivatives and fusions of the polypeptide of SEQ ID NO: 1 to induce hypothermia can be determined using methods well known in the art (see Example 1 below).

In one embodiment of the first aspect of the invention, the polypeptide is for use in the treatment or prevention of neuronal damage in the central nervous system.

By "treatment or prevention of neuronal damage" we mean that the polypeptide of the invention is capable of preventing or inhibiting (at least in part) one or more symptom, signal or effect constituting or associated with neuronal damage. For example, the polypeptide may be used to prevent or reduce the death of neuronal cells in the central nervous system. Additionally, or alternatively, the polypeptides of the invention may be used to alleviate or prevent neurological dysfunction due to ischemic brain damage

It will be appreciated by persons skilled in the art that the polypeptides of the invention are not for use in the treatment or prevention of reperfusion injury per se, e.g. by metabolic therapies (such as those disclosed in WO 00/666142 and WO 00/66138). Rather, an essential feature of the polypeptides of the present invention is their use to induce or maintain hypothermia, which in turn provides a neuroprotective effect.

In a further embodiment of the invention, the polypeptide is for use in the treatment or prevention of acute brain injury.

For example, the polypeptides of the invention may be for use in the treatment or prevention of neuronal damage due to ischemia.

Thus, the invention provides polypeptide-based therapeutic agents for use in the treatment of acute brain injury following an ischemic episode, wherein said agents are capable of inducing hypothermia in vivo (preferably following IV or sub-cut administration)

The polypeptides of the invention are of particular use where the subject is suffering from or has recently suffered from a stroke, a brain trauma, a cardiac arrest, spinal cord injury or asphyxia.

By "recently" in this context, we mean the subject has experienced such an ischemic episode within the last 24 hours, and preferably within the last 12 hours, 6 hours, 4 hours, 2 hours, or one hour or less.

It will be appreciated by persons skilled in the art that the polypeptides of the invention may be used in combination with one or more additional hypothermic treatments, such as external and/or internal surface cooling.

For example, the polypeptides of the invention may be used in combination with nasopharyngeal cooling and/or cold saline infusion. Alternatively, or in addition, the polypeptides of the invention may be used in combination with vanilloid receptor agonists and/or cannabinoid receptor agonists (see WO 2008/040361 , the disclosures of which are incorporated herein by reference).

Alternatively, or in addition, the polypeptides of the invention may be used in combination with adenosine receptor agonists (for example, see Yang et a/., 2009, Am J Physiol Heart Circ Physiol. 296(4):H 1141-9) or fever reducing agents such as regulators of prostaglandin synthesis (for example, see Morrison et al., 2008, Central control of thermogenesis in mammals.Exp Physiol. 93:773-97).

Persons skilled in the art will further appreciate that the uses and methods of the present invention have utility in both the medical and veterinary fields. Thus, the polypeptide medicaments may be used in the treatment of both human and non-human animals (such as horses, dogs and cats).

Preferably, however, the patient is human.

The degree of hypothermia necessary to treat, reduce or prevent neuronal damage following an ischemic episode may be determined by the doctor or physician administering the polypeptide of the invention. In one embodiment, the polypeptide is capable of inducing a fall of at least TC in core body temperature of the subject, for example a fall of at least 2⁰C, 2.5⁰C, 3°C, 4°C, 5°C or more. It will be appreciated that such a reduction in temperature may be measured either relative to the body temperature before administration of the polypeptide of the invention or relative to body temperature following administration of a vehicle control.

In another embodiment, the polypeptide of the invention is capable of inducing a hypothermic effect having a duration of at least 10 minutes following a single administration, for example at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 120 minutes or more. However, it will be appreciated that repeated administrations or continuous infusions may provide a more sustained hypothermic effect. In a still further embodiment, the polypeptide of the invention has an in vivo half-life in humans of at least 10 minutes following IV and/or sub-cutaneous administration, for example at least 15 minutes or at least 20 minutes or more.

In one particularly preferred embodiment of the invention, the polypeptide has comparable or even improved hypothermia-inducing efficacy in subjects suffering from neuronal damage due to an ischemia compared to its efficacy in healthy subjects.

By "comparable hypothermia-inducing efficacy" in this context we mean that a given dose of the polypeptide induces a quantitatively similar hypothermic effect in both normal and ischemic subjects. Such efficacy comparisons may be determined in a suitable animal model (see Example 1).

As indicated above, the invention encompasses polypeptides comprising or consisting of SEQ ID NO: 1 , as well as fragments, variants, fusions and derivatives of this amino acid sequence which retain (at least in part) the hypothermia-inducing activity of exendin-4.

In one embodiment of the first aspect of the invention, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 1.

The term 'amino acid' as used herein includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the 'D' form (as compared to the natural 'L' form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).

When an amino acid is being specifically enumerated, such as 'alanine' or 'Ala' or 'A', the term refers to both L-alanine and D-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid. In accordance with convention, the amino acid sequences disclosed herein are provided in the N-terminus to C-terminus direction.

In one embodiment, the polypeptides of the invention comprise or consist of L-amino acids.

Where the polypeptide comprises an amino acid sequence according to SEQ ID NO: 1, it may comprise additional amino acids at its N- and/or C- terminus beyond those of SEQ ID NO: 1 , for example, the polypeptide may comprise additional amino acids at its C- terminus. Likewise, where the polypeptide comprises a fragment, variant or derivative of an amino acid sequence according to SEQ ID NO: 1 , it may comprise additional amino acids at its N- and/or C- terminus.

In a further embodiment the polypeptide comprises or consists of a fragment of the amino acid sequence according to SEQ ID NO: 1.

In one embodiment the polypeptide fragment comprises or consists of a fragment of the amino acid sequence according to SEQ ID NO: 1. Thus, the polypeptide may comprise or consist of at least 10 contiguous amino acid of SEQ ID NO: 1 , for example at least 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37 or 38 contiguous amino acid of SEQ ID NO: 1. Advantageously, the fragment comprises or consists of at least 36 contiguous amino acid of SEQ ID NO: 1.

In a further embodiment the polypeptide fragment commences at an amino acid residue selected from amino acid residues 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29 of SEQ ID NO:1.

However, alternatively/additionally, the polypeptide fragment may terminate at an amino acid residue selected from amino acid residues 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38 and 39 of SEQ ID NO:1.

Hence, the polypeptide fragment may comprise or consist of amino acids 3 to 8 and/or 15 to 32 of SEQ ID NO: 1. For example, the polypeptide fragment may comprise or consist of amino acids 1 to 30 of SEQ ID NO: 1 (for example, see Runge et al., 2007, Biochemistry 46:5830).

It will be appreciated by persons skilled in the art that the polypeptide of the invention may alternatively comprise or consist of a variant of the amino acid sequence according to SEQ ID NO: 1 (or fragment thereof). Such a variant may be a non-naturally occurring variant.

By 'variants' of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes retain, at least in part, the hypothermia-inducing activity of the said polypeptide.

Such variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides (see example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2000, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference).

In one embodiment, the variant has an amino acid sequence which has at least 50% identity with the amino acid sequence according to SEQ ID NO: 1 or a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (as described in Thompson et al., 1994, Nuc. Acid Res. 22:4673-4680, which is incorporated herein by reference). The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.

Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine local sequence alignments.

In a further embodiment, the variant is as described in US 2009/0005312, the disclosures of which are incorporated herein by reference (in particularly, see Formulae Il and III therein).

In one particularly preferred embodiment, the variant comprises or consists of an amino acid sequence of SEQ ID NO: 2:

His-Xaa2-Xaa3-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Glu-Glu-Glu-

Ala-Val-Arg-Leu-Phe-lle-Glu-Trp-Leu-Lys-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Xaa36-

Pro-Pro-Ser-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46

[SEQ ID NO:2]

wherein

Xaa2 is GIy, Ser, VaI, Ala or aminoisobutyric acid (Aib);

Xaa3 is GIu or Asp; Xaa10 is Leu or VaI;

Xaa12 is Lys or Ser;

Xaa13 is GIn, GIu or Tyr;

Xaa14 is Met or Leu;

Xaa28 is Asn, Ser or Asp; Xaa36 is Pro or absent; Xaa40 is an amide group, Lys, AEEA-MPA-modified Lys or is absent; Xaa41 is Lys or absent; Xaa42 is Lys or absent; Xaa43 is Lys or absent; Xaa44 is Lys or absent;

Xaa45 is Lys or absent; and

Xaa46 is an amide group or is absent

or a fragment, derivative or fusion thereof (or a fusion of said fragment or derivative) which retains the hypothermia-inducing activity of a polypeptide having the amino acid sequence of SEQ ID NO:1.

In the above definition, "AEEA" corresponds to aminoethoxy ethoxy acetic acid and "MPA" corresponds to maleimide proprionic acid.

Thus, the variant may comprise or consist of an amino acid sequence of SEQ ID NO: 2.

For example, the variant may comprise or consist of an amino acid sequence selected from the group consisting of:

AU-08: HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:3];

AU-11: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGA-PPSKKKKKK

[SEQ ID NO:4];

AU-12: HGEGTFTSDLSKEMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:5];

AU-U: HGEGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPS

[SEQ ID NO:6];

AU-15: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSZ, where Z=Lys-(AEEA-MPA)-(CONH2) [SEQ ID IMO:7];

AU-17: HAEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:8];

AU-18: HGEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:9];

AU-19: HVEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:10]; AU-20: HΛ/ibEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:11] and

AU-28: HGEGTFTSDLSKQLEEEAVRLFIEWLKSGGPSSGAPPPS

[SEQ ID NO:12].

In one preferred embodiment, the above exemplary peptides comprise a C-terminal amide moiety.

For the avoidance of doubt, the variant may not be GLP-1.

In a further embodiment of the first aspect of the invention, the polypeptide comprises or consists of a fusion protein.

By 'fusion' of a polypeptide we include an amino acid sequence corresponding to SEQ ID NO: 1 (or a fragment or variant thereof) fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of said polypeptide are also included in the scope of the invention.

