AU2005202153A1 - Urotensin-II agonists - Google Patents

Description

0 Ci oD oD

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicants: THE ADMINISTRATORS OF THE TULANE EDUCATIONAL FUND SOCIETE DE CONSEILS DE RECHERCHES ET D'APPLICATIONS SCIENTIFIQUES, S.A.S.

Invention Title: UROTENSIN-II AGONISTS The following statement is a full description of this invention, including the best method of performing it known to me/us:

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UROTENSIN-I AGONISTS Field of the Invention The invention relates to urotensin-II polypeptide agonists and methods of their use.

In s Background of the Invention I Urotensin-II (U-II) is a cyclic neuropeptide with potent cardiovascular effects. Originally isolated from the caudal neurosecretory system of teleost fish, the primary structure of U-II has been established for several species of vertebrates, including various fish species, frogs, and humans. Sequence analysis of various U-U peptides from different species has revealed that, while the N-terminal region is highly variable, the C-terminal cyclic region of U-U is strongly conserved. Indeed, this cyclic region, which is responsible for the biological activity of U-1I, is fully conserved from fish to humans (Coulouran, et al, Proc. Natl. Acad. Sci. USA (physiology), 95:15803-15808 (1998)). The fact that evolutionary pressure has acted to fully conserve the biologically active sequence of U-II suggests that this polypeptide plays an important role in human physiology.

The cyclic region of U-U1 includes six amino acid residues (-Cys- Phe-Trp-Lys-Tyr-Cys- (SEQ ID NO: and is structurally similar to the biologically important central region of somatostatin- 14 (-Phe-Trp-Lys-Thr- (SEQ ID NO: However, molecular cloning and sequence analysis of the carp preprourotensin II gene suggests that U-II and somatostatin are not derived from a common ancestor (Ohsako, e al., J. Neurosci., 6:2730-2735 (1986)).

In fish, U-II peptides have been shown to exhibit several activities, o including general smooth muscle contracting activity, although responses vary C between species and vascular beds (Davenport, and Maquire, Trends in Pharmacological Sciences, 21:80-82 (2000); Bern, el al., Recent Prog.

Horn. Res., 45:533-552 (1995)). Fish U-II has also been shown to possess constrictor activity in mammals, including major arteries in rats, but the receptor(s) mediating these peptide actions are not fully characterized.

itn Recent studies have reported that an orphan human SG-protein-coupled receptor, homologous to the rat GPRI4 and expressed C 10 predominantly in cardiovascular tissue, functions as an U-lI receptor (Ames, O et al., Nature, 401:282-286 (1999)). Fish (goby) and human U-Il 0 Cl reportedly bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates (Ames, et al., supra). The potency of vasoconstriction of U-II is substantially greater than that of endothelin- making human U-II one of most potent mammalian vasoconstrictors currently known. In vivo, human U-UI markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction (Ames, et al., supra).

Since human U-II-like immunoreactivity is found within cardiac and vascular tissue (including coronary atheroma), U-U is believed to influence cardiovascular homeostasis and pathology ischemic heart disease and congestive heart failure). Furthermore, the detection of U-Il immunoreactivity within spinal cord and endocrine tissues suggests that U-lI may have additional activities, including modulation of central nervous system and endocrine function in humans (Ames, et al., supra). Indeed, a number of maladies have been potentially linked to an excess or an under expression of U-II activity, including ischemic heart failure, hypotension, portal hypertension, angina pectoris, variceal bleeding, myocardial infarction, ulcers, and certain psychological and neurological disorders. Thus, there is a strong need for the development of potent compounds capable of modulating U-ll activity, including U-II inhibitors or antagonists.

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Summary of the Invention The invention provides a Urotensin-II agonist polypeptide, and variants I thereof, having the formula Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH (SEQ ID NO:3).

