WO1993025580A1 - Cyclic peptides that modulate endothelin activity - Google Patents

Abstract

Cyclic peptides that modulate or alter the activity of the endothelin family of peptides and pharmaceutically acceptable salts and esters of the peptides are provided. More particularly, cyclic pentapeptides and cyclic hexapeptides and pharmaceutically acceptable salts and esters thereof that specifically inhibit the activity of endothelin are provided. Preferred cyclic peptides have the formulae: cyclo(X?1-X2-X3-X4¿-D-Trp) or pharmaceutically acceptable salts or esters thereof in which X1 is any amino acid, X2 is a hydrophobic amino acid, X3 is a hydrophobic D-amino acid, Gly or β-Ala, and X4 is a hydrophobic amino acid, β-Ala, Aib, Gly, D-His, L-His, D-His-Gly or Leu; cyclo(X1-L-Phe-X3-X4-X5) in which X1 is D-Tyr, D-Asp or D-Glu, X3 is His, D-His, β-Ala-D-His or Gly-D-His, X4 is Gly or β-Ala and X5 is D-Trp or N-Me-D-Trp; cyclo(X1-X2-L-Pro-X4 -L-Trp) in which X1 is D-Ala, Aib or Gly, X2 is D-Val, D-Leu, D-Ile, D-Ala or D-Gln, and X4 is D-Asp, D-Glu or D-Ser; cyclo(X?1-X2-X3-X4¿-L-Trp) in which X1 is D-Leu, D-Val, D-Ile or D-Ala, X2 is L-Val, D-Ile, D-Leu or D-Ala, X3 is D-Pro, D-Ala, D-Val or D-Ile, and X4 is L-Asp, L-Glu, L-Tyr or L-Ser. Pharmaceutical compositions containing the peptides and methods of treatment of diseases, including cardiovascular and respiratory diseases, using the compositions are also provided.

Description

CYCLIC PEPTIDES THAT MODULATE ENDOTHELIN ACTIVITY FIELD OF THE INVENTION

The present invention relates to compounds that modulate or alter the activity of the endothelin family of peptides. More particularly, compounds that inhibit the activity of endothelin and that thereby possess therapeutic utility are provided. BACKGROUND OF THE INVENTION

The vascular endothelium releases a variety of vasoactive substances, including the endothelium-derived vasoconstrictor peptide, endothelin (ET) (see, e.g., Vanhoutte et al. (1986) Annual Rev. Physiol. 48: 307-320; Furchgott and Zawadski (1980) Nature 288: 373-376). Endothelin- 1 , which is a potent twenty-one amino acid peptide vasoconstrictor that was originally identified in the culture supernatant of porcine aortic endothelial cells (see, Yanagisawa et aL (1988) Nature 332: 41 1-415), is the most potent vasopressor known. It is produced by numerous cell types, including the cells of the endothelium, trachea, kidney and brain. Endothelin is synthesized as a precursor of 203 amino acids, called preproendothelin, containing a signal sequence which is cleaved by an endogenous protease to produce a 38 (human) or 39 (porcine) amino acid peptide. This intermediate, referred to as big endothelin, is processed to the mature biologically active form in vivo by a putative endothelin-converting enzyme (ECE; see, e.g., Kashiwabara et aL (1989) FEBS Lttrs. 247: 337-340), which appears to be a metal- dependent neutral protease. Processing is required for induction of physiological responses (see, e.g., von Geldern et aL (1991 ) Peptide Res. 4: 32-35). In porcine aortic endothelial cells, the 39 amino acid intermediate, big endothelin, is hydrolyzed at the Trp ¹-Val²² bond to generate endothelin-1 and a C-terminal fragment. A similar cleavage occurs in human cells from a 38 amino acid intermediate. • Three distinct endothelin isopeptides, endothelin-1 , endothelin-2 and endothelin-3, that exhibit potent vasoconstrictor activity have been identified. Each induces vasoconstriction with a potency order of endothelin-2 > endothelin-1 > endothelin-3. Another family of peptides, sarafotoxins, a group of peptide toxins from the venom of the snake Atractaspis einoadensis that cause severe coronary vasospasm in snake bite victims, have structural and functional homology to endothelin- 1 and bind competitively to the same cardiac membrane receptors (Kloog et aL (1989) Trends Pharmacol. Sci. 10: 212-214). The family of three isopeptides endothelin-1 , endothelin-2 and endothelin-3 are encoded by a family of three genes (see, Inoue et aL (1989) Proc. Natl. Acad. Sci. USA 86: 2863-2867; see, also Saida et aL (19891J. Biol. Chem. 264: 14613-14616). The nucleotide sequences of the three human genes are highly conserved within the region encoding the mature 21 amino acid peptides. Endothelin-2 is (Trp⁶, Leu⁷) endothelin-1 and endothelin-3 is (Thr²,Phe⁴,Thr\Tyr⁶,Lys⁷,Tyr¹⁴) endothelin-1. These peptides are, thus, highly conserved at the C- terminal ends. In addition, endothelin is highly conserved among species. Release of endothelins from cultured endothelial cells is modulated by a variety of chemical and physical stimuli and appears to be regulated at the level of transcription and/or translation. For example, gene expression of endothelin-1 is increased by adrenaline, thrombin and Ca²⁺ ionophore. The production and release of endothelin from the endothelium is stimulated by angiotensin II, vasopressin and other factors, such as endotoxin and cyclosporin (see, Brooks et aL (1991 ) Eur. J. Pharm. 194: 1 15-117), and is inhibited by nitric oxide. Endothelial cells appear to secrete short-lived endothelium-derived relaxing factors (EDRF), such as nitric oxide or a related substance (Palmer et aL (1987) Nature 327: 524-526), when stimulated by vasoactive agents, such as acetylcholine and bradykinin. Endothelin-induced vasoconstriction is also attenuated by atrial natriuretic peptide (ANP).

The endothelin peptides exhibit numerous biological activities in vivo and in vitro. Endothelin provokes a strong and sustained vasoconstriction in vivo in rats and in vitro in isolated vascular smooth muscle preparations; it also provokes the release of eicosanoids and endothelium-derived relaxing factor (EDRF) from perfused vascular beds. Intravenous administration of endothelin-1 and in vitro addition to vascular and other smooth muscle tissues produces long-lasting pressor effects and contraction, respectively (see, e.g., Bolger at aL (1991 ) Can. J. Physiol. Pharmacol. 69: 406-413). For example, in isolated vascular strips, endothelin-1 is a potent (EC₅₀ = 4 x 10^"10 M) and slow acting, but persistent, contractile agent, in vivo, a single dose elevates blood pressure in about 20 to 30 minutes. Endothelin-induced vasoconstriction is not affected by antagonists to known neurotransmitters or hormonal factors, but is abolished by calcium channel antagonists. The effect of calcium channel antagonists, however, is most likely the result of blockage of calcium influx, since calcium influx appears to be required for the long-lasting contractile response to endothelin. Endothelin also mediates renin release, stimulation of ANP release and induces a positive inotropic action in guinea pig atria. In the lung, endothelin-1 acts as a potent bronchoconstrictor (Maggi et aL (1989) Eur. J. Pharmacol. 160: 179-182). Endothelin increases renal vascular resistance, decreases renal blood flow, and decreases glomerular filtrate rate. It is a potent mitogen of glomerular mesangial cells and invokes the phosphoinoside cascade in such cells (Simonson et aL (1990) J. Clin. Invest. 85: 790-797).

There are specific high affinity binding sites (K_d's in the range of 2- 6 x 10^~10 M) for the endothelins in the vascular system and in other tissues, including the intestine, heart, lungs, kidneys, spleen, adrenal glands and brain. Binding is not inhibited by catecholamines, vasoactive peptides, neurotoxins or calcium channel antagonists. Endothelin binds and interacts with receptor sites that are distinct from other autonomic receptors and voltage dependent calcium channels. Competitive binding studies indicate that there are multiple classes of receptors with different affinities for the endothelin isopeptides.

DNA clones encoding two distinct endothelin receptors, designated ET_A and ET_B, have been isolated (Arai et aL (1990) Nature 348: 730-732; Sakurai et aL (1990) Nature 348: 732-735). Based on the amino acid sequence of the proteins encoded by the cloned DNA, it appears that each receptor contains seven membrane spanning domains and exhibits structural similarity to G-protein-coupled membrane proteins. Messenger RNA encoding both receptors has been detected in a variety of tissues, including heart? lung, kidney and brain. The distribution of receptor subtypes is tissue specific (Martin et aL (1989) Biochem. Biophvs. Res. Commun. 162: 130-137). ET_A receptors appear to be selective for endothelin-1 and are predominant in cardiovascular tissues. ET_B receptors are predominant in noncardiovascular tissues, including the central nervous system and kidney, and interact with the three endothelin isopeptides (Sakurai et aL (1990) Nature 348: 732-734). In addition, the ET_A receptors, which are endothelin-1 -specific, occur on smooth muscle and are linked to vasoconstriction; whereas ET_B receptors are located on the vascular endothelium and are linked to vasodilation (Takayanagi et al. (1991 ) FEBS Lttrs. 282: 103-106). The activity of the endothelin isopeptides varies in different tissues by virtue of the distribution of receptor types and the differential affinity of each isopeptide for each receptor type. For example, endothelin-1 inhibits ¹²⁵l-labelled endothelin-1 binding in cardiovascular tissues 40-700 times more potently than endothelin-3. ¹²⁵l-labelled endothelin-1 binding in non-cardiovascular tissues, such as kidney, adrenal gland and cerebellum, is inhibited to the same extent by endothelin-1 and endothelin-3, which indicates that cardiovascular tissues are rich in ET_A receptors and non-cardiovascular tissues are rich in ET_B receptors.