The fusion may comprise a further portion which confers a desirable feature on the said polypeptide of the invention; for example, the portion may be useful in augmenting or prolonging the hypothermic effect. For example, in one embodiment the fusion comprises human serum albumin or similar protein (as disclosed in US 2009/0005312, the disclosures of which are incorporated herein by reference).

Alternatively, the fused portion may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art. The moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art. In one particular embodiment, the fusion is a chimeric protein comprising or consisting of an amino acid sequence corresponding to a fragment of SEQ ID NO: 1 and an amino acid sequence corresponding to a fragment of GLP-1.

For example, the fusion may comprise or consist of a C-terminal receptor binding region of GLP1 and an N-terminal fragment of SEQ ID NO: 1. An example of such a chimeric protein comprises or consists of an amino acid sequence of SEQ ID NO: 9:

AU-18: HGEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:9]

A polypeptide according to any one of the preceding claims wherein the polypeptide, or fragment, variant, fusion or derivative thereof, comprises or consists of L-amino acids.

In a further embodiment of the first aspect of the invention, the polypeptide comprises or consists of one or more amino acids that are modified or derivatised.

Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, f-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications. It will be further appreciated by persons skilled in the art that peptidomimetic compounds may also be useful. Thus, by 'polypeptide' we include peptidomimetic compounds which

are have ^R-afrtHftflammatef* activity of the polypeptide of SEQ ID NO: 1. The term ■peptidomimetic' refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent.

For example, the polypeptides of the invention include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptϊdomimetics may be made using methods known in the art, for example such as those described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, which is incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, the polypeptide of the invention may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -y(CH₂NH)- bond in place of the conventional amide linkage.

In a further alternative, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it may be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond.

It will be appreciated that the polypeptide may conveniently be blocked at its N- or C- terminus so as to help reduce susceptibility to exoproteolytic digestion.

A variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have also been used to modify mammalian peptides. In addition, a presumed btoactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges, for example see Veber er al., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell et a/., 1983, Biochem. Biophys. Res. Comm. 111: 166, which are incorporated herein by reference. In one preferred embodiment, however, the polypeptide of the invention comprises one or more amino acids modified or derivatised by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

It will be appreciated by persons skilled in the art that the use of a fragment, variant, fusion or derivative as described above may provide a functional advantage relative to a polypeptide of SEQ ID NO:1.

By "functional advantage" in this context we mean that the fragment, variant, fusion or derivative exhibits an altered property relative to the polypeptide of SEQ ID NO: which renders it better suited to clinical use.

For example, the functional advantage may be selected from the following group:

(a) Increased receptor affinity for GLP1 receptors

Such increase affinity for GLP1 receptors may be achieved by modifying one or more of the amino acid residues of SEQ ID NO:1 which interact with the receptor (such as E15, V19, R20, F22, 123, L26, K27 and/or S32).

Alternatively, chimeric polypeptides may be used which include the GLP1 receptor binding region of another protein (such as GLP1 itself).

(b) Reduced vulnerability to DPP-IV degradation

Such reduced vulnerability to DPP-IV degradation may be achieved using methods well known in the art, for example N-terminal modifications (such as maleimide conjugation) and/or the use of non-naturally occurring amino acids (such as aminoisobutyric acid, Aib).

(c) Decreased rate of renal clearance

Such decreased rate of renal clearance (i.e. increased in half-life in vivo) may be achieved using methods well known in the art, for example by fusion, conjugation or crosslinking to human serum albumin (as described in US 2009/0005312) and/or by PEGylation.

For example, the half life in vivo (preferably in humans following IV and/or sub- cutaneous administration) may be increased to at least 30 minutes, for example at least 40 minutes, 50 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours or 24 hours or more.

It will be appreciated by persons skilled in the art that the polypeptides of the invention may be of any suitable length. Preferably, the polypeptides are between 10 and 100 amino acids in length, for example between 20 and 60, 30 and 50, 35 and 45, or 38 and

40 amino acids in length. However, where the polypeptide is a fusion protein, e.g. with human serum albumin, its length may be considerably longer, for example at least 100 amino acids, 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids or 600 amino acids.

In one embodiment, the polypeptide is 39 amino acids in length.

In a further embodiment, the polypeptide is linear.

In an additional embodiment, the polypeptide is a recombinant polypeptide.

In one particularly preferred embodiment, the polypeptide comprises an amide group at its C-terminus.

A second, related aspect of the invention provides an isolated polypeptide comprising or consisting of an amino acid sequence according to SEQ ID NO:2 above, with the proviso that the isolated polypeptide does not consist of an amino acid sequence according to SEQ ID NO:1.

In one embodiment, the polypeptide is selected from the group consisting of

AU-12: HGEGTFTSDLSKEMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:6];

AU-U: HGEGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPS

[SEQ ID NO:7]; AU-18: HGEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:9]; AU-19: HVEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NCMO];

AU-20: HΛ/fcEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:11]; and

AU-28- HGEGTFTSDLSKQLEEEAVRLFIEWLKSGGPSSGAPPPS

[SEQ ID NO:12].

Advantageously, the above polypeptides comprise an amide group at its C-terminus.

A third aspect of the invention provides an isolated nucleic acid molecule (such as a DNA or RNA molecule) encoding a polypeptide according to the second aspect of the invention.

A fourth aspect of the invention provides a vector, for example as expression vector, comprising a nucleic acid molecule according to the third aspect of the invention

A fifth aspect of the invention provides a host cell comprising a vector according to the fourth aspect of the invention.

The polypeptides of the invention, as well as nucleic acid molecules, vectors and host cells for producing the same, may be made using methods well known in the art (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York, the relevant disclosures in which document are hereby incorporated by reference).

Alternatively, the polypeptides of the invention may be synthesised by known means, such as liquid phase and solid phase synthesis (for example, t-Boc solid-phase peptide synthesis and BOP-SPPS).

It will be appreciated by persons skilled in the art that the present invention also includes pharmaceutically acceptable acid or base addition salts of the above described polypeptides. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate [i.e. 1 ,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.

Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the polypeptides. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine- (meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.

It will be further appreciated that the polypeptides of the invention may be lyophilised for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilisation method (e.g. spray drying, cake drying) and/or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate. Preferably, the lyophilised (freeze dried) polypeptide loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (prior to lyophilisation) when rehydrated.

A sixth aspect of the invention provides a pharmaceutical composition comprising a polypeptide according to the first or second aspects of the invention together with a pharmaceutical acceptable buffer, diluent, carrier, adjuvant or excipient. Additional compounds may be included in the compositions, including, chelating agents such as EDTA, citrate, EGTA or glutathione. The antimicrobial/pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. The pharmaceutical compositions may be lyophilised, e.g., through freeze drying, spray drying, spray cooling, or through use of particle formation from supercritical particle formation.

By "pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the hypothermia-inducing activity of the polypeptide of the invention. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed ., Pharmaceutical Press (2000), he disclosures of which are incorporated herein by reference).

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine,

Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO,

BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The term "diluent" is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the peptide in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the polypeptide of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as polyvinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

The polypeptides of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery of polypeptide agents.

In one embodiment, the pharmaceutical compositions of the invention may be in the form of a liposome, in which the polypeptide is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations is can be found in for example US 4,235,871 , the disclosures of which are incorporated herein by reference.. The pharmaceutical compositions of the invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microshperes. Preparations of such microspheres can be found in US 5,851 ,451 and in EP 0 213 303, the disclosures of which are incorporated herein by reference...

In a further embodiment, the pharmaceutical compositions of the invention are provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the peptide. The polymers may also comprise gelatin or collagen.

Alternatively, the polypeptides may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of the invention may include ions and a defined pH for potentiation of action of the polypeptides. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.

The pharmaceutical compositions according to the invention may be administered via any suitable route known to those skilled in the art. Thus, possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, parenteral, vaginal and rectal. Also administration from implants is possible. In one preferred embodiment, the pharmaceutical compositions are administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intrastemally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are conveniently used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Thus, the pharmaceutical compositions of the invention are particularly suitable for parenteral, intravenous, intracerebroventricular, intratechal and/or sub-cutaneous administration.

Alternatively, the pharmaceutical compositions may be administered intranasally or by inhalation (for example, in the form of an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas). In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active polypeptide, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

The pharmaceutical compositions will be administered to a patient in a pharmaceutically effective dose. A 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art. The administration of the pharmaceutically effective dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the does may be provided as a continuous infusion over a prolonged period.

The polypeptides can be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used. Preferably, the formulation comprises the active agent at a concentration of between 0.1 μM and 1 mM, more preferably between 1 μM and 500 μM, between 500 μM and 1 mM, between 300 μM and 700 μM, between 1 μM and 100 μM, between 100 μM and 200 μM, between 200 μM and 300 μM, between 300 μM and 400 μM, between 400 μM and 500 μM and most preferably about 500 μM. Thus, the pharmaceutical formulation may comprise an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to induce or maintain hypothermia.

It will be appreciated by persons skilled in the art that the pharmaceutical compositions of the invention may be administered alone or in combination with other therapeutic agents used in the treatment of or as a consequence of ischemic episodes (for example, thrombolytic ("clot busting") agents such as tissue plasminogen activator, epinephrine, vasopressin, antiarrhythmic agents such as amiodarone, and aspirin).

A seventh aspect of the invention provides the use of a polypeptide according to the first or second aspects of the invention in the preparation of a medicament for inducing or maintaining hypothermia in a subject in need thereof.

In one embodiment, the medicament is for the treatment or prevention of neuronal damage in the central nervous system. For example, the medicament may be for use in the treatment or prevention of acute brain injury and/or the treatment or prevention of neuronal damage due to ischemia.

Preferably, the medicament is for inducing or maintaining hypothermia in a subject who is suffering from or has recently suffered from a stroke, a brain trauma, a cardiac arrest, spinal cord injury or asphyxia.