0C- The polypeptide of the present invention is capable of altering U-II activity and can affect the binding of U-II to a receptor. Thus, this polypeptide may be Iadministered to a subject as a means for preventing or treating medical or psychological conditions characterized by under expression of Urotensin-II activity.

The present invention also provides pharmaceutical compositions that include a therapeutically effective amount of a polypeptide in combination with a pharmaceutically acceptable carrier. Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The composition can be adapted for the mode of administration and can be in the form of a pill, tablet, capsule, spray, powder, or liquid.

Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.

Definitions By "polypeptide" is meant any peptide (including cyclic peptides) or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer 0 C chains, generally referred to as proteins. Polypeptides may contain amino acids 0 other than the 20 gene-encoded amino acids. "Polypeptides" include amino

C

acid sequences modified either by natural processes, or by chemical c modification techniques which are well known in the art. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.

The notations used herein for the polypeptide amino acid residues are ec v those abbreviations commonly used in the art. The less common abbreviations SAbu, Cpa, Nle, Pal, Tie, Dip, 4-Fpa, and Nal stand for 2-amino-butyric acid, pchloroPhenylalanine, norleucine, 3-pyridyl-2-alanine, tert-leucine, 2,2o diphenylalanine, 4-fluoro-phenylalanine. and 3-(2-naphthyl)-alanine or 3-(1naphthyl)-alanine, respectively By "alkyl" is meant an aliphatic branched or straight chain hydrocarbon group. An alkyl is optionally substituted with one or more substituents which may be the same or different, and include, but are not limited to, halo, cycloalkyl, hydroxy, alkoxy, amino, carbamoyl, acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkyloxycarbonyl, or heteroaralkyloxycarbonyl groups. Representative alkyl groups include, but are not limited to, methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n-propyl, ipropyl, n-butyl, t-butyl, n-pentyl, 3-pentyl. methoxyethyl, and carboxymethyl.

By "lower alkyl" is meant a branched or straight chain alkyl group having less than 11 carbon atoms, preferably a alkyl. o By "acyl" is meant a group having the structure R- wherein R is H or an alkyl group as described herein. By "lower acyl" is meant an acyl group having less than I carbon atoms (either branched or straight chain), preferably between 1-8 carbon atoms R is H or a lower alkyl).

y By "lower alkanoyl" is meant an acyI group as described above wherein 0 SR is a lower alkyl.

By "aryl" is meant a monocyclic or bicyclic aromatic group contaninng from 6 to 12 carbons in the ring portion, preferably 6- 10 carbons in the ring portion, such as phenyl, napthyl or tetrahydronaphthyl. By "arylalkyl" is meant an alkyl group as described herein having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.

By "pharmaceutically acceptable salt" is meant non-toxic acid addition 0 salts or metal complexes which are commonly used in the pharmaceutical n 10 industry. Examples of acid addition salts include organic acids such as acetic, O lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like.

By "variant" is meant a polypeptide that differs from a reference polypeptide, but retains essential properties. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions, in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polypeptides may be made by mutagenesis techniques or by direct synthesis.

-6iy-s Generally, the variant differs From the reference polypeptide by Sconservative amino acid substitutions, whereby a residue is substituted by another with like characteristics acidic, basic, aromatic, etc.). Typical substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic icsidues Asp and Glu: among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.

n By "subject" is meant an animal or human suffering from a U-U-related physiological or psychological condition. The subject may be a mammal, Sincluding, but not limited to, humans and non-human mammals such as in 10 primates, dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, and the like.

By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to an administered animal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18" 1 edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.