Endothelin-1 plasma levels in healthy individuals, as measured by radioimmunoassay (RIA), are about 0.26-5 pg/ml. Blood levels of endothelin-1 and its precursor, big endothelin, are elevated in shock, myocardial infarction, vasospastic angina, kidney failure and a variety of connective tissue disorders. Increased levels of circulating endothelin are present in patients with pulmonary hypertension. In patients undergoing hemodialysis or kidney transplantation or suffering from cardiogenic shock, myocardial infarction or pulmonary hypertension, endothelin levels as high as 35 pg/ml have been observed (see, Stewart et aL (1991 ) Annals Internal Med. 1 14: 464-469). The levels of endothelin at the endothelium/smooth muscle interface are probably much higher because endothelin-1 likely acts as a local, rather than a systemic, regulating factor.

Endothelin agonists and antagonists

Because of the numerous physiological effects of endothelin, it appears that it has an important physiological function, and, thus, may play a critical role in some pathophysiological conditions, including asthma, hypertension, pulmonary hypertension, renal failure, asthma, endotoxin shock and vasospasm (see, Saito et aJL (1990) Hypertension ^" 15: 734-738; Tomita et aL (1989) N.Enol.J. Med. 321 : 1 127; Kurihara et aL (1989) J. Cardiovasc. Pharmacol. 1 3(SUPPI. 5): S13-S17); Morel et aL (1989) Eur. J. Pharmacol. 167: 427-428). Because endothelin is associated with these and other disease states, more detailed knowledge of the function and structure of the endothelin peptide family should provide insight in the progression and treatment of such conditions.

Studies of structural analogs of endothelin have been conducted in order to gain insight into its role in the patho-physiology of cardiovascular disorders, such as hypertension, atherosclerosis, cerebral and coronary vasospasm, asthma and renal failure. Such studies have demonstrated the importance of the two S-S bonds, the C-terminal Trp and the cluster of charged residues Asp⁸-Lys⁹-Glu¹⁰ (see, Nishikori et L (1991 ) Neurochem. Int. 18: 535-539) for vasoconstriction activity. Thus, compounds that can interfere with or potentiate endothelin- associated activities, such as endothelin-receptor interaction and vasoconstrictor activity, are of interest.

A limited number of compounds that exhibit endothelin antagonistic activity have been identified. In particular, a fermentation product of Streptomvces misakiensis. designated BE-18257B, has been identified as an ET_A receptor antagonist. BE-18257B is a cyclic pentapeptide, cyclo(D-Glu-L-Ala-allo-D-lle-L-Leu-D-Trp), which inhibits ¹²⁵l- labelled endothelin-1 binding in cardiovascular tissues in a concentration- dependent manner (IC₅₀ 1.4 μM in aortic smooth muscle, 0.8 μM in ventricle membranes and 0.5 μWA in cultured aortic smooth muscle cells), but fails to inhibit binding to receptors in tissues in which ET_B receptors predominate at concentrations up to 100 μWΛ. Cyclic pentapeptides related to BE-18257B, such as cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ- 123), have been synthesized and shown to exhibit activity as ET_A receptor antagonists (see, U.S. Patent No. 5, 1 14,918 to Ishikawa et al.; see, also, EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (October 7, 1991 )). Studies that measure the inhibition by these cyclic peptides of endothelin-1 binding to endothelin-specific receptors indicate that these cyclic peptides bind preferentially to ET_A receptors.

The analog [Ala¹'^3,11-¹⁵]endothelin-1 , in which the four Cys residues are replaced with Ala, inhibits ¹²⁵l-endothelin-1 binding to cerebral membranes, in which ET_B receptors predominate (Hiley et aL (1989) Trends Pharmacol. Sci 10: 47-49). This peptide and certain truncated forms of endothelin-1 elicit endothelium-dependent vasorelaxation of precontracted porcine pulmonary arteries to an extent that parallels the respective binding affinities of each form for ET_B (Saeki et aL (1991 ) Biochem. and Biophvs Res. Commun. 179: 286-292).

Endothelin antagonists and agonists as therapeutic agents To aid in understanding the physiological role of endothelin, there is a need to identify compounds that modulate or alter endothelin activity. Compounds that modulate endothelin activity, particularly compounds that act as specific antagonists or agonists, may not only aid in elucidating the function of endothelin, but may be therapeutically useful. In particular, compounds that specifically interfere with the interaction of endothelin peptides with the ET_A, ET_B or other receptors should aid in the design of therapeutic agents, and may themselves be useful as disease specific therapeutic agents.

In particular, compounds that specifically interfere with the interaction of endothelin peptides with ET_A, ET_B or other receptors should be useful in identifying essential characteristics of endothelin peptides, may aid in the design of therapeutic agents, and may be useful as disease specific therapeutic agents.

The studies and methods discussed above regarding the selection and design of compounds that provide insights into the structure and function of endothelin and into the structure of compounds that modulate the activity the endothelin peptides provide an imprecise non-systematic approach to this problem. In order to gain further insight into the biology of endothelin, its role in disease, and the identification of pharmaceuticals for treatment of such diseases, there is a need for additional compounds that have the ability to specifically modulate the biological activity of endothelin.

Therefore, it is an object herein to provide compounds that have the ability to modulate the biological activity of one or more of the endothelin isopeptides. It is another object herein, to provide compounds that have activity as specific endothelin antagonists and agonists. It is also an object herein to provide compounds that specifically interact with or inhibit the interaction of endothelin peptides with ET_A or ET_B receptors. It is also an object herein to provide compounds that are useful for treatment of disorders that are mediated by the action of endothelin. It is also an object herein to provide methods for distinguishing between ET_A and ET_B receptors and methods for identifying and purifying endothelin- specific receptors. SUMMARY OF THE INVENTION Cyclic peptides that modulate the activities of endothelin peptides are provided. The cyclic peptides provided herein are pentapeptides, hexapeptides and heptapeptides that contain an L-Trp residue, a D-Trp residue or a derivative of D- or L-Trp, such as N-Me-Trp. Pharmaceutically acceptable salts, esters and other derivatives of the peptides are also provided. The cyclic peptides provided herein modulate the activity of one or more members of the endothelin family of peptides. In particular, cyclic peptides that inhibit or interfere with the interaction of endothelin with endothelin-specific receptors or with endothelin-mediated biological responses and thereby act as specific endothelin antagonists are provided.

Cyclic peptides, or pharmaceutically acceptable salts, esters or other derivatives of the peptides, containing between 5 and 7 residues including either a D-Trp or an L-Trp residue are provided. Selected cyclic peptides have been synthesized and their biological activity as modulators of endothelin-1 activity has been assessed.