An eighth aspect of the invention provides a method for inducing or maintaining hypothermia in a subject in need thereof, the method comprising administering to the patient a therapeutically-effective amount of a polypeptide according to the first or second aspects of the invention.

Preferably, the subject is human.

Conveniently, the subject is conscious during administration of the polypeptide (e.g. following a stroke).

In one embodiment, the method is for the treatment or prevention of neuronal damage in the central nervous system. For example, the method may be for use in the treatment or prevention of acute brain injury and/or the treatment or prevention of neuronal damage due to ischemia.

Preferably, the method is for inducing or maintaining hypothermia in a subject who is suffering from or has recently suffered from a stroke, a brain trauma, a cardiac arrest, spinal cord injury or asphyxia.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

Figure 1. Dose dependent hypothermic response of AU-01 in a rat model of experimental stroke

Figure 2. Time course of the changes in body temperature of a rat subjected to experimental stroke and injected twice with either saline of AU-01.

Figure 3. Changes in body temperature of rats subjected to experimental stroke and treated with continuous infusion of saline or AU-01.

Figure 4. Changes in core temperature following repeated i.v. injections of AU-01.

Figure 5. Effect on body temperature of rats subjected to experimental stroke and treated with negative control peptide AU-02.

Figure 6. The effect of a bolus dose of AU-01 followed by a continuous infusion in rats subjected to 10 min of transient global cerebral ischemia.

Figure 7. The effect on body temperature of AU-08 injected i.p. at the end of ischemia and 3 hrs later into rats subjected to transient experimental stroke.

Figure 8. (a) The effect on body temperature of AU-19 and AU-20 injected i.p. at the end of ischemia and 3 hrs later into rats subjected to transient experimental stroke, (b) The effect on body temperature of AU-01 and AU-20 injected iv. at the end of ischemia and 3 hrs later into rats subjected to transient experimental stroke.

Figure 9. (a) The effect of AU-13, AU-23, AU-24, AU-25, AU-26, AU-27 on core temperature in rats subjected to experimental stroke, (b) The effect on body temperature of AU-28 injected i.p. or i.v. at the end of ischemia and 3 hrs later into rats subjected to transient experimental stroke, (c) The effect on body temperature of AU- 12and AU-14 injected i.p. at the end of ischemia and 3 hrs later into rats subjected to transient experimental stroke.

Figure 10. The effect on body temperature of AU-17 and AU-18 injected i.p. at the end of ischemia and 3 hrs later into rats subjected to transient experimental stroke.

Figure 11. The effect of AU-11 on core temperature in rats subjected to 2 hr tMCAO.

Figure 12. Comparison between the hypothermic effect of AU-01 and AU-15 given i.v. at the end of ischemia.

Figure 13. The effect of AU-3,4,5,6,7 on core temperature of rats subjected to 2hrs of tMCAO.

Figure 14. The effect of AU-10, 16 on core temperature of rats subjected to 2hrs of tMCAO.

Figure 15. The effect of AU-02, 03, 04, 05 and 07 on core temperature of healthy rats.

Figure 16. The effect of AU-09, 10, 13, 16 and 21 on core temperature of healthy rats

Figure 17. The effect of AU-23, 24, 25, 26 and 27 on core temperature of healthy rats.

Figure 18. The effect of AU-15, 18, 19, 20 and 28 on core temperature of healthy.

Figure 19. The effect of AU-08,11,12,14 and 17 on core temperature of healthy rats. Figure 20. The effect of AU-01 on core temperature of rats subjected to 2hrs of tMCAO and the temperature of AU-01 treated rats housed in an incubator to compensate temperature loss.

Figure 21. Infarct size of rats subjected to 2hrs of tMCAO and treated with saline, AU-01 and AU-01 with concomitant housing in an incubator, respectively.

Figure 22. Neurological score on the rotating pole (a) and the grip test (b) of rats subjected to 2hrs of tMCAO and treated with saline, AU-01 and AU-01 with concomitant housing in an incubator, respectively.

Figure 23. Damage in the hippocampal CA1 subfield of rats subjected to 10min of global cerebral ischemia and subsequent AU-01 and vehicle treatment, respectively.

Figure 24. The effect of combination therapy of AU-01 treatment and intravenous ice- cold saline infusion of core temperature of rats subjected to 2 hrs of tMCAO.

Figure 25. Damage in the hippocampla CA1 subfield of rats subjected to 10min of global cerebral ischemia and subsequent AU-01 in combination with intravenous infusion of ice cold saline (i.s.)

Figure 26 shows the body (rectal) temperature of rats subjected to 90 minutes of experimental stroke.

Figure 27 shows the effect of Ex-4 treatment at one day after 90 minutes of experimental stroke.

Figure 28 shows the time course of body temperature in one rat treated with Ex-4 (5μg/kg Lp.).

Figure 29 shows the effect of Ex-4 treatment on body temperature in non-injured animals. EXAMPLES

EXAMPLE 1: Hypothermic effect of an exemplary polypeptide of the invention (designated "AU-01"; see SEQ ID NO:1)

1. Dose response study

1.1 Experimental procedure

1.1.1 Animals. All animal experiments were approved by the Regional Ethical Committee. Male Wistar rats (Taconic A/S, Copenhagen, Denmark) weighing (290-36Og) were used. All animals were fasted overnight prior to the experiments with free access to water.

1.1.2 Materials. AU-01 was from California Peptide Research lnc and dissolved in 0.1% bovine serum albumin in saline.

1.1.3 Administration of compounds. Bolus injections were always 1 ml/kg. Intraperitoneal (i.p.) injections were placed into the middle lower quadrant of the abdomen.

1.1.4 Temperature measurements. Core temperature was measured by introducing a lubricated thermistor probe (Elab, Copenhagen, Denmark) into the rectum. A stable temperature reading was obtained within 10 sec.

1.1.5 Experimental stroke - occlusion of the middle cerebral artery (MCA). Transient occlusion of the middle cerebral artery (tMCAO) was conducted as described by Koizumi et al., 1992 and Zhao et al., 1994. The animals were immersed into a gas mixture of 4% isoflurane in nitrous oxide:oxygen (35:65) in a glass jar, and thereafter anestesia (decreasing isoflurane concentration to 2%) was continued while the rat was spontaneously breathing on a mask during surgery. Body temperature during surgery was maintained by a heating pad at 37°C±0.5°C. A midline incision was made and the scull bone exposed. A laser Doppler probe (Perimed, ) was glued onto the scull to monitor the blood flow in the territory of MCA. A tail artery catheter was placed to measure mean arterial blood pressure. Temperature, blood flow, and blood pressure were monitored and recorded by a Macintosh PPC7600 computer. The rat was subsequently placed on the back and an incision made in the neck. Heparin was injected and blood gases were analyzed to ensure physiological stability of the animal. The right common and external carotid arteries were exposed and ligated. The internal carotid artery was clamped and an incision made in the vessel. While observing the cortical blood flow, a nylon filament (0.25mm diameter) was introduced approximately 19mm into the internal carotid artery until a sudden drop in the laser Doppler signal was seen indicating occlusion of the middle cerebral artery. All wounds were closed and pain relieved by Marcain (Astra-Zeneca) (2.5mg/ml) infusion into the wounds. The animals were allowed to wake up in a cage. After 1.5 h the animal was re-anesthetized the wound opened and while the occluding filament was removed the laser Doppler signal recorded. The right MCA territory was considered reperfused if there was an immediate increase in blood flow as recorded by the laser Doppler. The neck and scull wounds were closed and the rat allowed to wake up, and placed in a home cage until further treatment. The first injection of compound or vehicle was immediately after removal of the filament.

1.2 Results

The HT inducing effect of AU-01 (i.p) in a rat model of stroke (figure 1) is dose dependent with a negligible effect on body temperature at 0.2 nmole/kg (n=3) (filled squares) compared to saline treated rats (n=3) (open squares) with maximal effect at 0.6 nmoles/kg (n=3) (open circles) and 1.2 nmoles/kg (n=4)

(filled circles).

2 Time course study for the hypothermic effect for AU-01 in the rat MCAO model

2.1 Experimental procedure

2.1.1 Rats were as described in Section 1.1.1 and materials as described in Section 1.1.2. Injections as described in Section 1.1.3. The stroke model was as described in Section 1.1.5 and temperature assessment as described in Section 1.1.4. 2.1.2 For continuous administrations, solutions were injected into by mini-osmotic pumps (Alzet 2001 D₁ Durect, Cupertino, CA, USA). The pumps containing 20OuI solution, delivered 8ul/h. They were filled with desired solution and placed in saline solution for 3hrs at 37°C prior to insertion into the peritoneum of the rat.

Hence there was approximately solution for 20hrs of infusion once the pump was inserted. After 28 hrs the rat was again anesthetized and the pump removed and checked for any remaining solution.

2.2 Results

Core temperature progressively decreases with time of administration of AU-01 (i.p) after induction of experimental stroke in the rat (Figure 2). In vehicle (saline) treated animals (n=6) core temperature (open circles) increases to approximately 38.5°C. A single dose of AU-01 (1.2 nmoles/kg i.p) (n=6) injected at the end of ischemia decreases core temperature (filled circles) to approximately 36°C which is maintained with an additional dose of AU-01 (1.2 nmoles/kg i.p) three hours after the end of ischemia (arrow).

Injecting a bolus dose of 1.2nmoles/kg i.p. immediately at the end of ischemia following 2hrs of MCAO, followed by a continuous infusion of AU-01 (0.2nmol/hή by means of mini-osmotic pumps implanted in the peritoneum decreased core temperature to below 35 ⁰C (figure 3, filled circles) within a 5hr period, while in saline treated animals, core temperature increased to 39°C (open circles). Hence, AU-01 decreased body temperature by 4°C at 5 hrs after the end of ischemia compared to saline treated animals.

