WO2009094137A2 - Urotensin-ii agonists - Google Patents

Abstract

The present invention features a novel class of polypeptides that have Urotensin-II agonist activity. The invention also features methods for treating physiological and psychological conditions characterized by an excess or under expression of Urotensin-II.

Description

UROTENSIN-II AGONISTS

Field of the Invention

The invention relates to urotensin-II polypeptide agonists and methods of their use.

Background of the Invention

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

The cyclic region of UII includes six amino acid residues (-Cys-Phe-Trp-Lys- Tyr-Cys- (SEQ ID NO: I)) and is structurally similar to the biologically important central region of somatostatin- 14 (-Phe-Trp-Lys-Thr- (SEQ ID NO:2)). However, molecular cloning and sequence analysis of the carp preprourotensin II gene suggests that UII and somatostatin are not derived from a common ancestor (Ohsako, S., et ah, J. Neurosci., 6:2730-2735 (1986)).

In fish, UII peptides have been shown to exhibit several activities, including general smooth muscle contracting activity, although responses vary between species and vascular beds (Davenport, A., and Maquire, J., Trends in Pharmacological

Sciences, 21:80-82 (2000); Bern, H.A., et al, Recent Prog. Horm. Res., 45:533-552

(1995)). Fish UII 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. Recent studies have reported that an orphan human G-protein-coupled receptor, homologous to the rat GPR 14 and expressed predominantly in cardiovascular tissue, functions as an UII receptor (Ames, H., et al., Nature, 401 :282- 286 (1999)). Fish (goby) and human UII reportedly bind to recombinant human GPR 14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human UII is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates (Ames, H., et al, supra). The potency of vasoconstriction of UII is substantially greater than that of endothelin-1, making human UII one of most potent mammalian vasoconstrictors currently known. In vivo, human UII markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction (Ames, H., et al, supra).

Since human Ull-like immunoreactivity is found within cardiac and vascular tissue (including coronary atheroma), UII is believed to influence cardiovascular homeostasis and pathology {e.g., ischemic heart disease and congestive heart failure). Furthermore, the detection of UII immunoreactivity within spinal cord and endocrine tissues suggests that UII may have additional activities, including modulation of central nervous system and endocrine function in humans (Ames, H., et al, supra). Indeed, a number of maladies have been potentially linked to an excess or an under expression of UII 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 UII activity.

Summary of the Invention The present invention features a novel class of cyclic polypeptides that have

UII agonist activity. The polypeptides of the invention are UII agonist peptides having the general formula:

R¹ -cyclo(AA' - AA²- AA³-AA⁴- AA⁵- AA⁶)-AA⁷-NH₂, wherein AA¹ is Cys, N-Me-Cys, or Pen;

AA² is Phe;

AA³ is Tip or N-Me-Trp; AA⁴ is Lys; AA⁵ is Tyr or Cpa; AA⁶ is Cys or Pen; AA⁷ is VaI, Ala, or Thr; and R¹ is Asp, N-Me- Asp, or succinyl.

In a preferred embodiment, the peptide of this invention according to the above Formula I has either one or more N-Me amino acid(s) or one or more Pen residue(s).

The following are examples of the UII agonist peptides of this invention as covered by the above Formula I:

Asp-cyclo(Cys-Phe-N-Me-Tφ-Lys-Tyr-Cys)-Val-NH₂; SEQ ID NO:3 Asp-cyclo(Pen-Phe-Tφ-Lys-Tyr-Cys)-Val-NH₂; SEQ ID NO:4 Asp-cyclo(Cys-Phe-Tφ-Lys-Tyr-Cys)-N-Me- VaI-NH₂; SEQ ID NO:5 succinyl-cyclo(Cys-Phe-Trp-Lys-Cpa-Cys)-Val-NH₂; SEQ ID NO:6 succinyl-cyclo(Cys-Phe-Trp-Lys-Tyr-Cys)-Thr-NH₂; SEQ ID NO: 7

Asp-cyclo(Cys-Phe-Tφ-Lys-Tyr-Cys)-Ala-NH₂; SEQ ID NO:8 Asp-cyclo(N-Me-Cys-Phe-Tφ-Lys-Tyr-Cys)- VaI-NH₂; SEQ ID NO: 9 Asp-cyclo(Cys-Phe-Tφ-Lys-Tyr-Pen)-Val-NH₂; and SEQ ID NO: 10 N-Me- Asp-cyclo(Cys-Phe-Tφ-Lys-Tyr-Cys)- VaI-NH₂; SEQ ED NO: 11 and variants thereof.