Among the cyclic peptides provided herein are those that have formula (I): cyclo(X¹-X²-X³-X⁴-D-Trp) (I) or pharmaceutically acceptable salts, esters and other derivatives of the peptides, in which X¹ is any amino acid; X² is a hydrophobic amino acid; X³ is a hydrophobic D-amino acid, Gly, or /ff-Ala, and X⁴ is a hydrophobic amino acid, preferably a D-amino acid, or is Ala, yff-Ala, Aib, Gly, D-His-gly or Leu, provided that X¹, X², X³ and X⁴ are selected such that the peptide of formula (I) is not selected from cyclo(D-Asp-Pro-D-Val-Leu-D-Trp); cyclo(D-Glu-L-Ala-allo-D-lle-L-Leu-D-Trp); cyclo(D-Glu-Ser-D- Val-Leu-D- Trp); cyclo(D-Cys(O₃Na)-Ala-D-Val-Leu-D-Trp); cyclo(D-Asp-Lys-D- Val- Leu-D-Trp); cyclo(D-Cys(O₃Na)-Pro-D- Val-Leu-D-Trp); cyclo(D-Cys(O₃Na)- Pro-D-Val-Nle-D-Trp); cyclo(D-Asp-Leu-D- Val-Leu-D-Trp); cyclo(D-Glu-Ala- D-Val-Leu-D-Trp); cyclo(D-Glu-Ala-D-Alle-Leu-D-Trp); cyclo(D-Glu-Pro-D- Val-Leu-D-Trp); cyclo(D-Asp-Gly-D- Val-Leu-D-Trp); cyclo(D-Asp-Ala-D- Val- Leu-D-Trp); cyclo(D-Asp-Leu-D-Val-Leu-D-Trp); cyclo(D-Asp-MeAla-D- Val- Leu-D-Trp); cyclo(D-Asp-Met-D-Val-Leu-D-Trp); cyclo(D-Asp-Trp-D-Val- Leu-D-Trp); cyclo(D-Asp-His-D-Val-Leu-D-Trp); cyclo(D-Asp-Arg-D-Val- Leu-D-Trp); cyclo(D-Asp-Orn-D- Val-Leu-D-Trp); cyclo(D-Asp-Gln-D-Val- Leu-D-Trp); cyclo(D-Asp-Asp-D- Val-Leu-D-Trp); cyclo(D-Asp-Cys(O₃Na)-D- Val-Leu-D-Trp); cyclo(D-Asp-Cys-D-Val-Leu-D-Trp); cyclo(D-Asp-Ser-D- Val-Leu-D-Trp); cyclo(D-Asp-Thr-D-Val-Leu-D-Trp); cyclo(D-Asp-Aia-D- Leu-Leu-D-Trp); cyclo(D-Asp-Ala-D-Thr-Leu-D-Trp); cyclo(D-Asp-Pro-D-lle- Leu-D-Trp); cyclo(D-Asp-Pro-D-Alle-Leu-D-Trp); cyclo(D-Asp-Pro-D-Nle- Leu-D-Trp); cyclo(D-Asp-Pro-D-Phg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Nva- Leu-D-Trp); cyclo(D-Asp-Ser-D-Val-Nlle-D-Trp); cyclo(D-Asp-Ser-D-Val- Met-D-Trp); cyclo(D-Asp-Asp-D-Val-Ala-D-Trp); cyclo(D-Asp-Ala-D-Ala- Ala-D-Trp); cyclo(D-Asp-Ala-D-Val-Pro-D-Trp); cyclo(D-Asp-Pro-D-Val-lle- D-Trp); cyclo(D-Asp-Pro-D-Val-Nlle-D-Trp); cyclo(D-Cys(O₃Na)-Cys(O₃Na)- D-Val-Leu-D-Trp); cyclo(D-Cys(O₃Na)-Pro-D-Alle-Leu-D-Trp); cyclo(D-Asp- Val-D- Val-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val-Nva-D-Trp); cyclo(D-Asp- Nlle-D-Val-Leu-D-Trp); cyclo(D-Asp-Pip-D-Val-Leu-D-Trp); cyclo(D-Asp- Phe-D-Val-Leu-D-Trp); cyclo(D-Cys(O₃Na)-Glu-D- Val-Leu-D-Trp); cyclo(D- Cys(O₃Na)-Lys-D- Val-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(CHO)); cyclo(D-Glu-Ala-D-Alle-Leu-D-Trp(CHO)); cyclo(D-Asp-Pro-D-Alle-Leu-D- Trp(CHO)); cyclo(D-Asp-Ser-D-Val-Nle-D-Trp(CHO)); cyclo(D-Asp-Met-D- Val-Leu-D-Trp(CHO)); cyclo(D-Asp-Pro-D-Val-Nva-D-Trp(CHO)); cyclo(D- Asp-Lys(CHO)-D-Val-Leu-D-Trp(CHO)); cyclo(D-Asp-Met(O)-D-Val-Leu-D- Trp(CHO)); cyclo(D-Asp(ONa)-Pro-DVal-Leu-D-Trp); cyclo(D-Asp-Pro-D- Pen-Leu-D-Trp); cyclo(D-Asp-Aib-D-Val-Leu-D-Trp); cyclo(D-Asp-Pro-Aib- Leu-D-Trp); cyclofD-Asp-Pro-ACgC-Leu-D-Trp); cyclo(D-Asp-Pro-AC₆C-Leu- D-Trp); cyclo(D-Asp-Sar-D- Val-Leu-D-Trp); cyclo(D-Asp-£-Ala-D-Val-Leu-D- Trp); cyclo(D-Asp-Pro-D-Thg-Leu-D-Trp);cyclo(D-Asp-Thz-D- Val-Leu-D- Trp); cyclo(D-Asp-Pro-D-Val-MeLeu-D-Trp); cyclo(D-Asp-MeMet-D-Val- Leu-D-Trp); cyclo(D-Asp-Sar-D-Thg-Leu-D-Trp); cyclo(D-Asp-CpGly-D-Thg- Leu-D-Trp); cyclo(D-Asp-Pro-D-Dpg-Leu-D-Trp); cyclo(D-Cys(O₃Na)-Pro-D- Thg-Leu-D-Trp(CHO)); cyclo(D-Cys(O₃Na)-Pro-D-Thg-Leu-D-Trp); cyclo(D- Asp-Met(O₂)-D-Val-Leu-D-Trp(CHO)); cyclo(D-Asp-PrGly-D-Thg-Leu-D- Trp); cyclo(D-Asp-trans-Hyp-D-Thg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Fug- Leu-D-Trp); cyclo(D-Asp-Pro-D-Cpg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Etg- Leu-D-Trp); cyclo(D-Asp-CmGly-D-Thg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val- His-D-Trp); cyclo(D-Asp-leGly-D- Val-Leu-D-Trp); cyclo(D-Asp-Pro-D- Val- Leu-D-Trp(COOCH₃)); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(COO^tBu)); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(O)); cyclo(D-Asp-MeAla-D-Val-Leu-D- Trp(O)); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(CH₂OOCH₃)); cyclo(D-Asp-Pro- D-Val-Leu-D-Trp(CH₂CONH₂)); cyclo(D-Asp-Pro-D-Val-Leu-D- Trp(CH₂CONHCH₃)); cyclo(D-Glu-Ala-D-Val-Leu-D-Nal); cyclo(D-Asp-trans- Hyp-D-Cpg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(CH₂COOH)); cyclo(D-Asp-Ala-D-Val-C₆al-D-Trp); cyclo(D-Asp-Pro-D-Val-Leu-D-Phe); or cyclo(D-Asp-Pro-D-Val-Leu-D-Tyr).

In particular, cyclic pentapeptides and hexapeptides of formula (I) in which X¹ is D-Tyr or D-Asp; X² is Phe, Ala or Pro; X³ is D-His, D-Ala, D-Val, Gly or 0-Ala; and X⁴ is D-His, L-His, Ala, £-Ala, Aib, Gly, D-His-gly or Leu, such that the resulting peptide is not cyclo(D-Asp-Ala-D-Ala-Ala- D-Trp), are provided. Cyclic pentapeptides and hexapeptides of formula (II): cyclo(X¹-L-Phe-X³-X -X⁵), (II) or pharmaceutically acceptable salts, esters and other derivatives of the peptides, in which X¹ is D-Tyr, D-Asp or D-Glu, X³ is His, D-His, β-Ala-D- His or gly-D-His; X⁴ is Gly or β-Ala and X⁵ is D-Trp or N-Me-D-Trp are also provided. Preferred peptides of formula II include: cyclo(X-L-Phe-His-Gly- D-Trp); cyclo(X-L-Phe-D-His-_/ff-Ala-D-Trp); and cyclo(X-L-Phe-D-His-Gly-N- Me-D-Trp); in which X is D-Tyr, D-Asp or D-Glu. In order to increase the solubility of the above cyclic peptides the Gly and β-Ala may be replaced by serine.

Cyclic peptides of formula (III): cyclo(X¹-X²-X³-X⁴-L-Trp) (III) and pharmaceutically acceptable salts and esters and other derivatives thereof in which X¹ is D-Ala, Aib, Gly, D-Val, D-Leu, D-Ile, D-Nva, D-Nle, or D-Alle; X² is D-Val, D-Leu, D-Ile, D-Ala, D-GIn, Gly, Aib, D-Nva, D-Nle, or D-Alle; X³ is L-Pro, Gly, Aib, L-Val, L-Leu, L-Nva, L-Nle, L-Alle or L-Hyp and X⁴ is D-Asp, D-Glu, D-Ser, D-Thr, D-Tyr, D-Cys(O₃H) or D-Pen(O₃H) are provided.

Preferred cyclic peptides of formula (III) or pharmaceutically acceptable salts, esters and other derivatives of the peptides include thosein which X¹ is D-Ala, Aib, or Gly; X² is D-Val, D-Leu, D-Ile, D-Ala or D-GIn; and X⁴ is D-Asp, D-Glu or D-Ser.

In addition, cyclic peptides that have L-Trp in the backbone and that have formula (IV): cyclo(X¹-X²-X³-X⁴-L-Trp) (IV) and pharmaceutically acceptable salts and esters and other pharmaceutically acceptable derivatives thereof in which X¹ is D-Leu, D- Val, D-Ile, D-Ala, Gly, Aib, D-Nva, D-Nle or D-Alle; X² is Val, lie, Leu, Ala, Gin, Gly, Aib, L-Nva, L-Nle or L^AIIe; X³ is D-Pro, D-Hyp, D-Ala, D-Val, D- lle, Gly, Aib, D-Nva, D-Nle or D-Alle; and X⁴ is L-Asp, L-Glu, L-Tyr, L-Ser, L-Thr, L-Cys(O₃H) or L-Pen(O₃H) are provided.