3 Comparison of intravenous and intraperitoneal administration of AU-01 on body temperature in the rat MCAO model

3.1 Experimental procedure

3.1.1 Rats were as described in Section 1.1.1 and materials as described in Section 1.1.2. Injections as described in Sections 1.1.3 and 2.1.2. The stroke model was as in 1.1.5 and temperature assessment as described in Section 1.1.4. 3.1.2 Intravenous administration of AU-01 was accomplished by a catheter inserted into the jugular vein positioned in the inferior vena cava tunellated through the skin of the back.

3.2 Results

The rat received bolus injections of 1.2 nmoles AU-01/kg i.v. every 30 minutes over a period of 5.5 hours (figure 4). Core temperature dropped to 34.2°C, i.e. of similar magnitude as that obtained by the i.p. route.

4 Negative control, hypothermic effect for AU-02 in MCAO model

4.1 Experimental procedure

Rats were as described in Section 1.1.1 and materials as described in Section 1.1.2. Injections as described in Section 1.1.2 The stroke model was as described in Section 1.1.5 and temperature assessment as described in Section 1.1.4.

The following negative control peptide was used (which corresponds to Exendin- 4(9-39):

AU-02: DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 [SEQ ID NO: 13]

4.2 Results

Rats (n=3) were injected with two doses of the AU-01 antagonist AU-02 (once immediately after the end of ischemia and a second time three hours later [see arrow]). There was a 0.5⁰C increase in core temperature following treatment with AU-02 (figure 5). 5. Hypothermic effect of AU-01 in a model of global brain ischemia

5.1 Experimental

5.1.1 Rats were as described in Section 1.1.1. Intraperitoneal injections as described in Section 1.1.3, continuous i.p. injections as described in Section 2.1.2. and i.v. injections as described in Section 3.1.2. The temperature assessment as described in Section 1.1.4.

5.1.2 Experimental global ischemia - mimicking cardiac arrest. Rats were anesthetized and then intubated with a PE200 catheter and then connected to a ventilator (model 7025, Ugo Basile) and continuously ventilated on 1.5% isoflurane in nitrous oxide/oxygen (70/30). Body temperature was maintained by heating pad at 37°C±0.5°C. A tail artery catheter was inserted for blood gas analysis and blood pressure monitoring, a tail vein catheter was placed for administration of drugs, and a silastic catheter (1.6 mm outer diameter, Sedat Inc, Irigny, France), introduced into the jugular vein for blood removal. An incision was made in the neck and both common carotid exposed and encircled with a thread. EEG needle electrodes positioned in each temporalis muscle. A muscle relaxant (Vercuronium, Organon, Oss, Holland), was provided 2mg/ml with a speed of

1ml/h. After a stabilization period of 20-30 min pCO2, pO2, pH was determined and if within normal range ischemia was initiated. This was accomplished, by occluding both carotid arteries with non-traumatic stainless steel clamps while concomitantly decreasing blood pressure to 50 mmHg by removal of approximately blood through the jugular vein catheter. The onset of ischemia was defined when EGG became isoelectric. After 10 minutes of ischemia the blood was reinfused, the clamps released and 0.5 ml of sodium bicarbonate (50mg/ml) is given i.v.. The Vecoronium administration was stopped immediately at the end of ischemia, the catheters removed and the wounds sutured and anaesthesia discontinued. The animals were allowed to wake up and upon when spontaneous breathing commenced the animals were extubated and move to the home cage. The brains were perfusion fixed at 4 days after the ischemic insult. Damage in the hippocampus was assessed by histopathology. Results

Rats subjected to 10 minutes of global cerebral ischemia using the 2-VO model, mimicking events in the brain occurring during cardiac arrest, strangulation or neonatal asphyxia, where either injected i.p. with a bolus dose of AU-01 (n=3) or vehicle (n=3) and the with a continuous i.p. infusion of AU-01 or vehicle by means of mini-osmotic pumps. The mean core temperature dropped to 35 °C within 3 hrs and to 34 ⁰C within 12hrs (figure 6).

EXAMPLE 2: Hypothermic effects ofAU-01 analogues

1. Hypothermic effects for AU-08 in the rat MCAO model

1.1 Experimental procedure

Rats were as described in Section 1.1.1 and materials as described in Section 1.1.2 of Example 1. Injections as described in Section 1.1.3 of Example 1. The temperature assessment as described in Section 1.1.4 of Example l and the stroke model was as described in Section 1.1.5 of Example 1.

1.2 Results

Figure 6 displays the changes in core temperature after i.p. injection of 1.2nmoles/kg (n=3) and 6nmoles/kg i.p. of AU-08 at the end of ischemia and three hours later. There was a moderate decrease in body temperature with the low dose but a marked decrease with the higher dose of AU-08 of comparable efficiency as AU-01.

2. Hypothermic effects of AU-19. AU-20 and AU-21 in the rat MCAO model

2.1 Experimental procedure

2.1.1 Rats were as described in Section 1.1.1 of Example 1. Injections were as described in Section 1.1.3 or 3.1.2 of Example 1. The temperature assessment was as described in Section 1.1.4 of Example 1 and the stroke model was as described in Section 1.1.5 of Example 1.

2.1.2 The compounds AU-19 (SEQ ID NO: 10), AU-20 (SEQ ID NO: 11), AU-21 were synthesized by Biopeptide lnc and dissolved in 0.1 % rat serum albumin.

AU-21: HGPGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

[SEQ ID NO: 14] 2.2 Results

2.2.1 AU-19, AU-20, AU-21 are C-terminally (amide) stabilized variants of AU-01 that are resistant to proteolytic cleavage. Treatment with 6 nmoles/kg AU-21 did not show any effect on body temperature, while AU-19 had a modest effect (figure

8a). When administered i.v AU-20 (6nmol/kg) (figure 8b) at the end of ischemia and 3 hrs later had a hypothermic effect comparable to that of AU-01 (6 nmol/kg i.v.).

3. Hypothermic effects of AU-12 (SEQ ID NO:5l AIM 3. AU-14 (SEQ ID NO:6), AU- 23 , AU-24. AU-25. AU-26. AU-27 and AU-28 (SEQ ID NO: 12) in the rat MCAO model

3.1 Experimental procedure

Rats were as described in Section 1.1.1 of Example 1. Injections were as described in Sections 1.1.3 and 3.1.2 of Example 1. The temperature assessment was as described in Section 1.1.4 of Example 1 and the stroke model was as described in Section 1.1.5 of Example 1.

Compounds and solutions as in 2.1.2 above.

AU-13: HGEGTFTSDLSKEMEEEAVRLFIEWLKDGGPSSGAPPPS-NH2

[SEQ ID NO: 15] AU-23: HGEGTFLSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

[SEQ ID NO: 16] AU-24: HGEGTFVSDLSKQLEEEAVRTFIEWLKNGGPSSGAPPPS-NH2

[SEQ ID NO: 17]

AU-25: HGEGTFVEDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 [SEQ ID NO: 18]

AU-26: HGEGNFTEDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

[SEQ ID NO: 19] AU-27: HGEGNFVEDLSKLLEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

[SEQ ID NO: 20] 3.2 Results

Rats were treated with either 1.2 or 6 nmoles/kg i.p. of AU-13, AU-23 AU-24, AU-

25, AU-26 and AU-27. There was no significant effect of core temperature by any of these compounds, figure 9a.

When rats subjected to experimental stroke of 2 hrs duration were treated with 6nmoles/kg i.p. of AU-28, no hypothermic effect was noticed, (figure 9b). When treated with 6 nmoles/kg i.v. a marked decrease of temperature was seen of similar magnitude as that seen with AU-01.

Figure 9c displays the changes in core temperature after i.p. injection of 1.2nmoles/kg (n=3) and 6nmoles/kg (n=1) i.p. of AU-12 and AU-14 at the end of ischemia and three hours later. There was no decrease in body temperature with the low dose but a marked decrease with the higher dose of AU-12 and AU-14.

4. Hypothermic effects of AU-17 and AU-18 in the rat MCAO model

4.1 Experimental procedure

Rats subjected to transient experimental stroke of 2hrs duration were as described in Section 1.1.1 of Example 1. Injections were as described in Section described in Section 1.1.3 of Example 1. The temperature assessment was as described in Section 1.1.4 of Example 1 and the stroke model was as described in Section 1.1.5 of Example 1.

Compounds as in 2.1.2 above.

4.2 Results

Rats were treated with 1.2 and 6 nmoles/kg of the chimeric variants of AU-01 and GLP-1, AU-17, AU-18. While AU-17 did not affect body temperature at 6 nmoles/kg, treatment with 6nmoles/kg of AU-18 but not 1.2nmoles/kg Lp. markedly decreased temperature, (figure 10).

EXAMPLE 3: Long-lasting hypothermic effect of AU-Oi analogues

1 Duration of hypothermic effect of AU-11 (SEQ ID NO: 4) the rat MCAO model

1.1 Experimental procedure

Rats were as described in Section 1.1.1 of Example 1. Injections were as described in Section 3.1.2 of Example 1 with a single injection at the end of the ischemic period. The temperature assessment was as described in Section 1.1.4 of Example 1. and the stroke model was as described in Section 1.1.5 of Example

1.

Compounds as in 2.1.2 of Example 2.

1.2 Results

AU-11 is more resistant to proteolytic degradation. Treatment of rats subjected to experimental stroke for 2hrs with AU-11 at a concentration of 6 but not 1.2 nmoles/kg i.p. induced a hypothermic response, Figure 11. This was not as pronounced as that of AU-01.

2 Duration of hypothermic effect of AU-15 (SEQ ID NO:7) and AU15-PC the rat MCAO model

2.1 Experimental procedure

2.1.1 Rats were as described in Section 1.1.1 of Example 1. Injections were as described in Section 3.1.2 of Example 1, though only one injection was performed at the end of ischemia. The temperature assessment was as described in Section 1.1.4 of Example 1. and the stroke model was as described in Section 1.1.5 of

Example 1.