The polypeptides of the present invention are capable of altering UII activity and can affect the binding of UII to a receptor. Thus, these polypeptides may be administered to a subject as a means for preventing or treating medical or psychological conditions characterized by a deficiency or under expression of Urotensin-II activity. Such conditions include, but are not limited to, ischaemic heart disease, congestive heart failure, portal hypertension, variceal bleeding, hypotension, angina pectoris, myocardial infarction, ulcers, anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation, and dyskinesias.

The present invention also provides pharmaceutical compositions that include a therapeutically effective amount of a polypeptide of Formula I 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 chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, or by chemical 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 those abbreviations commonly used in the art. The less common abbreviations Pen and Cpa stand for penicillamine and p-chlorophenylalanine, respectively.

By "pharmaceutically acceptable salt" is meant non-toxic acid addition salts or metal complexes which are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, 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. Generally, the variant differs from the reference polypeptide by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics (e.g., acidic, basic, aromatic, etc.). Typical substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.

By "subject" is meant an animal or human suffering from a UII-related physiological or psychological condition. The subject may be a mammal, including, but not limited to, humans and non-human mammals such as 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^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA, U.S.A.

Detailed Description

It was found that the minimum portion of the UII sequence which retained full biological activity was the octapeptide Asp-cyclo(Cys-Phe-Trp-Lys-Tyr-Cys)-Val- OH (SEQ ID NO: 12), which corresponds to hUII(4-l l). This octapeptide actually possesses greater potency than the full human and fish UII sequences in inducing rat aorta contraction and in binding to this tissue. Based on this parent sequence, a series of novel cyclic polypeptides have been synthesized which have UII agonist activity. Some of these peptides were discovered to have from moderate to high affinity for UII receptors and were tested for their contractile activity on circular strips of rat upper thoracic aorta which undergo potent tonic contractions in response to UII agonist peptides.

The polypeptides of the invention are UII agonist peptides having the general formula: R¹ -CVcIo(AA¹ -AA²- AA³- AA⁴- AA⁵- AA⁶)-AA⁷-NH₂, wherein

AA¹ is Cys, N-Me-Cys, or Pen; AA² is Phe;

AA³ is Tip or N-Me-Trp; AA⁴ is Lys;

AA⁵ is Tyr or Cpa;

AA⁶ is Cys or Pen;

AA⁷ is VaI, Ala, or Thr; and

R¹ is Asp, N-Me-Asp, or succinyl. The UII analogs have agonist activities as evidenced by their ability to stimulate contractions of rat upper thoracic aorta muscle in vitro, as does UII itself.

The polypeptides of the invention are capable of modulating UII activity and are, therefore, useful for treating physiological and psychological conditions related to a deficiency or under expression of UII activity within a subject. Such conditions include, for example, acute heart failure, 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 a deficiency or under expression of UII activity, one approach to treatment is to administer to a subject in need thereof a compound which activates UII (agonist), optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to activate the function of UII.

A therapeutically effective amount of a polypeptide of Formula I, or a variant or pharmaceutically acceptable salt-thereof, can be administered orally, parenterally (e.g., 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 example, in Remington's Pharmaceutical Sciences (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA, U.S.A. Compositions 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 preparation. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient. These may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents (e.g., 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 nonaqueous 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 copolymers may be used to control the release of the compounds. Other 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 compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium 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 may 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 UII- related condition being treated will also have an impact on the dosage level. Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses. Preferably, the general dosage range is between 250 μg/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 well known in the art. In general, the precise therapeutically effective dosage will be determined by the attending physician in consideration of the above identified factors. 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 synthesis of short peptides is well known in the art. See, e.g., Stewart et al, Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984) and Bodanszky, M. et al, The Practice of Peptide Synthesis (Springer- Verlag, 1984). The peptides of the present invention can be synthesized according to standard peptide synthesis methods known in the art and exemplified in Examples 1 and 2 below.