Preferred among the peptides of formula (IV) or pharmaceutically acceptable salts, esters and other derivatives of the peptides are those in which X¹ is D-Leu, D-Val, D-Ile, or D-Ala; X² is Val, lie, Leu or Ala; X³ is D-Pro, D-Ala, D-Val or D-Ile; and X⁴ is L-Asp, L-Glu, L-Tyr or L-Ser are provided. Peptides of formulas: cyclo(D-Leu-L-Val-D-Pro-L-Asp-L-Trp); cyclo(D-Leu-L-Val-D-Pro-L-Tyr-L-Trp); and cyclo(D-Leu-L-Val-D-Pro-L-Ser- L-Trp) are among the preferred peptides of formula (IV). More preferred cyclic peptides are those of the above peptides of formulas (l)-(IV) that inhibit the interaction of endothelin- 1 with ET_A receptors at an IC₅₀ of less than about 100 μM, and preferably less than about 50 μM and more preferably, less than about 10 μWΛ, but that do not inhibit binding of endothelin-1 to ET_B receptors at concentrations of about 100 μWΛ or less. Others of the most preferred peptides are those that interact with ET_B receptors at concentrations at which they do not interact with ET_A receptors.

The cyclic peptides can be used in methods for identification and isolation of specific endothelin receptors and in aiding in delineating the structure, function and biological activities mediated by endothelin. The peptides also should have use in pharmaceutical compositions as obstetric agents, including promoting closure of umbilical vessels, contraceptive agents, agents for the treatment of menstrual disorders, including amenorrhea and dysmenorrhea, wound healing agents, agents for the physiological regulation of blood pressure and treatment of vascular disorders, agents for neuroendocrine regulation, agents for treatment of cardiovascular diseases, and as agents for the treatment of other diseases listed herein and known to involve an endothelin peptide. Pharmaceutical compositions containing effective concentrations of one or more of the cyclic peptides, or pharmaceutically acceptable salts or esters of the peptides, for the treatment of hypertension, bronchoconstriction, asthma, shock, ocular hypertension, cardiovascular disease, menstrual disorders, wounds, glaucoma and other conditions that are in some manner mediated by an endothelin peptide or that involve vasoconstriction are also provided.

In particular, the compositions containing therapeutically effective concentrations of one or more of the cyclic peptides of formulas (I), (II), (III) and (IV) formulated for oral, intravenous, local and topical application for the treatment of hypertension, cardiovascular diseases, cardiac diseases, including myocardial infarction, respiratory diseases, including asthma, inflammatory diseases, ophthalmologic diseases, gastroenteric diseases, renal failure, endotoxin shock, anaphylactic shock, hemorrhagic shock, and other diseases in which endothelin mediated physiological responses are implicated are provided. Methods for treatment of hypertension, cardiovascular diseases, cardiac diseases including myocardial infarction, respiratory diseases and inflammatory diseases, including asthma, ophthalmologic diseases, menstrual disorders, gastroenteric diseases, renal failure, endotoxin shock, anaphylactic shock, hemorrhagic shock, and other diseases in which endothelin mediated physiological responses are implicated, by administering effective amounts of the compositions are also provided.

In particular, methods of treatment of diseases, including hypertension, pulmonary hypertension, asthma, shock, ocular hypertension, glaucoma, menstrual disorders, erythropoietin-mediated vasoconstriction, obstetric conditions and other conditions or disorders that are in some manner mediated by an endothelin peptide or that involve vasoconstriction, by administering an effective amount of the pharmaceutical compositions that contain effective concentrations of one or more of the cyclic peptides of formulas (l)-(IV), or pharmaceutically acceptable salts or esters of the peptides, are provided. The effective amounts and concentrations are those that are effective for ameliorating any of the symptoms of the any of the disorders. Preferred methods of treatment are methods for treatment of hypertension and endotoxin shock. in practicing the methods, effective amounts of compositions containing therapeutically effective concentrations of the compounds formulated for oral, intravenous, local and topical application for the treatment of hypertension, cardiovascular diseases, cardiac diseases, including myocardial infarction, respiratory diseases, including asthma, inflammatory diseases, ophthalmologic diseases, gastroenteric diseases, renal failure, immunosuppressive-mediated renal vasoconstriction, erythropoietin-mediated vasoconstriction, endotoxin shock, anaphylactic shock, hemorrhagic shock, and other diseases in which endothelin mediated physiological responses are implicated are administered to an individual exhibiting the symptoms of one or more of these disorders. The amounts are effective to ameliorate or eliminate one or more symptoms of the disorders.

Methods using the cyclic peptides for the identification and isolation of endothelin receptor subtypes are also provided. In particular, methods for detecting, distinguishing and isolating endothelin receptors using the cyclic peptides of formulas (I), (II), (III) and (IV) are provided. In practicing the method for identifying endothelin receptors, one or more of the cyclic peptides is linked to a support and used in methods of affinity purification of receptors. By selecting the cyclic peptides with particular specificities, distinct subclasses of endothelin receptors may be identified. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference.

As used herein, endothelin (ET) peptides include peptides that have substantially the amino acid sequence of endothelin-1 , endothelin-2, endothelin-3 and that act as potent endogenous vasoconstrictor peptides. As used herein, an endothelin agonist is a compound that potentiates or exhibits a biological activity associated with or possessed by an endothelin peptide.

As used herein, an endothelin antagonist is a drug or an antibody designed to inhibit endothelin-stimulated vasoconstriction and contraction and other endothelin-mediated physiological responses. The antagonist may act by interfering with the interaction of the endothelin with an endothelin-specific receptor or by interfering with the physiological response to or bioactivity of an endothelin isopeptide, such as vasoconstriction. Thus, as used herein, an endothelin antagonist interferes with endothelin-stimulated vasoconstriction or other response or interferes with the interaction of an endothelin with an endothelin- specific receptor, such as ET_A receptors, as assessed by assays known to those of skill in the art. The effectiveness of potential agonists and antagonists can be assessed using methods known to those of skill in the art. For example, endothelin agonist activity can be identified by its ability to stimulate vasoconstriction of isolated rat thoracic aorta or portal vein ring segments (Borges et L (1989) "Tissue selectivity of endothelin" Eur. J. Pharmacol. 165: 223-230). Endothelin antagonist activity can be assessed by the ability to interfere with endothelin-induced vasoconstriction.

As used herein, the biological activity or bioactivity of endothelin includes any activity induced, potentiated or influenced by endothelin in vivo. It also includes both the ability to bind to particular receptors and to induce a functional response, such as vasoconstriction. These activities include, but are not limited to, vasoconstriction, vasorelaxation and bronchodilation. For example, ET_B receptors appear to be expressed in vascular endothelial cells and may mediate vasodilation and other such responses; whereas ET_A receptors, which are endothelin-1 -specific, occur on smooth muscle and are linked to vasoconstriction Any assay known to those of skill in the art to measure or detect such activity may be used to assess such activity (see, e.g.. Spokes et a_L (19989) J. Cardiovasc. Pharmacol. 1 3(SUPPI. 5):S191-S192: Spinella et aL(1991 ) Proc. Natl. Acad. Sci. USA 88: 7443-7446; Cardell et a (1991 ) Neurochem. Int. 18:571-574); and the Examples herein).

As used herein, the IC₅₀ refers to an amount, concentration or dosage of a particular test compound that achieves a 50% of inhibition of a maximal response, such as binding of endothelin to tissue receptors, in an assay that measures such response. As used herein, EC₅₀ refers to an dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.

As used herein, a peptidomimetic is a compound that mimics the conformation and certain stereochemical features of the biologically active form of a particular peptide. In general, peptidomimetics are designed to mimic certain desirable properties of a compound, but not the undesirable properties, such as flexibility, that lead to a loss of a biologically active conformation and bond breakdown. Peptidomimetics may be prepared from biologically active compounds by replacing certain groups or bonds that contribute to the undesirable properties with bioisosteres. Bioisosteres are known to those of skill in the art. For example the methylene bioisostere CH₂S has been used as an amide replacement in enkephalin analogs (see, e.g., Spatola (1983) pp. 267-357 in Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins. Weistein, Ed. volume 7, Marcel Dekker, New York). Morphine, which can be administered orally, is a compound that is a peptidomimetic of the peptide endorphin.

As used herein, non-peptidic compounds refers to compounds that do not include more than two linked amino acids and that include linkages other than peptide bonds among the constituent groups.

As used herein, pharmaceutically acceptable salts, esters or other derivatives of the peptides include any such salts, esters or derivatives that may be readily prepared using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs.

As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use as contraceptive agents.

As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

As used herein, an endothelin-mediated condition is a condition that is caused by abnormal endothelin acitivity or one in which compounds that inhibit endothelin activity have therapeutic use. Such diseases include, but are not limited to hypertension, cardiovascular disease, asthma, inflammatory diseases, ophthalmologic disease, menstrual disorders, obstetric conditions, gastroenteric disease, renal failure, endotoxin shock, anaphylactic shock, or hemorrhagic shock. Endothelin-mediated conditions also include conditions that result from therapy with agents, such as erythropoietin and immunosuppressants, that elevate endothelin levels.