2.1.2 Synthesis of AU-15PC. AU-15 was conjugated to rat serum albumin (Sigma- Aldrich) by incubating 1mM AU-15 in 25% rats serum albumin solution at 37⁰C for 30 minutes. 2.2 Results

2.2.1 AU-15 given i.v. at a single dose of 6nmoles/kg at the end of the ischemic period, induced a hypothermic effect of similar magnitude as AU-01 of the same dose and route of administration, but was more long-lasting. AU-01 and AU-15 had a nadir of 35.4 ⁰C and 35.5 ⁰C, respectively. At 6 hours after the injection of the compounds, the AU-01 treated animals returned to temperatures seen in vehicle treated animals (approximately 38 ⁰C), while the AU-15 treated animals remained at temperatures below 36.5°C, Figure 12.

2.2.2 The hypothermic response of AU-15PC was dose dependent. At a dose of 6nom/kh i.v. temperature dropped to around 36⁰C and remained for at least 6hrs. At a dose of 12 nmol/kg i.v. temperature dropped to around 35⁰C. At a dose of 24 nmol/kg temperature further dropped 34,2-34,5°C.

EXAMPLE 4: Study of the ability of GLP-1 and GLP-1 analogues to induce hypothermia

1 Hypothermic effects of GLP-1 (AU-03) and analogues thereof (AU-04, AU-05. AU-06. AU-07) in the rat MCAO model

1.1 Experimental procedure

Rats were as described in Section 1.1.1 and materials as described in Section 1.1.2 of Example 1. Injections as described in Section 1.1.3 of Example 1. The temperature assessment as described in Section 1.1.4 of Example 1 and the stroke model was as described in Section 1.1.5 of Example 1.

The following polypeptides were used:

AU-03: HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[SEQ ID NO: 21]

AU-04: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA [SEQ ID NO: 22]

AU-05: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[SEQ ID NO: 23]

AU-06: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

[SEQ ID NO: 24]

AU-OT. HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2

[SEQ ID NO: 25]

1.2 Results

Rats subjected to 2 hrs of tMCAO were treated with either 1.2 nmol/kg i.p. of AU- 03, AU-04, AU-05, AU-06, AU-07 at the end of ischemia and three hours later did not display any hypothermia inducing effect (figure 13). 2 Hypothermic effects of GLP-1 analogues (AU-10. AU-16) in the rat MCAO model

2.1 Experimental procedure

Rats were as described in Section 1.1.1 and materials as described in Section 1.1.2 of Example 1. Injections as described in Section 1.1.3 of Example 1. The temperature assessment as described in Section 1.1.4 of Example 1 and the stroke model was as in 1.1.5 described in Section Example 1.

The following polypeptides were used

AU-10: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[SEQ ID NO: 26]

AU-16: HAibEGTFTSDVSSYLEGQAAKEFIAWLVKAibR-NH2

[SEQ ID NO: 27]

2.2 Results

Rats subjected to 2hrs of tMCAO treated with either 1.2 nmol/kg i.p. of AU-10 or AU-16 (1.2 or 72 nmoles/kg) at the end of ischemia and three hours later, did not display any persistent hypothermia inducing effect (figure 14).

EXAMPLE 5: Comparison of hypothermic effect in healthy rats versus MCAO stroke model rats

1 Hypothermic effects for AU-01 , AU-01 analogues and GLP-1 analogues in fasted rats (AU01 to 05. 07 to 21. 23 to 28^

1.1 Experimental procedure

Animals were as described in Section 1.1.1 in Example 1. Compounds were as described in Section 1.1.2 in Example 1. Injections were performed as described in Section 1.1.3 in example. 1. One injection was performed and the temperature measured over a three hour period.

The following new polypeptide was used:

AU-09: HADGSFSDEMNTILDNLATRDFINWLIQTKITD

[SEQ ID NO: 28]

1.2 Results

Healthy (i.e. non-stroke model) rats treated with saline, AU-02 (1.2 nmoles/kg), AU-03 (1.2 nmoles/kg), AU-04 (1.2 nmoles/kg), AU-05 (1.2 nmoles/kg), AU-07 (1.2 nmoles/kg and 60 nmoles/kg), AU-09 (1.2 nmoles/kg), AU-10 (1.2 nmoles/kg), AU-13 (1.2 nmoles/kg), AU-16 (25 nmoles/kg and 600 nmoles/kg), AU-21 (6 nmoles/kg and 30 nmoles/kg), AU-23 (1.2 nmoles/kg and 6 nmoles/kg),

AU-24 (1.2 nmoles/kg and 6 nmoles/kg), AU-25 (1.2 nmoles/kg and 6 nmoles/kg), AU-26 (6 nmoles/kg) and AU-27 (120 nmoles/kg), respectively, did not decrease below 37°C at any time point (figure 15 to 17). Similar treatments in rats subjected to stroke also did not induce a decrease in body temperature (see figures 5, 9, 13 and 14).

Treatment with AU-01 at a dose of 1.2 nmoles/kg and AU-15, AU-18, AU-19, AU- 20 and AU-28 at a dose of 6nmol/kg decreased core temperature to a nadir of 36°C (figure 18). The effect of the compounds on core temperature is similar to the effect seen in the stroke-injured animals (see figures 1 , 8, 10, 12). In contrast, treating rats with AU-08, AU-11, AU-12, and AU-14 with 1.2 nmol/kg induced a decrease in core temperature to below 37°C (figure19). Rats subjected to stroke required higher doses of these compounds to induce hypothermia (see figures 7, 9 and 11).

AU-17 induced hypothermia in healthy rats (figure 19), while hypothermia was not induced in rats subjected to stroke with this dose of AU-17. However, it is anticipated that a higher dose and/or IV administration will induce hypothermia in rats subjected to stroke.

EXAMPLE 6: Histological study of the neuroprotective effects of hypothermic induction using AU-01

1 Histology and neurology results for AU-01 treated stroke induced rats

1.1 Experimental procedure

1.1.1 Rats were as described in Section 2.1 of Example 1 . They received either saline or AU-01 as a bolus dose of 1.2 nmol/kg and then an infusion of 0.4nmol/hr for approximately 20hrs. In addition one experimental group was performed where rats were treated exactly as those receiving AU-01 but were in addition placed in an incubator with a humidified 25-37°C air to compensate for the temperature loss induced by AU-01 treatment. After removal of the mini-osmotic pump 24-26 hrs after the end of ischemia, the animals were allowed to recovery for 7 days.

They were then tested for neurological function and then perfusion fixed for histopathology.

1.1.2 Neurological scoring: The rorating pole test. The rotating pole test reveals dysfunction of coordination and sensori-motor function (Rickhag et al., 2008,

J Cereb Blood Flow Metah. 28(3):551-62). The pole (length 1500mm, diameter 40mm and elevation 700mm) rotates at 10 rotations per minute. The ability to cross this pole was graded: 6- the animal traverses pole without any foot slips; 5- the animal traverses pole with few foot slips; 4- the animal crosses pole with 50% slipping of the foot steps; 3- the animal crosses the pole while jumping with both hindlimbs; 2- the animal falls off during crossing; 1- the animal remains embraced to the pole unable to cross and then falls off; 0- the animal falls off immediately. Normal score is 5 and 6.

1.1.3 Grip Strength Test: Forelimb strength was measured using the Grip Strength Test Meter GS3 (BIOSEB, France). Rats voluntary gripped a grid either with the healthy or paralyzed forelimb and pulled it backward. Strength of each limb was assessed and the maximal data out of 3 trials was taken for analysis. 1.1.4 Histopathological evaluation: Rats were anesthetized and subsequently tracheostomized and ventilated on 3% isoflurane in nitrous oxide:oxygen (30/70). The brain were transcardially perfused with approximately 100 ml saline by means on a peristaltic pump and subsequently with either 4% paraformalin (MCAO model) The PF-fixed brains were removed from the scull and were post fixed over night in the fixation solution. Thereafter they were placed in 25% sucrose solution and stored in the refrigerator until further processing. The brains were serially sectioned at 40um. Sections were collected at a distance of 1 mm along the rostro-caudal axis (10 levels in total) and stained immunihistochemically using NeuN antibody uniquely recognizing neuronal epitopes. The stained sections were glassmounted and the infarct size measured by computer assisted evaluation of the brain region not stained with NeuN. The total infarct volume was calculated using the damaged brain areas at the 10 brain levels.

1.2 Results

1.2.1 Figure 20 shows the AU-01 treated animals (n=3), the vehicle (0.1%RSA in saline) treated animals (n=3) and the temperature compensated animals (n=3). AU-01 induces hypothermia below 36⁰C within 1hr and the temperature stays below 36⁰C for at least 24hrs with several hours of body temperature below 35°C.

1.2.2 Figure 21 shows the infarct size (mm³) evaluated at 7 day of recovery after 2hrs of tMCAO of the AU-01 treated animals (n=3), the vehicle (0.1%RSA in saline) treated animals (n=3) and the temperature compensated animals (n=3). A decrease in infarct size by approximately 20% can bee seen in the AU-01 treated animals, while there is no difference between vehicle treated animals and AU-01 treated animals that had been temperature compensated in the incubator.

1.2.3 Figure 22 shows the neurological score at 7 days of recovery after 2hrs of tMCAO. In the rotating pole test (a), saline treated animals show a marked neurological dysfunction (mean score 1.3). The AU-01 treated animals show significantly less neurological deficit (mean score 3.7), while the temperature compensate AU-01 treated animal have a score of 0.4. In the grip test (b) a similar improvement by AU-01 treatment is seen. The treated animals retain their grip strength, while vehicle treated animals lost 70% and the temperature compensated animals 50% of the grip strength.

1.2.4 Thus, it is evident that the neuroprotective effects of AU-01 arise as a consequence of the induction of hypothermia rather than by any direct biological effect of the polypeptide itself. In rats which AU-01 -induced hypothermia was compensated by keeping the rat warm in an incubator (i.e. such that a reduction in body temperature was avoided), the neuroprotective effects of AU-01 were absent.