The present invention is illustrated by the following examples, which are in no way intended to be limiting of the invention.

• Example 1: Preparation of Asp-cyclo(Cys-Phe-N-Me-Trp-Lys-Tyr-Cys)- VaI-NH₂ SEQ ID NO:3 Methylbenzhydrylamine polystyrene resin (1.0 g, 0.5 mmol) in the chloride ion form was placed in the reaction vessel of a CS Biosystems automatic peptide synthesizer programmed to perform the following reaction cycle: (a) wash with methylene chloride; (b) neutralize with 10% diisopropylamine in DMF; (c) wash with methylene chloride; (d) the neutralized resin was shaken with Boc-Val and diisopropylcarbodiimide coupling reagent (1 mmol each) in methylene chloride for 1 hour followed by washing of the resin with methylene chloride; and (e) removal of the Boc protecting group by treatment with 33% trifluoractetic acid in methylene chloride.

The following amino acids were then coupled successively by the same procedure: Boc-Cys(MeBzl), Boc-Tyr(ClZ), Boc-Lys(ClZ), Boc-Trp. After deblocking the Trp α-amino group, the resin was suspended in DCM (20 mL). To this suspension, collidine (3 equiv.) and o-nitrobenzenesulfonyl chloride (3 equiv.) were added and the mixture was shaken for 2 hours. The resin was then subjected to two

DCM washes and a DMF wash. Protection was monitored qualitatively by the ninhydrin test. The o-nitrobenzenesulfonamide protected resin was suspended in

DMF (20 mL), to which MTBD (3 equiv.) and methyl 4-nitrobenzenesulfonate were added. The mixture was shaken for ¹A hour and the resin was subjected to DMF wash (4 washes). Once the desired residue was methylated, the resin was again suspended in DMF (20 mL). DBU (3 equiv.) and 2-mercaptoethanol (3 equiv.) were added to the suspension and the mixture was agitated for ¹A hour to remove the NPS protecting group. The resin was then thoroughly washed with DMF (5 washes).

The following amino acids were then coupled successively by the procedures (a) through (d): Boc-Phe, Boc-Cys(MeBzl), Boc-Asp(Bzl). The peptide was cleaved from the resin support with simultaneous side-chain deprotection by acidolysis using anhydrous hydrogen fluoride containing the scavenger anisole (-30% v/v) for 45 minutes at 0 °C. The peptides were cyclized in 90% acetic acid (-600 mL) with a slight excess of I₂ (15 min). Excess I₂ was then removed by the addition of ascorbic acid, solvent was reduce in vacuo and the crude peptides was subjected to HPLC purification.

• Example 2: Preparation of Asp-cyclo(Pen-Phe-Trp-Lys-Tyr-Cys)- VaI-

NH₂ SEQ ID NO:4

Methylbenzhydrylamine polystyrene resin (1.0 g, 0.5 mmol) in the chloride ion form was placed in the reaction vessel of a CS Biosystems automatic peptide synthesizer programmed to perform the following reaction cycle: (a) wash with methylene chloride; (b) neutralize with 10% diisopropylamine in DMF; (c) wash with methylene chloride; (d) the neutralized resin was shaken with Boc-Val and diisopropylcarbodiimide coupling reagent (1 mmol each) in methylene chloride for 1 hour followed by washing of the resin with methylene chloride; and (e) removal of the Boc protecting group by treatment with 33% trifluoractetic acid in methylene chloride.