By "aromatic amino acid" is meant an amino acid that contains an aromatic group. In preferred embodiments, the aromatic amino acid has the following formula:

H

2 C-Ar

-N-CH

X =0° (Formula II), where X represents a bond or H, and Ar is a moiety containing an O optionally substituted aromatic ring. Examples of Ar, include but are not C limited to, the following structures wherein represents n optional substituents and n is 0, 1, 2, or 3: Yn eci Yn Yn

Y

Yn N

H

Yn and HN+ N

NH

CH

3 -8- In preferred embodiments, each substituent Y independently represents NO,, O CN, CI, Br, I, F, Me, COR 4

COOR

4 or OR 4 groups, where R' is H or C,-C, C alkyl. Examples of aromatic amino acids include, but are not limited to, Phe, ct Cpa, Trp, Pal, His, 3-Nal, 3-pyridyl-Ala, 4-pyridyl-Ala, 2,4-dichloro-phe, pentafluoro-Phe, p-Z-Phe, and o-Z-Phe, wherein Z is selected from the group consisting of Me, CI, Br, F, OH, OMe, and NO,.

Detailed Description We found that the minimum portion of the U-II sequence which O retained full biological activity was the octapeptide Asp-c[Cys-Phe-Trp-Lys- 10 Tyr-Cys]-Val-OH (SEQ ID NO: which corresponds to hUII(4-7). This 0 octapeptide actually possess greater potency than the full human and fish U-II sequences in inducing rat aorta contraction and in binding to this tissue.

Based on this parent sequence, a series of cyclic octapeptides have been synthesized which have U-II antagonist activity. These peptides were discovered to have moderate affinity for U-II receptors and were able to block U-II-induced phasic contracts in circular rat thoracic aorta strips. The polypeptides of the present invention have the general formula: cyclo[AA 2

-AA'-AA'-AA

5 -AA'-Cys]-AA 7

-R

2 (Formula wherein AA' is the L isomer of an aromatic amino acid; AA 2 is the L or D isomer of Cys; AA is an L isomer of an aromatic amino acid; AA 4 is the L or D isomer of Trp; AA' is the L or D isomer of Lys, N-Me-Lys, or Orn; AA 6 is the L or D isomer of Val, Thr, Leu, lie, tert-Leu, Abu, Nle, or an aromatic amino acid; AA 7 is the L or D isomer of Val, Thr, Leu, lie, tert-Leu, Abu, Nle, or an aromatic amino acid; R' is H, a lower alkyl, lower alkanoyl, or a lower acyl; a is I or 2; and R 2 is OH, OR', or NHR', where R3 is H, a lower alkyl, or arylalkyl.

SOne of the most potent U-ll inhibitors tested was the SRIF antagonist 0 O Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide (SEQ ID NO: which had an IC, of about 100 nM and Kd of 240. Another potent U-II antagonist was Cpa-c[D-Cys-Phe-Trp-Lys-Thr-Cys]-Val-NH, (SEQ ID NO: 5) which had an

IC,

5 of about 2nM. Other SRIF antagonists that were tested are summarized in Example 2 below (see table 1).

n The polypeptides synthesized are capable of modulating U-II activity and are, therefore, useful for treating physiological and psychological o conditions related to either an excess of or an under expression of U-II activity within a subject. Such conditions include, for example, acute heart failure, O hypotension, hypertension, angina pectoris, variceal bleeding, myocardial infarction, ulcers, and certain psychological and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation and dyskinesias.

If the condition stems from an excess of U-II activity, one approach to treatment is to administer to a subject in need thereof an inhibitor compound (antagonist), optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function of U-11. Alternatively, for treating conditions related to under expression of U-II activity, a compound which activates U-II (agonist) is administered.

A therapeutically effective amount of a polypeptide of Formula 1, or a variant or pharmaceutically acceptable salt-thereof, can be administered orally, parenterally intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasally, vaginally, rectally, sublingually or topically, in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.

Methods well known in the art for making formulations are found, for o example, in Remington's Pharmaceutical Sciences edition), ed. A.

C' Gennaro, 1990, Mack Publishing Company, Easton, PA. Compositions t intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions.

The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable Ipreparation. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound C' 10 is admixed with at least one inert pharmaceutically acceptable carrier or Sexcipient. These may include, for example, inert diluents, such as calcium C carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules.

These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.

Formulations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, or emulsions. Examples of suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate.

Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene -11tIy copolymers may be used to control the release of the compounds. Other o potentially useful parenteral delivery systems for the polypeptides of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the Scompositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which O can be dissolved in sterile water or some other sterile injectable medium V) 10 immediately before use.

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to active substances, excipients such as coca butter or a suppository wax. Compositions for nasal or sublingual administration are also prepared with standard excipients known in the art.

Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or spray, or as a gel.

The amount of active ingredient in the compositions of the invention can be varied. One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the polypeptide being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient. In addition, the severity of the U-I-related condition being treated will also have an impact on the dosage level. Generally, dosage levels of between 0. t gg/kg to 100 mg/kg of body weight are administered daily as a -12in) single dose or divided into multiple doses. Preferably, the general dosage 0 O range is between 250 zg/kg to 5.0 mg/kg of body weight per day. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are Ce l well known in the art. In general, the precise therapeutically effective dosage will be determined by the attending physician in consideration of the above r 10 identified factors.

O The polypeptides of the invention can be administered in a sustained release composition, such as those described in, for example, U.S. Patent Nos.

5,672,659 and 5,595,760. The use of immediate or sustained release compositions depends on the type of condition being treated. If the condition consists of an acute or over-acute disorder, a treatment with an immediate release form will be preferred over a prolonged release composition.

Alternatively, for preventative or long-term treatments, a sustained released composition will generally be preferred.

Polypeptides of the present invention can be prepared in any suitable manner. The polypeptides may be isolated from naturally occurring sources, recombinantly produced, or produced synthetically, or produced by a combination of these methods. The synthesis of short peptides is well known in the art. See e.g. Stewart el al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984). The peptides of the present invention can be synthesized according to standard peptide synthesis methods known in the art and exemplified in Example I below.

Brief Description of the Drawings Fig. 1 is a graph showing concentration-dependent constriction of isolated endothelium-preserved rat thoracic aorta rings by human urotensin II-(1-I 1)- OH (filled square); human urotensin II-(4-11)-OH (open circles); and human urotensin II-(4-1 I)-NH 2 (filled triangles). Responses are shown relative to 100 mM KC1 (accepted as 100%). Values are mean and vertical bars represent S.E.M. mean, n= 3. -13- The present invention is illustrated by the following examples, which O are in no way intended to be limiting of the invention.

C(

Example 1: Preparation of Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa- S amide Step t: Preparation of Boc-4-chlorophenylalanine-S-methylbenzyl-D-cysteineitn 3-pyridyl-2-alanine-D-tryptophan-N -benzyloxycarbonyl-lysine-valine-S- CN methylbenzyl-cysteine-4-chlorophenylalanine-benzhydrylamine resin.

0 in Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc., 0 SLouisville, KY) (1.2 g, 0.5 mmole) in the chloride ion form was placed in the reaction vessel of an Advanced ChemTech peptide synthesizer (Model 200) programmed to perform the following reaction cycle: methylene chloride; 33% trifluoroacetic acid in methylene chloride (2 times for 1 min. and min. each); methylene chloride; ethanol; methylene chloride; triethylamine in chloroform.

The neutralized resin is stirred with Boc-4-chlorophenylalanine and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for I hour and the resulting amino acid resin in then cycled through steps through (f) in the above wash program. The following amino acids (1.5 mmole) are then coupled successively by the same procedure: Boc-S-methylbenzyl-Cys, Boc- Val, Boc-N'-benzyloxycarbonyl-lysine, Boc-D-Trp, Boc-Pal, and Boc-Smethylbenzyl-D-Cys and Boc-4-chlorophenylalanine. After washing and drying, the completed resin weighed about 2.0 g.

-14- Step 2: Deprotection and cleavage from resin.