As used herein an effective amount of a compound for treating a particular disease, is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms assoicated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Typically repeated administration is required to achieve the desired amelioration of symptoms.

As used herein, pharmaceutically acceptable salts, esters or other derivatives of the compounds include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs. For example, hydroxy groups can be esterified or etherified and nitro groups can be reduced to the amine.

As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as gel electrophoresis, high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of peptides to produce substantially pure peptides are known to those of skill in the art. A substantially pure compound may, however, be a mixture of isomers, including stereoisomers. In such instances, further purification might increase the specific activity of the compound. As used herein, biological activity refers the m. vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.

As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or pro¬ perties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceu¬ tically active compound is known, can design prodrugs of the compound (see, e.g.. Nogrady (1985) Medicinal Chemistry A Biochemical Approach. Oxford University Press, New York, pages 388-392. For example, succinyl-sulfathiazole is a prodrug of sulfathiazole that exhibits altered transport characteristics.

As used herein, hydrophobic amino acids include Ala, Val, Leu, Me, Pro, Phe, Trp, and Met and any other non-naturally occurring amino acids, including as the corresponding D isomers of the hydrophobic amino acids, that have similar hydrophobic properties. It is also understood that certain amino acids may be replaced by substantially equivalent non- naturally occurring variants thereof, such as D-Nva, D-Nle, D-Alle, and others listed with the abbreviations below or known to those of skill in this art.

As used herein, the abbreviations for amino acids and protective groups are in accord with their common usage and the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 1 1 : 1726). Each naturally occurring L-amino acid is identified by the standard three letter code or the standard three letter code with the prefix "L-"; the prefix "D-" indicates that the stereoisomeric form of the amino acid is D. Other abbreviations used herein include: Aib for 2-amino-2- methylpropionic acid, β-Ala for β-alanine, σ-Aba for L-σ-aminobutanoic acid; D-σ-Aba for D-σ-aminobutanoic acid; Ac₃c for 1-aminocyclopropanecarboxylic acid; Ac₄c for 1 -aminocy- clobutanecarboxylic acid; Ac₅c for 1-aminocyclopentanecarboxylic acid; Ac₆c for 1 -aminocyclohexanecarboxylic acid; Ac₇c for 1 -aminocyclohep- tanecarboxylic acid; D-Asp(ONa) for sodium D-aspartate; D-Bta for D-3-(3-benzo[b]thienyl)alanine; C₃al for L-3-cyclopropylalanine; C₄al for L-3-cyclobutylalanine; C₅al for L-3-cyclopentylalanine; C₆al for L-3-cyclohexylalanine; D-Chg for D-2-cyclohexylglycine; CmGly for N-(carboxymethyl)glycine; D-Cpg for D-2-cyclopentylglycine; CpGly for N-cyciopentylglycine; Cys(O₃Na) for sodium L-cysteate; D-Cys(O₃H) for D-cysteic acid; D-Cys(O₃Na) for sodium D-cysteate; D-Cys(O₃Bu₄N) for tetrabutylammonium D-cysteate; D-Dpg for D-2-(1 ,4-cyclohexadienyl)- glycine; D-Etg for (2S)-2-ethyl-2-(2-thienyl)glycine; D-Fug for D-2-(2-furyl)glycine; Hyp for 4-hydroxy-L-proline; leGly for N-[2-(4-imida- zolyl)ethyl]glycine; alle for L-L-alloisoleucine; D-alle for D-alloisoleucine; D- Itg for D-2-(isothiazolyl)glycine; D-fertLeu for D-2-amino-3,3-dimethylbu- tanoic acid; Lys(CHO) for N⁶-formyl-L-lysine; MeAla for N-methyl-L-ala- nine; MeLeu for N-methyl-L-leucine; MeMet for N-methyl-L-methionine; Met(O) for L-methionine sulfoxide; Met(O₂) for L-methionine sulfone; D- Nal for D-3-(1 -naphthyl)alanine; Nle for L-norleucine; D-Nle for D-nor- leucine; Nva for L-norvaline; D-Nva for D-norvaline; Orn for L-ornithine; Orn(CHO) for N⁵-formyl-L-ornithine; D-Pen for D-penicillamine; D-Phg for D-phenylglycine; Pip for L-pipecolinic acid; 'PrGly for N-isopropylglycine; Sar for sarcosine; Tha for L-3-(2-thienyl)alanine; D-Tha for D-3(2-thienyl)- alanine; D-Thg for D-2-(2-thienyl)glycine; Thz for L-thiazolidine-4-carboxy- lic acid; D-Trp(CHO) for Nⁱⁿ-formyl-D-tryptophan; D-trp(O) for D-3-(2,3-di- hydro-2-oxoindol-3-yl)alanine; D-trp((CH₂)_mCOR¹) for D-tryptophan substituted by a -(CH₂)_mCOR¹ group at the 1 -position of the indole ring; Tza for L-3-(2-thiazolyl)alanine; D-Tza for D-3-(2-thiazolyl)alanine; D-Tzg for D-2-(thiazolyl)glycine; Bzl for benzyl; DMF for N,N-dimethylformamide; Boc for ferf-butoxycarbonyl; TFA for trifluoroacetic acid; HF for hydrogen fluoride; HFIP for hexafluoroisopropanol; HPLC for high performance liquid chromatography; FAB-MS for fast atom bombardment mass spectrometry; DCM for dichloromethane, Bom for benzyloxymethyl; Pd/C is palladium catalyst on activated charcoal; BOP for bensotriazol-1-yloxy- tris(dimethylamino) phosphonium hexafluorophosphate; DIC for diisopropylcarbodiimide; DCC for N,N'-dicyclohexylcarbodiimide; and (For) for formyl. A. Cyclic peptides that modulate the activity of endothelin

Two groups of peptides based on cyclic peptide backbones structures cyclo(D-Ala-L-Ala-D-Ala-L-D-Ala) and cyclo(D-Ala-L-Ala-D-Ala-L- Ala-L-Ala) are provided. The cyclic peptides, or pharmaceutically acceptable salts, esters or other derivatives of the peptides, contain between 5 and 7 residues. The first group includes peptides that have the cyclic peptide backbone cyclo(D-Ala-L-Ala-D-Ala-L-Ala-D-Ala) and include a D-Trp residue in place of a D-Ala residue. The second group includes peptides that either have the backbone cyclo(D-Ala-L-Ala -D-Ala- (L or D)-Ala) or cyclo(D-Ala-L-Ala-D-Ala-L-Ala-L-Ala) and include an L-Trp in place of an L-Ala.