2. Histology of the hippocampus of rats subjected to global ischemia

2.1 Experimental procedure

2.1.1 The rats used in this study were those described in Part 4 of Example 1.

2.1.2 Histopathological evaluation: Rats were anesthetized and subsequently tracheostomized and ventilated on 3% isoflurane in nitrous oxide:oxygen (30/70). The brains were transcardially perfused with approximately 100 ml saline by means on a peristaltic pump and subsequently with 4% formalin (global ischemia model) solution in phosphate buffer. The brains were sectioned and paraffin embedded. The neuronal damage was assessed at the level of the dorsal hippocampus on 6um sections stained with Celestin blue. Damaged cells were counted and damaged expressed as % of normal cell counts.

2.2 Results

Figure 23 shows the mean damage in the CA1 region of the hippocampus of vehicle treated animals (n=3) and animals treated with AU-01 (n=3). Damage in the vehicle treated animals was 70% while in the AU-01 treated animals it decreased to 30% damage. EXAMPLE 7: Hypothermic effect of combination therapy comprising A U-01

1.1 Experimental procedure

1.1.1 Rats and procedures were as described in 4.1.1 of Example 1.

1.1.2 Cold saline solutions are standard initial treatments of cardiac arrest patients that are to induce whole body hypothermia using surface or intravascular cooling.

Rats subjected to ischemia as in 4.1.2 received a 10 ml intravenous injection of ice-cold saline over 5 minutes starting at 60 min after the end of the ischemic episode

1.2 Results

1.2.1 Figure 24 shows the progressive decrease in body temperature of a rat treated with a bolus injection of AU-01 followed by continuous infusion i.p. After injection of the cold saline solution (arrow) the temperature decreased from a relative plateau of 35⁰C to a nadir value of 33.3 ⁰C at the end of the intravenous injection. After this the temperature rose to 34 ⁰C where it stabilized.

1.2.2 Hence, combination therapy of AU-01 and ice-cold saline further enhances the speed of the decrease in body temperature.

1.2.3 Figure 25 photomicrographs of the dorsal rat hippocampus CA1 subfield of (a) an AU-01 treated animal (n=3) and (b) a vehicle treated animal. In the two AU-01 treated animals there were less than 5% damaged neurons compared to 70% damage in the vehicle treated animals.

EXAMPLE 8: Exemplary embodiment of the invention

INTRODUCTION

The present invention relates to the use of Exendin-4 (Ex4) and similar substances in the induction and/or maintainance of hypothermia for treatment of acute brain injury caused by decrease in blood flow (ischemia), decrease in blood oxygen tension (hypoxia) or physical injury, particularly stroke, brain trauma, cardiac arrest and asphyxia in mammals, possibly in combination with other hypothermic treatments.

US Patent No. 6,284,725 teaches that individuals in need of treatment of ischemia- related reperfusion are treated, preferably intravenously, with a composition which includes a compound which binds to a receptor for the glucagon-like peptide-1. The invention relates to both the method and compositions for such treatment. US Patent No. 6,284,725 is specifically related to the situation in which the organ tissue is the myocardium.

There is a need in the art for an improved method of treating acute brain injury, particularly stroke, brain trauma, cardiac arrest and asphyxia in the human.

SUMMARY

Accordingly, an object of the present invention is to mitigate, alleviate or eliminate one or more of the above-identified deficiencies and disadvantages singly or in any combination.

In an aspect, there is provided a method of inducing and/or maintaining hypothermia in a patient, such as a mammal, exposed to brain injury, comprising administration of a therapeutically effective amount to the patient of a composition having a component that binds to a receptor for glucagon-like peptide-1 (GLP-1 ).

In an embodiment, the method may further comprise exposing said mammal to other hypothermic treatment, such as external surface cooling or internal surface cooling. The hypothermic treatment may comprise nasopharyngeal cooling and/or cold saline infusion. The administration and the other hypothermic treatment may take place simultaneously. In another embodiment, the composition may comprise a substance included in the group comprising: exendin-4; a human glucagon-like peptide (GLP)-i-albumin recombinant protein (Albugon); CJC-1131 ; liraglutide; and oxyntomodulin.

In another aspect, there is provided a use of a substance as mentioned above for the preparation of a of a medicament for the induction and/or maintainance of hypothermia for treatment of acute brain injury, particularly stroke, brain trauma, cardiac arrest and asphyxia. The use may be combined with other hypothermic treatments.

Further objects, features and advantages of the invention will become apparent from the following detailed description of embodiments of the invention with reference to Figures 25 to 28.

DETAILED DESCRIPTION OF EMBODIMENTS

Below, several embodiments of the invention will be described with references to the drawings. These embodiments are described in illustrating purpose in order to enable a skilled person to carry out the invention and to disclose the best mode. However, such embodiments do not limit the invention. Moreover, other combinations of the different features are possible within the scope of the invention.

Acute brain injury, such as stroke, cardiac arrest, neonatal asphyxia, and brain trauma, is major causes of death and adult disability in the world. For example, stroke, a condition where brain damage is caused by occlusion of one or several brain blood vessels, afflicts approximately 15 Million persons yearly worldwide, causing 5 Million deaths and leaving 5 Million severely disabled. Brain trauma is the main cause of disability of the young adult.

Brain injury may be caused by at least one of: a decrease in blood flow (ischemia), decrease in blood oxygen tension (hypoxia) and physical injury.

At present there is no therapy that protects the brain cells against damage caused by acute brain injury such as stroke, transient cardiac arrest, brain trauma and asphyxia apart from recanalization of occluded vessels by thrombolysis following embolic stroke. Deep hypothermia has been used in neuro- and cardiac surgery since the 40's. In such protocols, body temperature is decreased to below 25 °C and though efficient in protecting the brain, deep hypothermia is not a feasible treatment in the out-of hospital or the acute clinical setting, because of the complicated procedures.

Decreasing body temperature from non-physiological high levels, called temperature management, or to below normal levels, called hypothermia, diminishes brain damage in experimental animal models of stroke, cardiac arrest, neonatal asphyxia, and brain trauma. This has lead to the introduction of mild hypothermia, i.e. decreasing body temperature to about 31 °C to 35⁰C, such as about 33°C, as a successful treatment of cardiac arrest in human patients, widely implemented in the intensive care units worldwide.

The implementation of mild hypothermia as a human therapy in other clinical conditions has sofar not been demonstrated. In a controlled study on brain trauma patients, treatment with mild hypothermia was ineffective or even detrimental. Likewise, in stroke, mild hypothermia was proven ineffective.

The fact that studies in animal models of stroke and trauma repeatedly demonstrated efficacy of brain protection by mild hypothermia may suggests that the lack of clinical effects in these conditions is due to late introduction of the cooling procedures, the slow cooling process and/or insufficient knowledge of the extent of cooling. Animal studies teach us that mild hypothermia has to be instituted rapidly, such as within hours, after conditions such as stroke or trauma, which today is difficult to attain in humans due to the logistics and the cumbersome procedures for induction of hypothermia.

These hypothermic treatment procedures of decreasing body temperature employed in the clinic encompass various ways of external surface cooling of the body surface by blankets containing cooled circulating water or by cooling the body with cooled air. Alternatively, internal surface cooling devices, such as metal rods or plastic tubing, are introduced into the veins of the body, and are subsequently cooled, decreasing blood temperature and subsequently body temperature to desired levels. Another alternative hypothermic treatment method is the introduction of cold saline solution into the blood stream of the patient, resulting in a very fast initial cooling of the body. A further alternative method is disclosed in US Patent No. US 7,189,253 B2, which discloses methods for cooling the brain and the body via balloons introduced into the nasal and adjacent cavities, so called nasopharyngeal cooling. Further hypothermic treatment methods may include introduction of cooling devices into cavities of the body. The surface cooling method is labor intensive and rather space requiring. The cooling blankets cover the body to an extent that limits the access to the patient. The use of the intravenous cooling method is interventional and requires treatment of the patient at intensive care units and also may cause unwanted bleeding in patients were thrombolytics are used as anti coagulation treatment.

As the core temperature progressively declines, the compensatory metabolic, adrenergic, and cardiovascular body responses attempt to maintain thermal homeostasis.

The mechanism by which mild hypothermia (33°C) is neuroprotective is still unknown. The protective effect is time and temperature dependent and hence involves multiple cellular and molecular mechanisms.

Protection is attained by decreasing brain temperature from 37°C to 31⁰C concomitantly with a decrease of brain metabolism by 30%. Still a lowering to a temperature of 33°C provides a robust neuroprotective effect, which cannot be explained by a moderate (15%) decrease in metabolism . Decreasing brain temperature decreases the release of glutamate, results in a reduction of reactive oxygen species, tissue acidosis and neuronal apoptosis.

A conspicuous correlation between the temperature dependence of actin dynamics and hypothermia suggests that the multiple actions of hypothermia is exerted through the actin cytoskeleton that regulates the activity of several organelles, receptors, channels and enzyme systems.

Incretins, such as glucagon-like polypeptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut hormones that stimulate insulin and suppress glucagon secretion and regulate food intake . GLP-1 (7-37) and GLP-1 (7-36)amide (the latter, two forms of GLP-1) act on G-protein-coupled receptors (GPCRs) in peripheral tissues and the central nervous systems by rapidly increasing the levels of intracellular cAMP and intracellular calcium. GLP-1 is removed from circulation by dipeptidyl peptidase-4 (DPP-4) degradation and/or renal clearance within 2 to 5 minutes.

Exendin-4, in short Ex-4, is a peptide with approximately 50% sequence homology with GLP-1 , and a potent agonist at the mammalian GLP-1 R. It is resistant to DPP-4 degradation and hence has a circulating half-life of about 60-90 min. Subcutaneously injected Ex-4 increases its plasma concentrations for 4-6 h.

Similarly as GLP-1 , Ex-4 has insulinotropic properties in rats and humans and has been approved by FDA for treatment of human type 2 diabetes. It has cell protective properties including neuroprotective properties. See US Patent No. 6,284,725, mentioned above, for a description of i.a. Ex-4 and other GLP-1 -analogues. The contents of US Patent No. 6,284,725 is incorporated in the present specification by reference.