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SThe resin described in Step 1 (1.0 g, 0.25 mmole) was mixed with

CA"

anisole (5 ml), dithiothretol (100 mg), and anhydrous hydrogen fluoride ml) at about 0°C and stilred for 45 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen, after which free peptide was precipitated and washed with ether. The crude peptide was then dissolved in 500 ml of 90% acetic acid. A concentrated solution of ,/MeOH was then added until a permanent brown color was observed. Excess I, was removed by the addition of ascorbic acid and the solution evaporated to a small volume which was applied to a column (2.5 x 90cm) of VYDACTM octadecylsilane o silica (10-15m). This was eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. Fractions were examined by thin layer chromatography and analytic high performance liquid chromatography and pooled to give maximum purity. Repeated lyophilization of the solution from water gave 125 mg of the desired product as a white, fluffy powder.

The product was found to be homogenous by HPLC and TLC. Amino acid analysis of an acid hydrolysate and matrix-assisted laser desorption MS confirmed the composition of the octapeptide. Other peptide of the invention may be made using an analogous procedure with appropriate reactants.

Example 2: Use of Rat Aorta Circular Strip for Assay U-II Antagonists Male Sprague-Dawley rats (250-350 which had been quarantined for 5-7 days prior to the experiments, were sacrificed by decapitation (experiments were approved by the Advisory Committee For Animal Resources, Tulane University School of Medicine). The thoracic aorta was dissected, freed from connective tissue, and cut into rings of about 1.5 mm in width. The rings were suspended in a 15 ml organ bath containing high potassium Kreb's solution O (9.15 g/L potassium chloride, 2.1 g/L sodium bicarbonate, 1.0 g/L glucose, C 0.16 g/L potassium phosphate monobasic. 0.14 g/L magnesium sulfate c(anhydr.), and 0.22 g/L calcium chloride (dihydr.)) Optimal tension was applied (0.2 g) to the tissues and the bath medium was maintained at 37°C and bubbled with a mixture of 95% CO,. Prior to mounting in the organ bath, selected preparations were rubbed with a Ce moistened cotton wool swab, in order to remove the endothelial cell layer, and the effect of this procedure was tested using an acetylcholine-relaxation test.

c 10 (Gibson, Br. J. Pharmacol. 91:205 (1987)). The aorta rings were allowed Sto equilibrate for 90 min. at the optimal tensions. During the equilibration l period, the bath solution was replaced every 15 min. Contractile responses of aortae rings to various concentrations of peptides were expressed in volts.

Changes in arterial smooth muscle tension were recorded isometrically using a force-displacement transducer (Radnoti), and a AcqKnowledge ACKI00 Version 3.2 (BIOPAC Systems, Inc., Santa Barbara, CA.) In siliconized glass tubes, peptides were dissolved in dionized water at a concentration of I /g/1pL (stock solution) and then diluted 1:10 with sterile BSA-saline solution BSA, fraction V, Sigma, St. Louis in 0.9% NaCI).

All peptide solutions were prepared fresh directly before the experiments.

Peptides in the concentration ranges of 10.6 to 10' M/L in a final volume of 16-80 pL were direcly introduced into the tested organ bath containing Krebs buffer continuously gassed with 95% 0, and 5% CO,, and the aorta rings at an optimal resting tension (1 0.2g). Peptide-induced changes in tension of the aorta rings were recorded by force-displacement transducers and processed by the computer system BIOPAC Inc., as described above. Each ring was exposed to one peptide concentration only.

16- Using assay techniques known in the art, we found that the minimally, 0 fully potent sequence of U-II was the octapeptide Asp-c[Cys-Phe-Trp-Lys-Tyr- Cl Cys]-Val-OH (SEQ ID NO: which was actually more potent than the full e3 human and fish sequences in inducing rat aorta contracts. Various somatostatin (SRIF) antagonists were discovered to have the ability to block Ull-induced phase contractions in the circular rat thoracic aorta strips. One of the most potent inhibitors was the SRIF antagonist Cpa-c[D-Cys-Pal-D-Trp- C Lys-Val-Cys]-Cpa-amide (SEQ ID NO: which had an IC,, of about 100 nM Sand a Kd of 240 nM. The polypeptide Cpa-c[D-Cys-Phe-Trp-Lys-Thr-Cys]- C( 10 ValNH 2 (SEQ ID NO:5) was also a strong U-II antagonist with an ICso of 2nM. Other 0 compounds that were tested are summarized in Table I below.