The following amino acids were then coupled successively by the same procedure: Boc-Cys(MeBzl), Boc-Tyr(ClZ), Boc-Lys(ClZ), Boc-Trp, Boc-Phe, Boc- Pen(MeBzl), Boc-Asp(Bzl). The peptide was cleaved from the resin support with simultaneous side-chain deprotection by acidolysis using anhydrous hydrogen fluoride containing the scavenger anisole (~30% v/v) for 45 minutes at 0 °C. The peptides were cyclized in 90% acetic acid (-600 mL) with a slight excess of I₂ (15 min). Excess I₂ was then removed by the addition of ascorbic acid, solvent was reduce in vacuo and the crude peptides was subjected to HPLC purification as described.

The following examples were made according to the appropriate procedure described hereinabove:

Example 3: Asp-cyclo(Cys-Phe-Trp-Lys-Tyr-Cys)-N-Me- VaI-NH₂; SEQ ID NO: 5

Example 4: succinyl-cyclo(Cys-Phe-Trp-Lys-Cpa-Cys)- VaI-NH₂; SEQ ID NO: 6

Example 5: succinyl-cyclo(Cys-Phe-Tφ-Lys-Tyr-Cys)-Thr-NH₂; SEQ ID NO:7

Example 6: Asp-cyclo(Cys-Phe-Trp-Lys-Tyr-Cys)-Ala-NH₂; SEQ ID NO:8

Example 7: Asp-cyclo(N-Me-Cys-Phe-Tφ-Lys-Tyr-Cys)-Val-NH₂; SEQ ID NO:9

Example 8: Asp-cyclo(Cys-Phe-Trp-Lys-Tyr-Pen)- VaI-NH₂; and SEQ E) NO:10

Example 9: N-Me- Asp-cyclo(Cys-Phe-Trp-Lys-Tyr-Cys)- VaI-NH₂. SEQ ID NO:11

Physical data for the compounds exemplified herein are summarized in Table 1 below.

[Table 1]

• Binding to Rat and Human UII Receptors o Receptor Expression

The cDNAs of rat and human UII receptors were cloned by polymerase chain reaction from rat brain and human placenta cDNA (Clontech, Palo Alto, CA, U.S.A.) respectively, using gene specific primers flanking full-length coding sequence (Rossowki, WJ, et al, Eur. J. Phamacol., 438:159-170 (2002)). The receptors were cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen, Carlsbad, CA, U.S.A.). The plasmids were transfected into Chinese hamster ovary cell line, CHO- Kl (American Type Culture Collection, Rockville, MD, U.S.A.), by the calcium phosphate method. Single cell clones stably expressing the rat or the human UII receptor were obtained by selecting transfected cells grown in cloning rings in RPMl 1640 media supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate containing 0.8 mg/ml G418 (Gibco BRL, Grand Island, NY, U.S.A.).

o Binding Assays

Membranes were prepared for radioligand binding studies by homogenization of the recombinant cells in 20 ml of ice-cold 50 mM Tris-HCl with a Brinkmann Polytron (Westbury, NY, U.S.A.) (setting 6, 15 seconds). The homogenates were washed twice by centrifugation (39,000 x g, 10 minutes), and the final pellets were resuspended in 50 mM Tris-HCl, containing 2.5 mM MgCl₂, 0.1 mg/ml bacitracin (Sigma, St. Louis, MO, U.S.A.) and 0.1% BSA. For assay, aliquots (0.4 ml) were incubated with 0.05 nM [¹²⁵I]Goby-urotensin II with and without 0.05 ml of unlabeled competing test peptides. After a 45-minute incubation (25 ⁰C), the bound [¹²⁵I]Goby- urotensin II was separated from the free by rapid filtration through GF/C filters (Brandel, Gaithersburg, MD, U.S.A.) which had been previously soaked in 0.5% polyethyleneimine. The filters were then washed three times with 5-ml aliquots of ice-cold 50 mM Tris-HCl and 0.1% bovine serum albumin and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Wallac LKB, Gaithersburg, MD, U.S.A.). Specific binding was defined as the total [¹²⁵I]Goby-urotensin II bound minus that bound in the presence of 1000 nM Goby- urotensin II (Bachem, Torrance, CA, U.S.A.).