In rats, 3 μg/kg Ex-4 given i.p reduced mean body temperature by 0.6 degrees for 4h. Also, 1 nmol/kg of Ex-4 provided intracerebroventricularly (i.c.v.) decreased body temperature by 2.5 -2 ⁰C, for several days. Moreover, in rats, fever caused by lipopolysaccharide, is enhanced by blocking the GLP-1 receptor with a specific antagonist. In addition, in rats, 10μg GLP-1 given i.p. decreased body temperature over a subsequent 2h period, which is blocked by a concomitant administration of a GLP-1 receptor antagonist.

No data have been published regarding the effect of Ex-4 on body temperature in models of experimental brain injury (ischemia, hypoxia or trauma).

According to embodiments of the present invention, it has been found that Ex 4 induces body hypothermia in rats with brain injury.

Figure 26 shows the body (rectal) temperature of rats subjected to 90 minutes of experimental stroke, in the form of a transient occlusion of the middle cerebral artery

(MCAO). One hour after MCAO body temperature was 38.9±0.4°C in rats to be exposed to Ex-4 (5μg/kg i.p) treatment (n=11) and 38.6 ±0.6°C in rats to be exposed to saline treatment (n=11). At one h after cessation of MCAO (and 30 minutes after intraperitoneal injection of Ex-4 and saline,) temperatures decreased to 37.4±1 and 38.3±0.8, respectively. At one h after cessation of MCAO (i.e. 1.5h after injections), temperature in the Ex-4 and saline treated rats were 37.2±0.4 and 38.1 ±0.8 ⁰C, respectively. Hence at 3.5 hours after onset of stroke, the Ex-4 treatment decreased body temperature by 1.7 C.

Figure 27 shows the effect of Ex-4 treatment at one day after 90 minutes of MCAO. Prior to treatment, the body core temperatures of the rats to be treated with the saline and Ex-

4 were 38.5±0.6 and 38.0±0.4, respectively, and not significantly different. One hour after

Ex-4 (5μg /kg i.p.) injection, body temperature decreased to 36.8±0.7°C. In vehicle

(saline) injected animals, body core temperature was 38.0±0.4°C. At 2 hours after injection, temperature decreased further to 36.1±0.4°C following Ex-4 treatment, and was 37.8±1.2 in vehicle treated animals. At 3h after injection temperature was 36.0±0.9 and

38.0±0.7 in Ex-4 and saline treated animals respectively. Hence Ex-4 treatment decreases body temperature by approximately 2°C within 2 hours after Ex-4 injection.

Figure 28 shows the time course of body temperature in one rat treated with Ex-4 (5μg /kg i.p.). Temperature drops by 2⁰C within 2h and by 4°C by 2h after injection.

Figure 29 shows the effect of Ex-4 treatment on body temperature in uninjured animals. Temperature decreased transiently by 1°C at 2h after injection. However, the differences were not statistically significant.

The above experiments were with intraperitoneal infusion of Ex-4. Experiments by others suggest that intracerebroventricular administration may be more efficient and requiring less concentrations to be therapeutically effective. Intravenous or subcutaneous administration may be used.

It is known that rats subjected to 90 minutes of transient middle cerebral artery occlusion (MCAO), and with a subsequent induction of hypothermia to 34°C by surface cooling starting 30 minutes after recovery of perfusion and with a duration of 4 hours, had significantly less brain damage compared to rats that were normothermic (37°C).

Body cooling to therapeutic levels (33-35⁰C) can be attained by Ex-4 treatment, according to the embodiments of the present invention shown in Figure 1-3. It is therefore feasible to use Ex-4 to induce and maintain hypothermia as a treatment of patients with brain damage. An alternative approach is to use Ex-4 treatment to induce hypothermia and use other conventional methods for maintaining hypothermia, such as nasopharyngeal cooling.

Since it is important to initate hypothermia as soon as possible, treatment with Ex-4 can be combined with other hypothermic treatment methods, such as i.v. infusion of cold saline or nasopharyngeal cooling in order to rapidly reach a therapeutic hypothermic temperature in the body and in the brain. The order of initiation of the different treatment modalities is dependent on the actual situation for the injured patient.

Including Ex-4 with cold saline solution and i.v. infusion thereof, would initiate hypothermic conditions rapidly. Then, nasopharyngeal cooling may be used for further cooling.

The hypothermic condition may be maintained by any combination of Ex-4 treatment and other hypothermic treatment methods. For example, nasopharyngeal cooling may be used for maintaing hypothermic condition, optionally with continued administration of Ex- 4.

The Ex-4 treatment can be performed with other analogues to GLP-1 , such as those mentioned below, or with GLP-1.

To rapidly initiate therapeutic hypothermia, patients may be provided with intravenous cold saline infusions.

Ex-4 treatment in combination with hypothermia induced by other means, such as surface cooling, cooling of the internal parts of the body, nasopharyngeal cooling or by cold solution infusions, could enhance the speed and efficacy of body cooling provided by Ex-4.

On the other hand, administration Ex-4 before or in conjuction with other hypothermia inducing means, as mentioned above, may enhance the cooling efficiency of said other cooling means, for example by blocking one or several of the body hypothermal defence mechanisms. Such blocking may also occur due to the brain injury. The mechanism whereby Ex-4 is causing body hypothermia is not fully understood. The Ex-4 effect is mediated by GLP-1 receptors in the CNS₁ present i.a. in the hypothalamus and in nuclei of the caudal brain stem. The brain stem nuclei seem to be particularly important for temperature control.

GLP-1 receptor activation by Ex-4 in local circuits in the hindbrain appears to decrease body temperature. This hypothermic effect may be counteracted by neuronal connections from forebrain structures. Hence in uninjured animals, the hypothermic effect of Ex-4 treatment may be transient and less prominent. In the ischemia damaged brain, the inhibitory influence of the forebrain on GLP-1 receptor activation may be depressed causing a more extensive induction of hypothermia by Ex-4.

Similar effects may be obtained with other GLP-1 -analogues than Ex-4, such as any substance that binds to a receptor for GLP-1. Some examples are: 1 ) Albugon, a human glucagon-Iike peptide (GLP)-i-albumin recombinant protein, which activates GLP-1 receptor (GLP-1 R)-dependent cAMP formation in BHK- GLP-1 R cells

2) CJC-1131 , a DPP-IV-resistant drug affinity complex (DAC) GLP-1 compound that conjugates to albumin in vivo 3) Liraglutide is a fatty acylated human DPP-IV-resistant GLP-1 analog that binds to albumin and exhibits a t1/2 of -1 1-15 h after parenteral administration in humans.

4) Oxyntomodulin (OXM).

Although the present invention has been described above with reference to specific embodiment, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and other embodiments than the specific above are equally possible within the scope of these appended claims.

In the claims, the term "comprises/comprising" does not exclude the presence of other elements or steps. Furthermore, although individually listed, a plurality of means, elements or method steps may be implemented by e.g. a single unit. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. The terms "a", "an", "first", "second" etc do not preclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way.

SUMMARY

Method of inducing hypothermia in a patient exposed to brain injury. A therapeutically effective amount of a composition having a component that binds to a receptor for glucagon-like peptide-1 (GLP-1) is administred to the patient. The component may be exendin-4 (Ex-4) or similar. The hypothermic Ex-4 method may be combined with other hypothermic treatments, such as external surface cooling or internal surface cooling, specifically nasopharyngeal cooling and cold saline infusion. Use of the substance for the preparation of a of a medicament for the induction of hypothermia for treatment of acute brain injury, particularly stroke, brain trauma, cardiac arrest and asphyxia.

Claims

1. A polypeptide capable of binding to a receptor for glucagon-like peptide-1 (GLP- 1 ) for use in inducing or maintaining hypothermia in a subject in need thereof,

wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 1:

AU-01: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS [SEQ ID NO: 1]

2. A polypeptide according to Claim 1 for use in the treatment or prevention of neuronal damage in the central nervous system.

3. A polypeptide according to Claim 1 or 2 for use in the treatment or prevention of acute brain injury.

4. A polypeptide according to any one of the preceding claims for use in the treatment or prevention of neuronal damage due to ischemia.

5. A polypeptide according to any one of the preceding claims wherein the subject is suffering from or has recently suffered from a stroke, a brain trauma, a cardiac arrest, spinal cord injury or asphyxia.

6. A polypeptide according to any one of the preceding claims wherein the polypeptide is for use in combination with one or more additional hypothermic treatments.

7. A polypeptide according to Claim 6 wherein the one or more additional hypothermic treatments include external and/or internal surface cooling.

8. A polypeptide according to Claim 6 or 7 wherein the one or more additional hypothermic treatments include nasopharyngeal cooling and/or cold saline infusion.

9. A polypeptide according to Claim 6 or 7 wherein the one or more additional hypothermic treatments is selected from the group consisting of vanilloid receptor agonists, cannabinoid receptor agonists adenosine receptor agonists and fever reducing agents such as regulators of prostaglandin synthesis.

10. A polypeptide according to any one of the preceding claims wherein the subject is human.

11. A polypeptide according to any one of Claims 1 to 9 wherein the subject is non- human.

12. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of inducing a fall of at least 1⁰C in core body temperature of the subject, for example a fall of at least 2°C, 2.5⁰C, 3°C, 4°C, 5°C or more.

13. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of inducing a hypothermic effect having a duration of at least 10 minutes, for example at least 20 minutes, 30minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 120 minutes or more.

14. A polypeptide according to any one of the preceding claims wherein the polypeptide has an in vivo half-life in humans of at least 10 minutes following IV administration, for example at least 15 minutes or at least 20 minutes or more.

15. A polypeptide according to any one of the preceding claims wherein the polypeptide has comparable efficacy in healthy subjects and in subjects suffering from neuronal damage due to an ischemia.

16. A polypeptide according to any one of the preceding claims wherein the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 1.

17. A polypeptide according to any one of Claims 1 to 15 wherein the polypeptide comprises or consists of a fragment of the amino acid sequence according to SEQ ID NO: !