0 Table 1. SRIF Antagonist IC(s (nM) against U-II Stimulation of Rat Aorta Phasic Contractions Polypeptide ICs, Nal-D-Cys-His-D-Trp-Lys-Val-Cys-D-Dip-NH, (SEQ ID NO: 6) 1800 4Fpa-D-Cys-Pal-D-Trp-Lys-Val-Cys-Nal-NH, (SEQ ID NO: 7) 1090 4Fpa-D-Cys-Pal-D-Trp-Lys-Tle-Cys-Nal-NH, (SEQ ID NO: 8) 100 Cpa-D-Cys-Tyr-D-Trp-Lys-Thr-Cys-Nal-NH, (SEQ ID NO: 9) 12 Cpa-D-Cys-Pal-D-Trp-Lys-Tle-Cys-Nal-NH (SEQ ID NO: 10) Cpa-D-Cys-Pal-Trp-Lys-Thr-Cys-Cpa-NH, (SEQ ID NO: 11) 2 17- O Example 3: Peptides C1 Human urotensin II-(1 1 l)-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]- Val-OH (SEQ ID No:12); human urotensin II-(4 11)-OH Asp-c[Cys-Phe-Trp- Lys-Tyr-Cys]-Val-OH)(SEQ ID NO:3) and human urotensin II-(4 11)-NH 2

(SEQ

ID NO: 13) were synthesized by standard solid phase methodologies on CS Bio (San Carlos, CA, USA) model CS 136 or Advanced ChemTech (Louisville, KY, USA) model 200 automatic peptide synthesizers and purified by reverse phase-high ¢C performance liquid chromatography (RP-HPLC) on C 18 bonded silica gel columns -(Dynamax-300A, 5 or 8jm, 21.4 x 250 mm). Samples of the purified peptides were 2 hydrolyzed in 4M methanesulphonic acid containing 0.2% 3-(2-aminoethyl)indole and subjected to amino acid analyses performed using an automatic HPLC system o (Varian, Walnut Creek, CA, USA). Molecular weights were determined by matrix 0, assisted laser desorption mass spectrometry using a LaserMat 2000 mass spectrometer (Finnegan MAT, San Jose, CA, USA) with substance P (1348.7 Da) as an internal standard.

Example 4: Animals and aorta ring contraction assays Male Sprague Dawley rats (250-350g) were sacrificed by decapitation. The thoracic aorta was dissected and freed from connective tissue and cut into in width rings which were vertically suspended in 15 ml organ bath containing normal Krebs solution containing: 118 mM NaC 1; 25 mM NaHCO 3 4.7 mM KC1; 1.2 mM KH 2

PO

4 1.2 mM MgSO 4 (anhydrous); 15 mM CaC1 2 (dihydrate); 11 mM D-glucose, or in high potassium Krebs solution (NaC1 was replaced by KC 1).

Optimal tension was applied (1 or 2g for tonic and 0.2 g for phasic contractions) and the bath medium was maintained at 37 0 C bubbled with a mixture of 95% 02-5%

CO

2 Some preparations, before being mounted in the organ bath, were rubbed with a moistened cotton wool swab in order to remove the endothelial cell layer and the effect of this procedure was tested using an acetylcholine-relaxation test. The aorta rings were allowed to equilibrate for 90 min at the optimal tensions prior to starting experiments. During the equilibration period, the bath solution was replaced every min. At the end of the equilibration period, tissue contractions were initiated using 100 mM KC 1 solution. Changes in arterial smooth muscle tension were recorded isometrically using a force-displacement transducer (Radnoti) and recorded using AcqKnowledge ACK100 Version 3.2 (BIOPAC Systems, Santa Barbara, CA). After washing and tissue recovery, aorta rings were exposed to single doses of freshly prepared peptide solution. Contractile responses of aorta rings in regular Krebs buffer were originally expressed in volts and finally calculated as the percentage of 18 O the contractile response to 100 mM KC 1 (accepted as 100%). In studies of human Cl urotensin II-induced phasic oscillations (Ca 2 transients) in high potassium Krebs buffer we evaluated the effect of human urotensin II concentration.