• Use of Rat Aorta Circular Strip for Assay UII Agonists

Male Sprague-Dawley rats (250-350 g), 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 (9.15 g/L potassium chloride, 2.1 g/L sodium bicarbonate, 1.0 g/L glucose, 0.16 g/L potassium phosphate monobasic, 0.14 g/L magnesium sulfate (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 ⁰C and bubbled with a mixture of 95% O₂ / 5% CO₂. Prior to mounting in the organ bath, selected preparations 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. (Gibson, A., Br. J. Pharmacol. 91 :205 (1987)). The aorta rings were allowed to equilibrate for 90 minutes, at the optimal tensions. During the equilibration period, the bath solution was replaced every 15 minutes. 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 ACKlOO Version 3.2 (BIOPAC Systems, Inc., Santa Barbara, CA.). hi siliconized glass tubes, peptides were dissolved in dionized water at a concentration of 1 μg/lμL (stock solution) and then diluted 1 :10 with sterile BSA- saline solution (0.1% BSA, fraction V, Sigma, St. Louis in 0.9% NaCl). All peptide solutions were prepared fresh directly before the experiments. Peptides in the concentration ranges of 10^"6 to 10^"12 M/L in a final volume of 16-80 μL were direcly introduced into the tested organ bath containing Krebs buffer continuously gassed with 95% O₂ and 5% CO₂ and the aorta rings at an optimal resting tension (1 - 0.2 g). 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.

The exemplified compounds were tested using the above-described assay methods and the results are summarized in Table 2 below.

[Table 2]

Equivalents

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.

Claims

What is claimed is:

1. A compound of the formula:

R¹ -CyClO(AA¹ -AA²- AA³- AA⁴-AA⁵- AA⁶)- AA⁷-NH₂, wherein

AA¹ is Cys, N-Me-Cys, or Pen;

AA² is Phe;

AA³ is Tip or N-Me-Trp;

AA⁴ is Lys; AA⁵ is Tyr or Cpa;

AA⁶ is Cys or Pen;

AA⁷ is VaI, Ala, or Thr; and

R¹ is Asp, N-Me-Asp, or succinyl; or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 , wherein said compound is:

Asp-cyclo(Cys-Phe-N-Me-Tφ-Lys-Tyr-Cys)-Val-NH₂; SEQ ID NO:3 Asp-cyclo(Pen-Phe-Trp-Lys-Tyr-Cys)- VaI-NH₂; SEQ ID NO:4 Asp-cyclo(Cys-Phe-Tφ-Lys-Tyr-Cys)-N-Me- VaI-NH₂; SEQ ID NO:5 succinyl-cyclo(Cys-Phe-Trp-Lys-Cpa-Cys)-Val-NH₂; SEQ ID NO:6 succinyl-cyclo(Cys-Phe-Trp-Lys-Tyr-Cys)-Thr-NH₂; SEQ ID NO:7 Asp-cyclo(Cys-Phe-Trp-Lys-Tyr-Cys)-Ala-NH₂; SEQ ID NO:8 Asp-cyclo(N-Me-Cys-Phe-Trp-Lys-Tyr-Cys)-Val-NH₂; SEQ ID NO:9 Asp-cyclo(Cys-Phe-Trp-Lys-Tyr-Pen)- VaI-NH₂; or SEQ ID NO: 10 N-Me- Asp-cyclo(Cys-Phe-Tφ-Lys-Tyr-Cys)-Val-NH₂; SEQ ID NO: 11 or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition comprising an effective amount of a compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

4. A method of eliciting an agonist effect from a Urotensin-II receptor in a subject in need thereof which comprises administering to said subject a therapeutically effective amount of a compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof.

5. A method of preventing or treating an abnormal condition characterized by a deficiency or under expression of Urotensin-II activity, said method comprising administering to said subject a therapeutically effective amount of a compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein said condition is selected from the group consisting of ischaemic heart disease, congestive heart failure, portal hypertension, variceal bleeding, hypotension, angina pectoris, myocardial infarction, ulcers, anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation, and dyskinesias.