Among the cyclic peptides provided herein are those that have formula (I): cyclo(X -X²-X³-X⁴-D-Trp) (I) or pharmaceutically acceptable salt, esters or other derivatives thereof in which X¹ is any amino acid; X² is a hydrophobic amino acid; X³ is a hydrophobic D-amino acid, Gly or β-Ala, and X⁴ is a hydrophobic amino acid, preferably a D-amino acid, or is β-Ala, Aib, Gly, D-His-gly or Leu, provided that X¹, X², X³ and X⁴ are selected such that the peptide of formula (I) is not cyclo(D-Asp-Pro-D-Val-Leu-D-Trp); cyclo(D-Glu-L-Ala- allo-D-lle-L-Leu-D-Trp); cyclo(D-Glu-Ser-D-Val-Leu-D-Trp); cyclo(D- Cys(O₃Na)-Ala-D-Val-Leu-D-Trp); cyclo(D-Asp-Lys-D- Val-Leu-D-Trp); cyclo(D-Cys(O₃Na)-Pro-D-Val-Leu-D-Trp); cyclo(D-Cys(O₃Na)-Pro-D-Val- Nle-D-Trp); cyclo(D-Asp-Leu-D-Val-Leu-D-Trp); cyclo(D-Glu-Ala-D-Val-Leu- D-Trp); cyclo(D-Glu-Ala-D-Alle-Leu-D-Trp); cyclo(D-Glu-Pro-D-Val-Leu-D- Trp); cyclo(D-Asp-Gly-D-Val-Leu-D-Trp); cyclo(D-Asp-Ala-D-Ala-Ala-D- Trp), cyclo(D-Asp-Ala-D- Val-Leu-D-Trp); cyclo(D-Asp-Leu-D- Val-Leu-D- Trp); cyclo(D-Asp-MeAla-D- Val-Leu-D-Trp); cyclo(D-Asp-Met-D-Val-Leu-D- Trp); cyclo(D-Asp-Trp-D-Val-Leu-D-Trp); cyclo(D-Asp-His-D-Val-Leu-D- Trp); cyclo(D-Asp-Arg-D- Val-Leu-D-Trp); cyclo(D-Asp-Orn-D-Val-Leu-D- Trp); cyclo(D-Asp-Gln-D-Val-Leu-D-Trp); cyclo(D-Asp-Asp-D-Val-Leu-D- Trp); cyclo(D-Asp-Cys(O₃Na)-D- Val-Leu-D-Trp); cyclo(D-Asp-Cys-D- Val- Leu-D-Trp); cyclo(D-Asp-Ser-D-Val-Leu-D-Trp); cyclo(D-Asp-Thr-D- Val- Leu-D-Trp); cyclo(D-Asp-Ala-D-Leu-Leu-D-Trp); cyclo(D-Asp-Ala-D-Thr- Leu-D-Trp); cyclo(D-Asp-Pro-D-lle-Leu-D-Trp); cyclo(D-Asp-Pro-D-Alle-Leu- D-Trp); cyclo(D-Asp-Pro-D-Nle-Leu-D-Trp); cyclo(D-Asp-Pro-D-Phg-Leu-D- Trp); cyclo(D-Asp-Pro-D-Nva-Leu-D-Trp); cyclo(D-Asp-Ser-D-Val-Nlle-D- Trp); cyclo(D-Asp-Ser-D-Val-Met-D-Trp); cyclo(D-Asp-Asp-D-Val-Ala-D- Trp); cyclo(D-Asp-Ala-D-Val-Pro-D-Trp); cyclo(D-Asp-Pro-D-Val-lle-D-Trp); cyclo(D-Asp-Pro-D-Val-Nlle-D-Trp); cyclo(D-Cys(O₃Na)-Cys(O₃Na)-D-Val- Leu-D-Trp); cyclo(D-Cys(O₃Na)-Pro-D-Alle-Leu-D-Trp); cyclo(D-Asp-Val-D- Val-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val-Nva-D-Trp); cyclo(D-Asp-Nlie-D- Val-Leu-D-Trp); cyclo(D-Asp-Pip-D-Val-Leu-D-Trp); cyclo(D-Asp-Phe-D-Val- Leu-D-Trp); cyclo(D-Cys(O₃Na)-Glu-D-Val-Leu-D-Trp); cyclo(D-Cys(O₃Na)- Lys-D-Val-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(CHO)); cyclo(D- . Glu-Ala-D-Alle-Leu-D-Trp(CHO)); cyclo(D-Asp-Pro-D-Alle-Leu-D-Trp(CHO)); cyclo(D-Asp-Ser-D-Val-Nle-D-Trp(CHO)); cyclo(D-Asp-Met-D-Val-Leu-D- Trp(CHO)); cyclo(D-Asp-Pro-D-Val-Nva-D-Trp(CHO)); cyclo(D-Asp- Lys(CHO)-D-Val-Leu-D-Trp(CHO)); cyclo(D-Asp-Met(O)-D-Val-Leu-D- Trp(CHO)); cyclo(D-Asp(ONa)-Pro-DVal-Leu-D-Trp); cyclo(D-Asp-Pro-D- Pen-Leu-D-Trp); cyclo(D-Asp-Aib-D-Val-Leu-D-Trp); cyclo(D-Asp-Pro-Aib- Leu-D-Trp); cyclofD-Asp-Pro-ACgC-Leu-D-Trp); cyclo(D-Asp-Pro-AC₆C-Leu- D-Trp); cyclo(D-Asp-Sar-D- Val-Leu-D-Trp); cyclo(D-Asp-β-Ala-D-Val-Leu-D- Trp); cyclo(D-Asp-Pro-D-Thg-Leu-D-Trp);cyclo(D-Asp-Thz-D-Val-Leu-D- Trp); cyclo(D-Asp-Pro-D-Val-MeLeu-D-Trp); cyclo(D-Asp-MeMet-D-Val- Leu-D-Trp); cyclo(D-Asp-Sar-D-Thg-Leu-D-Trp); cyclo(D-Asp-CpGly-D-Thg- Leu-D-Trp); cyclo(D-Asp-Pro-D-Dpg-Leu-D-Trp); cyclo(D-Cys(O₃Na)-Pro-D- Thg-Leu-D-Trp(CHO)); cyclo(D-Cys(O₃Na)-Pro-D-Thg-Leu-D-Trp); cyclo(D- Asp-Met(O₂)-D-Val-Leu-D-Trp(CHO)); cyclo(D-Asp-PrGly-D-Thg-Leu-D- Trp); cyclo(D-Asp-trans-Hyp-D-Thg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Fug- Leu-D-Trp); cyclo(D-Asp-Pro-D-Cpg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Etg- Leu-D-Trp); cyclo(D-Asp-CmGly-D-Thg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val- His-D-Trp); cyclo(D-Asp-leGly-D- Val-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val- Leu-D-TrptCOOCHaJJj cycloJD-Asp-Pro-D-Val-Leu-D-TrpfCOOΕu)); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(O)); cyclo(D-Asp-MeAla-D-Val-Leu-D- Trp(O)); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(CH₂OOCH₃)); cyclo(D-Asp-Pro- D-Val-Leu-D-Trp(CH₂CONH₂)); cyclo(D-Asp-Pro-D-Val-Leu-D- Trp(CH₂CONHCH₃)); cyclo(D-Glu-Ala-D-Val-Leu-D-Nal); cyclo(D-Asp-trans- Hyp-D-Cpg-Leu-D-Trp); cyclo(D-Asp-Pro-D-Val-Leu-D-Trp(CH₂COOH)); cyclo(D-Asp-Ala-D-Val-C₆al-D-Trp); cyclo(D-Asp-Pro-D-Val-Leu-D-Phe); or cyclo(D-Asp-Pro-D-Val-Leu-D-Tyr) .

Preferred cyclic peptides include cyclic pentapeptides and cyclic hexapeptides of formula (I) in which X¹ is D-Tyr or D-Asp; X² is Phe, Ala or Pro; X³ is D-His, D-Ala, D-Val, Gly or β-Aia; and X⁴ is D-His, L-His Ala, β-Ala, Aib, Gly, D-His-gly or Leu, provided that X¹, X², X³ and X⁴ are not selected such that the peptide of formula (I) is cyclo(D-Asp-Pro-D-Val-Leu- D-Trp), cyclo(D-Asp-Ala-D- Val-Leu-D-Trp), cyclo(D-Asp-Phe-D-Val-Leu-D- Trp), cyclo(D-Asp-Ala-D-Ala-Ala-D-Trp) or cyclo(D-Asp-Pro-D-Val-His-D- Trp), are provided.

More preferred compounds of formula (I) include: cyclo(D-Tyr-Phe- D-His-Gly-D-Trp); cyclo(D-Tyr-Phe-D-His-β-Ala-D-Trp); cyclo(D-Tyr-Ala-D- Ala-Ala-D-Trp); cyclo(D-Asp-Ala-D-His-Ala-D-Trp); cyclo(D-Asp-Ala-D-Val- Aib-D-Trp); cyclo(D-Asp-Pro-D-Ala-Aib-D-Trp); cyclo(D-Asp-Ala-D-His-Leu- D-Trp); and cyclo(D-Tyr-Phe-Gly-D-His-Gly-D-Trp).

Most preferred peptides of the above include: cyclo(D-Tyr-Ala-D- Ala-Ala-D-Trp) and cyclo(D-Tyr-Phe-D-His-β-Ala-D-Trp).

Cyclic pentapeptides and cyclic hexapeptides of formula (II) are also provided: cyclo(X¹-L-Phe-X³-X⁴-X⁵) (II) in which X¹ is D-Tyr, D-Asp or D-Glu, X³ is His, D-His, β-Ala-D-His, or gly- D-His; X⁴ is Gly, D-His or β-Ala and X⁵ is D-Trp or N-Me-D-Trp.

Preferred peptides of formula (II) include: cyclo(X-L-Phe-His-Gly-D- Trp); cyclo(X-L-Phe-D-His-β-Ala-D-Trp); cyclo(X-L-Phe-D-His-Gly-N-Me-D- Trp); cyclo(D-Tyr-Phe-β-Ala-D-His-D-Trp); cyclo(X-L-Phe-β-Ala-D-His-Gly- D-Trp); cyclo(X-L-Phe-Gly-D-His-Gly-D-Trp); and cyclo(D-Asp-Ala-D-Val- Aib-σ-Me-D-Trp, in which X is D-Tyr, D-Asp or D-Glu. Cyclic peptides of formula (III): cyclo(X¹-X²-X³-X⁴-L-Trp) (III! and pharmaceutically acceptable salts and esters and other derivatives thereof in which X¹ is D-Ala, Aib, Gly, D-Val, D-Leu, D-Ile, D-Nva, D-Nie, or D-Alle; X² is D-Val, D-Leu, D-Ile, D-Ala, D-GIn, Gly, Aib, D-Nva, D-Nle, and D-Alle; X³ is L-Pro, Gly, Aib, L-Val, L-Leu, L-Nva, L-Nle, L-Alle, or L- Hyp and X⁴ is D-Asp, D-Glu, D-Ser, D-Thr, D-Tyr, D-Cys(O₃H), or D- Pen(O₃H) are provided.

Among the preferred peptides of formula (III) and pharmaceuticaly acceptable salts and esters thereof are those in which X¹ is D-Ala, Aib, or Gly; X² is D-Val, D-Leu, D-Ile, D-Ala or D-GIn; and X⁴ is D-Asp, D-Glu, or D-Ser. When X² is D-GIn, the peptides of formula (III) should preferentially bind to ET_B receptors.