18. A polypeptide according to Claim 17 wherein the fragment comprises or consists of at least 10 contiguous amino acid of SEQ ID NO: 1 , for example at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37 or 38 contiguous amino acid of SEQ ID NO: 1.

19. A polypeptide according to Claim 17 or 18 wherein the fragment commences at an amino acid residue selected from amino acid residues 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 and 29 of SEQ ID NO:1.

20. A polypeptide according to any one of Claims 17 to 19 wherein the fragment terminates at an amino acid residue selected from amino acid residues 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38 and 39 of SEQ ID NO:1.

21. A polypeptide according to any one of Claims 17 to 20 wherein the fragment comprises or consists of amino acids 3 to 8 and/or 15 to 32 of SEQ ID NO: 1.

22. A polypeptide according to any one of Claims 1 to 15 wherein the polypeptide comprises or consists of a variant of the amino acid sequence according to SEQ

ID NO: 1.

23. A polypeptide according to Claim 22 wherein the variant is a non-naturally occurring variant.

24. A polypeptide according to Claim 22 or 23 wherein the variant has an amino acid sequence which has at least 50% identity with the amino acid sequence according to SEQ ID NO: 1 or a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

25. A polypeptide according to any one of Claims 22 to 24 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO: 2:

His-Xaa2-Xaa3-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Glu- Glu-Glu-Ala-Val-Arg-Leu-Phe-lle-Glu-Trp-Leu-Lys- Xaa28-Gly-GIy-Pro-Ser-Ser-

Gly-Ala-Xaa36-Pro-Pro~Ser-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46

[SEQ ID NO:2]

wherein

Xaa2 is GIy, Ser, VaI, Ala or aminoisobutyric acid (Aib);

Xaa3 is GIu or Asp;

Xaa10 is Leu or VaI;

Xaa12 is Lys or Ser; Xaa13 is Gln, Glu or Tyr;

Xaa14 is Met or Leu

Xaa28 is Asn, Ser or Asp;

Xaa36 is Pro or absent;

Xaa40 is an amide group, Lys, AEEA-MPA-modified Lys or is absent; Xaa41 is Lys or absent;

Xaa42 is Lys or absent;

Xaa43 is Lys or absent;

Xaa44 is Lys or absent;

Xaa45 is Lys or absent; and Xaa46 is an amide group or is absent

or a fragment, derivative or fusion' thereof (or a fusion of said fragment or derivative) which retains the hypothermia-inducing activity of a polypeptide having the amino acid sequence of SEQ ID NO:1.

26. A polypeptide according to Claim 25 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO: 2.

27. A polypeptide according to Claim 25 or 26 wherein the variant comprises or consists of an amino acid sequence selected from the group consisting of: AU-08: HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:4]; AU-11: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGA-PPSKKKKKK

[SEQ ID NO:5];

AU-12: HGEGTFTSDLSKEMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:6]; AU-14: HGEGTFTSDLSKQMEEEAVRLFIEWLKDGGPSSGAPPPS

[SEQ ID NO:7];

AU-15: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSZ, where Z=Lys-(AEEA-MPA)-(CONH2) [SEQ ID NO:8];

AU-17: HAEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:9];

AU-18: HGEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS [SEQ ID NO:10];

AU-19: HVEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:11]; AU-20: HΛ/jbEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:12]; and

AU-28: HGEGTFTSDLSKQLEEEAVRLFIEWLKSGGPSSGAPPPS [SEQ ID NO:13].

28. A polypeptide according to any one of the preceding claims wherein the polypeptide comprises or consists of a fusion protein.

29. A polypeptide according to Claim 28 comprising or consisting of an amino acid sequence corresponding to a fragment of SEQ ID NO: 1 and an amino acid sequence corresponding to a fragment of GLP-1.

30. A polypeptide according to Claim 29 comprising or consisting of an amino acid sequence of SEQ ID NO: 9:

AU-18: HGEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NCCIO]. 31. A polypeptide according to any one of the preceding claims wherein the polypeptide, or fragment, variant, fusion or derivative thereof, comprises or consists of L-amino acids.

32. A polypeptide according to any one of the preceding claims wherein the polypeptide, or fragment, variant, fusion or derivative thereof, comprises one or more amino acids that are modified or derivatised.

33. A polypeptide according to Claim 32 wherein the one or more amino acids are modified or derivatised by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

34. A polypeptide according to any one of the preceding claims wherein the fragment, variant, fusion or derivative exhibits a functional advantage relative to a polypeptide of SEQ ID NO:1.

35. A polypeptide according to Claim 34 wherein the functional advantage is selected from the group consisting of:

(a) Increased receptor affinity for GLP1 receptors;

(b) Reduced vulnerability to DPP-IV degradation; and

(c) Decreased rate of renal clearance.

36. A polypeptide according to any one of the preceding claims wherein the polypeptide is between 10 and 100 amino acids in length, for example between 20 and 60, 30 and 50, 35 and 45, or 38 and 40 amino acids in length.

37. A polypeptide according to Claim 36 wherein the polypeptide is 39 amino acids in length.

38. A polypeptide according to any one of the preceding claims wherein the polypeptide is linear.

39. A polypeptide according to any one of the preceding claims wherein the polypeptide is a recombinant polypeptide.

40. A polypeptide according to any one of the preceding claims wherein the polypeptide comprises an amide group at its C-terminus.

41. An isolated polypeptide comprising or consisting of an amino acid sequence according to SEQ ID NO:2, with the proviso that the isolated polypeptide does not consist of an amino acid sequence according to SEQ ID NO:1.

42. A polypeptide according to Claim 41 wherein the polypeptide is selected from the group consisting of:

AU-18: HGEGTFTSDVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS [SEQ ID NO:10];

AU-19: HVEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:11];and AU-20: H/tøEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[SEQ ID NO:12]; and

AU-28: HGEGTFTSDLSKQLEEEAVRLFIEWLKSGGPSSGAPPPS [SEQ ID NO:13].

43. A polypeptide according to Claim 41 or 42 wherein the polypeptide comprises an amide group at its C-terminus.

44. An isolated nucleic acid molecule encoding a polypeptide according to any one of Claims 41 to 43.

45. A vector comprising a nucleic acid molecule according to Claim 44.

46. A vector according to Claim 45 wherein the vector is an expression vector.

47. A host cell comprising a vector according to Claim 45 or 46.

48. A pharmaceutical composition comprising a polypeptide as defined in any one of Claims 1 to 40 together with a pharmaceutically acceptable excipient, diluent or carrier.

49. A pharmaceutical composition according to Claim 48 suitable for parenteral administration.

50. A pharmaceutical composition according to Claim 49 suitable for intravenous, intracerebroventricular, intratechal and/or sub-cutaneous administration.

51. Use of a polypeptide according to any one of Claims 1 to 40 in the preparation of a medicament inducing or maintaining hypothermia in a subject in need thereof.

52. A use according to Claim 51 wherein the medicament is for the treatment or prevention of neuronal damage in the central nervous system.

53. A use according to Claim 51 or 52 wherein the medicament is for use in the treatment or prevention of acute brain injury.

54. A use according to any one of Claims 51 to 53 wherein the medicament is for use in the treatment or prevention of neuronal damage due to ischemia.

55. A use according to any one of Claims 51 to 54 wherein the subject is suffering from or has recently suffered from a stroke, a brain trauma, a cardiac arrest, spinal cord injury or asphyxia.

56. A method for inducing or maintaining hypothermia in a subject in need thereof, the method comprising administering to the patient a therapeutically-effective amount of a polypeptide according to any one of Claims 1 to 40.

57. A method according to Claim 56 wherein the subject is human.

58. A method according to Claim 56 or 57 wherein the subject is conscious during administration of the polypeptide.

59. A method according to any one of Claims 56 to 58 wherein the medicament is for the treatment or prevention of neuronal damage in the central nervous system.

60. A method according to any one of Claims 56 to 59 wherein the medicament is for use in the treatment or prevention of acute brain injury.

61. A method according to any one of Claims 56 to 60 wherein the medicament is for use in the treatment or prevention of neuronal damage due to ischemia.

62. A method according to any one of Claims 56 to 61 wherein the subject is suffering from or has recently suffered from a stroke, a brain trauma, a cardiac arrest, spinal cord injury or asphyxia.

63. Method of inducing and/or maintaining hypothermia in a patient, such as a mammal, exposed to brain injury, comprising administration of a therapeutically effective amount to the patient of a composition having a component that binds to a receptor for glucagon-like peptide-1 (GLP-1).

64. The method according to claim 63, further comprising exposing said mammal to other hypothermic treatment, such as external surface cooling or internal surface cooling.

65. The method according to claim 64, wherein said hypothermic treatment comprises nasopharyngeal cooling and/or cold saline infusion.

66. The method according to claim 64 or 65, wherein said administration and said other hypothermic treatment takes place simultaneously.

67. The method according to any one of Claims 63 to 66, wherein said composition comprises a substance included in the group comprising:

exendin-4; a human glucagon-like peptide (GLP)-i-albumin recombinant protein (Albugon); CJC-1131 ; liraglutide; and oxyntomodulin.

68. Use of a substance as defined in Claim 67, for the preparation of a medicament for the induction and/or maintenance of hypothermia for treatment of brain injury, particularly stroke, brain trauma, cardiac arrest and asphyxia.

69. The use according to claim 68, wherein the use is combined with hypothermic treatment according to claim 65 or 66.

70. A polypeptide substantially as described herein with reference to the description and figures.

71. A nucleic acid molecule as described herein with reference to the description and figures.

72. A vector as described herein with reference to the description and figures.

73. A host cell as described herein with reference to the description and figures.

74. A pharmaceutical composition substantially as described herein with reference to the description and figures.

75. Use of a polypeptide substantially as described herein with reference to the description and figures.

76. A method for inducing or maintaining hypothermia as described herein with reference to the description and figures.

77. A method for diagnosis substantially as described herein with reference to the description and figures.