o0 5 Table 2. In vitro inhibition of 25 I]urotensin II (goby) binding to transfected rat and human urotensin II receptors by goby, human urotensin II and human urotensin II-(4- 1 1)-OH/NH 2 fragments and peptide stimulation of tonic contractions of rat aorta c rings.

0 Peptide Ki (nM) ECo (nM) rU-II a hU-IIb Rat aorta o _Rings Cl Ala-Gly-Thr-Ala-Asp-c 2.41 0.21 1.78 0.18 [Cys-Phe-Trp-Lys-Tyr-Cys]- Val-OH (SEQ ID NO:14) Glu-Thr-Pro-Asp-c 2.34 0.85 1.68 0.31 0.73 0.13 [Cys-Phe-Trp-Lys-Tyr-Cys]- Val-OH (SEQ ID NO:12) Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]- 1.33 0.29 1.20 0.30 0.16 0.10 Val-OH (SEQ ID NO:3) Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]- 1.02 0.22 1.43 0.29 0.37 0.05 Val-NH 2 (SEQ ID NO: 13) a Rat urotensin II.

b Human urotensin

II.

Addition of human urotensin II-(1-l11)-OH at doses of 0.3-10 nM to the rat aorta-rings incubated in regular Kregs medium produced slow developing tonic contractions lasting for over 30 min with an EC5o value of 0.73 0.13 nM. (Fig. 1 and Table Elimination of the first three N-terminal amino acids (Glu-Thr-Pro) from human urotensin II created a more potent analogue in this assay, displaying an value of 0.16 0.09 nM (P 0.02), while the amidation of the C-terminus of the human urotensin II-(4-11) did not significantly affect its biological activity 2 0 (Table Both human and goby urotensin II inhibited binding of [1 25 1]UII (goby) to the urotensin II binding sites with similar Ki values of 1.68 0.31 and 1.78 0.18 nM, respectively (Table 2).

19 o Equivalents 0 Although the present invention has been described with reference to preferred embodiments, one skilled in the art can easily ascertain its essential characteristics and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the present invention.

All publications and patents mentioned in this specification are herein incorporated by reference.

o In the claims which follow and in the preceding description of the 0 invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, ie. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It is to be clearly understood that although prior art publication(s) are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art in Australia or in any other country.

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Claims (6)

1. A urotensin-II agonist polypeptide, or functional variant thereof, said polypeptide having the formula: Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]- Val-OH (SEQ ID NO: or a pharmaceutically acceptable salt thereof.

2. A method of modulating the effect of a Urotensin-II (U-II) rr n peptide, said method comprising administering to a subject the polypeptide, or Ifunctional variant thereof, of claim 1, or a pharmaceutically acceptable salt thereof.

3. The method of claim 2, wherein said modulating comprises Sincreasing the effect of said U-II peptide.

4. A method of preventing or treating an abnormal condition characterized by an under expression of Urotensin-II activity, said method comprising administering to a subject a therapeutically effective amount of the polypeptide, or functional variant thereof, of claim 1, or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition comprising a polypeptide, or a functional variant thereof, of claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein said carrier is selected from the group consisting of saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Dated this 17 th day of May 2005 THE ADMINISTRATORS OF THE TULANE EDUCATIONAL FUND SOCIETE DE CONSEILS DE RECHERCHES ET D'APPLICATIONS SCIENTIFIOUES, S.A.S. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 21