In particular, the following peptides of formula (III) are preferred: cyclo(D-Ala-D-Val-L-Pro-D-Asp-L-Trp); cyclo(D-Ala-D-Val-L-Pro-D-Glu-L- Trp); cyclo(-D-Ala-D-Val-L-Pro-D-Ser-L-Trp); cyclo(D-Ala-D-Gln-L-Pro-D- Asp-L-Trp); cyclo(D-Ala-D-Gln-L-Pro-D-Glu-L-Trp-D-Ala-D-Gln); cyclo(D- Ala-D-Gln-L-Pro-D-Ser-L-Trp); cyclo(Gly-D-Val-L-Pro-D-Asp-L-Trp); cyclo(Gly-D-Val-L-Pro-D-Glu-L-Trp); cyclo(Gly-D-Val-L-Pro-D-Ser-L-Trp); cyclo(Gly-D-Gln-L-Pro-D-Asp-L-Trp); cyclo(Gly-D-Gln-L-Pro-D-Glu-L-Trp); cyclo(Gly-D-Gln-L-Pro-D-Ser-L-Trp); cyclo(Aib-D-Gln-L-Pro-D-Asp-L-Trp); cyclo(Aib-D-Gln-L- Pro-D-Glu-L-Trp); cyclo(Aib-D-Gln-L-Aib-D-Gln-L-Pro-D- Ser-L-Trp); cyclo(Aib-D-Gln-L-Pro-D-Asp-L-Trp); cyclo(Aib-D-Gln-L-Pro-D- Glu-L-Trp); and cyclo(Aib-D-Gln-L-Pro-D-Ser-L-Trp). Cyclic peptides having formula (IV): cyclo(X¹-X²-X³-X⁴-L-Trp) (IV) and pharmaceutically acceptable salts and esters and other pharmaceutically acceptable derivatives thereof in which X¹ is D-Leu, D- Val, D-Ile, D-Ala, Gly, Aib, D-Nva, D-Nle or D-Alle; X² is Val, lie, Leu, Ala, Gin, Gly, Aib, L-Nva, L-Nle or L-Alle; X³ is D-Pro, D-Hyp, D-Ala, D-Val, D- lle, Gly, Aib, D-Nva, D-Nle or D-Alle; and X⁴ is L-Asp, L-Glu, L-Tyr, L-Ser, L-Thr, L-Cys(O₃H), or L-Pen(O₃H) are provided.

Among the preferred peptides of formula (IV) and pharmaceuticaly acceptable salts and esters thereof are those in which X¹ is D-Leu, D-Val, D-Ile, or D-Ala; X² is L-Val, L-lle, L-Leu or L-Ala; X³ is D-Pro, D-Ala, D-Val or D-Ile; and X⁴ is L-Asp, L-Glu, L-Tyr or L-Ser. Peptides of formulas: cyclo(D-Leu-L-Val-D-Pro-L-Asp-L-Trp), cyclo(D-Leu-L-Val-D-Pro-L-Tyr-L- Trp), and cyclo(D-Leu-L-Val-D-Pro-L-Ser-L-Trp) are among the more preferred peptides of formula (IV). More preferred cyclic peptides are any of the cyclic peptides that inhibit binding of endothelin-1 to ET_A receptors at an IC₅₀ of less than or equal to about 100 μM, and preferably less than 50 μM and more preferably, less than 10 μM, but that do not inhibit binding of endothelin- 1 to ET_B receptors at concentrations of 100 μM or less. Such cyclic peptides include, but are not limited to: cyclo(D-Tyr-Phe-His-β-Ala-D-Trp) and cyclo(D-Asp-Ala-D-Ala-Ala-D-Trp) .

Other preferred cyclic peptides are any of the peptides that interact with ET_B receptors at a lower concentration than with ET_A receptors. Such peptides should include those of formula (III) in which X² is D-GIn. B. Synthesis of the cyclic peptides.

The cyclic peptides may be synthesized by any method for synthesis of cyclic peptides known to those of skill in the art. Such methods for peptide synthesis are known in the art. Some of the cyclic peptides have been synthesized as described in the Examples by solid phase peptide synthesis on 1 % crosslinked polystyrene with an oxime resin (see, De Grado et aL (1980) J. Org. Chem. 45: 1295-1300). The peptides were synthesized using the Boc synthesis strategy with benzyl (Bzl) type side chain protection. Activation of the amino acid derivatives was accomplished with diisopropylcarbodiimide (DIC).

Cleavage of the synthesized peptide from the resin and cyclizatiόn was effected in one step (see, Osapay et L (1991 ) Techniques in Protein Chemistry 2: 2-1 1 ; Osapay et aL (1990) Tetrahedron Lttr. 43: 6121 - 6124; and Osapay et aL (1990) J. Amer. Chem. Soc. 1 12: 6046-6051 ) in which cleavage is initiated by the free N-terminal amino group, which attacks the C-terminal carboxy group on the oxime linker. The reaction was carried out in about ten-fold excess acetic acid, which catalyzes the cyclization reaction. In instances in which solubility in the reaction mixture was low, cleavage was effected in DCM or DCM/DMF. In order to favor intrachain reaction over interchain reaction, the substitution level on the resin was low, about 0.2 mmol/g. This was accomplished by substituting the resin with fewer amino acid groups than oxime groups. Free oxime groups were capped using trimethyl acetic anhydride, which was more effective for capping than normal acetic anhydride. Side chains of the of the crude peptides were cleaved either by catalytic hydrogenation in 10% acetic acid in methanol resuspended in TFE/HFIP or by HF with thioanisole/indol. Trp was deprotected using 20% piperidine in DM. Crude deprotected cyclic peptides were purified by HPLC using a

Waters (Bedford, MA) cartridge system (25 mm x 10 cm/hr) with Novapak™ C₁₈ packing and a gradient composed of 0.1 % TFA/water and 0.1 % TFA/CH₃CN. The final products were lyophilized and analyzed by HPLC, FAB-MS and amino acid analysis. C. Evaluation of the bioactivity of the cyclic peptides

After synthesis the bioactivity of the cyclic peptides may be evaluated. Standard physiological, pharmacological and biochemical procedures are available for testing the cyclic peptides to identify the peptides that possess any biological activities of an endothelin peptide or the ability to interfere with or inhibit endothelin peptides.

(1 ) Screening Compounds for the Ability to Modulate the Activity of an Endothelin Peptide

After synthesis, the cyclic peptides may be tested for the ability to modulate the activity of endothelin-1 . Numerous assays are known to those of skili in the art for evaluating the ability of compounds to modulate the activity of endothelin (see, e^, EP 0436189 Al to BANYU PHARMACEUTICAL CO., LTD. (October 7, 1991 ); Borges et al. (1989) Eur. J. Pharm. 165: 223-230; Filep et al. (1991 ) Biochem. Biophvs. Res. Commun. 177: 171-176). In vitro studies may be corroborated with in vivo studies (see, e^, EP 0436189 A1 to BANYU PHARMACEUTICAL CO., LTD. (October 7, 1991 )) and pharmaceutical activity thereby evaluated.

The properties of a potential antagonist may be assessed as a function of its ability to inhibit an endothelin induced activity in vitro using a particular tissue, such as rat portal vein and aorta as well as rat uterus, trachea and vas deferens (see e.g.. Borges, R., Von Grafenstein, H. and Knight, D.E., Tissue selectivity of endothelin, Eur. J. Pharmacol 1_65_:223-230, (1989)). The ability to act as an endothelin antagonist in vivo can be tested in hypertensive rats, ddy mice or other recognized animal models (see, Kaltenbronn et aL (1990) J. Med. Chem. 33:838- 845, see, also EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (October 7, 1991 ); see, also Bolger et aL (1983) J. Pharmacol. Exp. Ther. 225291 -309). Using the results of such animal studies, pharmaceutical effectiveness may be evaluated and pharmaceutically effective dosages determined.

For example, endothelin activity can be identified by the ability to stimulate vasoconstriction of isolated rat thoracic aorta or portal vein ring segments (Borges et aL (1989) "Tissue selectivity of endothelin" Eur. J. Pharmacol. 165: 223-230). To perform the assay, the endothelium is abraded and ring segments mounted under tension in a tissue bath and treated with endothelin in the presence of the cyclic peptide. Changes in endothelin induced tension are recorded. Dose response curves may be generated and used to provide information regarding the relative inhibitory potency of the cyclic peptide. Other tissues, including heart, skeletal muscle, kidney, uterus, trachea and vas deferens, may be used for evaluating the effects of a particular test compound on tissue contraction.

Endothelin isotype specific antagonists may be identified by the ability of a test compound to interfere with endothelin binding to different tissues or cells expressing different endothelin-receptor subtypes, or to interfere with the biological effects of endothelin or an endothelin isotype (Takavanaαi et al. (1991 ) Reo. Peo. 32: 23-37, Panek et al. (1992) Biochem. Biophys. Res. Commun. 183: 566-571 ). For example, ET_B receptors are expressed in vascular endothelial cells, possibly mediating the release of prostacyclin and endothelium-derived relaxing factor (De Nucci et aL (1988) Proc. Natl. Acad. Sci. USA 85:9797). ET_A receptors are not detected in cultured endothelial cells, which express ET_B receptors.

The binding of compounds or inhibition of binding of endothelin to ET_B receptors can be assessed by measuring the inhibition of endothelin- 1 -mediated release of prostacyclin, as measured by its major stable metabolite, 6-keto PGF_1σ, from cultured bovine aortic endothelial cells (see, e.g.. Filep et aL (1991 ) Biochem. and Biophvs Res. Commun. 177: 171 -176). Thus, the relative affinity of the cyclic peptides for different endothelin receptors may be evaluated by determining the inhibitory dose response curves using tissues that differ in receptor subtype. A potential agonist may also be evaluated using in vitro and in vivo assays known to those of skill in the art.

Using such assays, the relative affinities of the cyclic peptides for ET_A receptors and ET_B receptors have been and can be assessed. Those that possess the desired properties, such as specific inhibition of binding of endothelin-1 , are selected. The selected cyclic peptides that exhibit desirable activities may be therapeutically useful and are tested for such use using the above-described assays from which in vivo effectiveness may be evaluated. Cyclic peptides that exhibit activities that correlate with in vivo effectiveness will then be formulated in suitable ^• pharmaceutical compositions and used without further modification.

The cyclic peptides also may be used in methods for identifying and isolating endothelin-specific receptors and as models "for the design of peptidomimetics. (2) Identification and Isolation of Endothelin Receptors

Methods for identifying and isolationg endothelin receptors are provided. In practicing one such method, one or more of the cyclic peptides is linked to a support and used in methods of affinity purification of receptors. By selecting the cyclic peptides with particular specificities, distinct subclasses of ET receptors may be identified.

One or more of the cyclic peptides may be linked to an appropriate resin, such as Affi-gel, covalently or by other linkage, by methods known to those of skill in the art for linking endothelin to such resins (see, Schvartz et al. (1990) Endocrinology 126: 3218-3222). The linked peptides can be those that are specific for ET_A or ET_B receptors or other subclass of receptors. For example, peptides of the formula cyclo(X¹-X²- L-Pro-X -L-Trp) in which X¹ is D-Ala, Aib, or Gly, X⁴ is D-Asp, D-Glu, or D-Ser, and X² is D-GIn should be ET_B specific and may be used for identification of receptors that have binding properties similar to ET_B receptors.

The resin is pre-equilibrated with a suitable buffer generally at a physiological pH (7 to 8). A composition containing solubilized receptors from a selected tissue are mixed with the resin to which the cyclic peptide is linked and the receptors are selectively eluted. The receptors can be identified and further characterized by testing them for binding to an endothelin isopeptide or analog or by other methods by which proteins are identified and characterized. Preparation of the receptors, the resin and the elution method may be performed by modification of standard protocols known to those of skill in the art (see, e.g., Schvartz et aL (1990) Endocrinology 126: 3218-3222).

A method for distinguishing specificities among endothelin receptors is also provided. In particular, ET_A and ET_B receptors may be identified by comparing the binding affinity of each receptor for the peptides of formula cyclo(X¹-X²-L-Pro-X⁴-L-Trp) in which X¹ is D-Ala, Aib, or Gly; X⁴ is D-Asp, D-Glu, or D-Ser, and X² is D-GIn with the binding affinity of such receptors for peptides in which X² is D-Val, D-Leu, D-Ile or D-Ala. Peptides in which X² is D-Val, D-Leu, D-Ile or D-Ala should preferentially interact with ET_A receptors compared to ET_B receptors. Peptides in which X² is Gin should preferentially interact with ET_B receptors.

Other methods for distinguishing receptor type based on differential affinity to any of the compounds provided herein are provided. Any of the assays described herein for measuring the affinity of selected compounds for endothelin receptors may also be used to distinguish receptor subtypes based on affinity for particular compounds provided herein. In particular, ET_A and ET_B receptors may be identified by measuring the binding affinity of the unknown receptor for a compound provided herein that has a known affinity for one receptor over the other. Such preferential interaction is useful for determining the particular disease that may be treated with a compound prepared as described herein. For example, compounds with high affinity for ET_A receptors and little or no affinity for ET_B receptors are candidates for use as hypertensive agents; whereas, compounds that preferentially interact with ET_B receptors are candidates for use as anti-asthma agents. Thus, by identifying receptors that selectively bind to cyclic peptides, new subclasses of endothelin receptors may be identified. Such information may then be used in the design of disease-specific analogs and peptidomimetics. D. Design and analysis of the biological activity of the peptidomimetics Peptidomimetic compounds, based on the structure and activity of previously evaluated cyclic peptides or chemically modified analogs thereof, may be designed by replacing residues of the cyclic peptides or endothelin analogs with residues that are bioisosteric with respect to the replaced residues. For example, by reference to available data bases, such as the Cambridge crystallographic data base, replacement residues which are bioisosteric with various residues of the compound of interest can be identified and used to replace the native residue in the compound of interest. Those of skill in the art can identify suitable bioisosteric moieties which can be used in place of the naturally occurring amino acid residues.

Identification of any flexible portions of the structure that should be replaced with suitable rigid or conformationally constrained bioisostere(s) is an important consideration in designing the peptidomimetic. Any portions or sections of the structure subject to degradation when the analog is administered may also be replaced with bioisosteres or equivalents that are not readily biologically degraded, and that maintain the desired binding between target peptides and receptors or peptidomimetics. Selected replacements will depend upon the mode of administration, which includes, oral administration, inhalation, topical application, intramuscular injection, intravenous injection, subcutaneous injection and other modes of administration known to those of skill in this art. Oral administration and parenteral administration are preferred herein. In addition, various substituents on the amide nitrogen and the σ-carbon can be bound to one another to form a cyclic structure to produce a constrained analog. Other constrained, cyclic structures may also be produced by linking other substituents. Since the replacement residues and the bonds in the constrained cyclic structures should not be recognized by the enzymes that degrade naturally occurring proteins, the chemically modified analogs typically are much more resistant to enzymatic cleavage than are the unmodified peptides from which they are derived. In addition, the wide range of possible replacement groups which can be used to modify the backbone and side' chains of peptides affords the opportunity to reduce the conformational flexibility of the parent structure. Thus, the possibility that the peptide will adopt conformation(s) other than the specifically desired conformation(s) can be substantially minimized by appropriate modification of the peptide. Once the desired analog, including backbone and side chain modification, as appropriate, has been identified, chemical synthesis using standard synthetic techniques will be undertaken. For a given analog, the skilled artisan can identify suitable synthetic approaches for the preparation of the peptidomimetic. E. Formulation of pharmaceutical compositions Effective amounts of one or more of the cyclic peptides of formulas

(l)-(IV) or pharmaceutically acceptable salts, esters or other derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle to produce pharmaceutical compositions.. The resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The amounts or concentrations are effective for delivery of an amount, upon administration, that ameliorates the symptoms of the endothelin-mediated disease. Typically, the compositions are formulated for single dosage administration. The effective amount is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined. Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

The active compounds can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Preferred modes of administration include oral and parenteral modes of administration. The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of serious toxic effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems (see, e^, EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (October 7, 1991 ); Borges et aL (1989) Eur. J. Pharm. 165: 223-230; Filep et aL (1991 ) Biochem. Biophvs. Res. Commun. 177: 171-176).

The concentration of active compound in the drug composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

Typically a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml/kg to about 50-100 μg/ml/kg body weight. The pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 2000 mg of compound per kilogram of body weight per day. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to' be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the needs of the treated individual and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. For example, if the compound is used for treating asthma or hypertension, it may be formulated with other bronchodilators and antihypertensive agents, respectively.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Patent No. 4,522,81 1 .

The active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, poiyorthoesters, polylactic acid and others. Methods for preparation of such formulations are known to those skilled in the art.

The compounds may be formulated for local or topical application, such as for topical application to the skin in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, may be formulated as 0.01 % - 10% isotonic solutions, pH about 5- 7, with appropriate salts. The compounds may be formulated as aeorsols for topical application, such as by inhalation (see, e.g.. U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma).

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES Synthesis of endothelin analogs The following endothelin analogs were synthesized:

⁽1⁾ cyclo(D-Tyr-Phe-D-His-Gly-D-Trp); (2) cyclo(D-Tyr-Phe-D-His-β-Ala-D-Trp); (3) cyclo(D-Glu-Ala-D-Val-Leu-D-Trp); (4) cyclo(D-Tyr-Phe-Gly-D-His-Gly-D-Trp); (5) cyclo(D-Asp-Pro-D-Val-Leu-D-Trp); (6): cyclo(D-Asp-Ala-D-Ala-Ala-D-Trp); (7): cyclo(D-Tyr-Ala-D-Ala-Ala-D-Trp); and (8): cyclo(D-Leu-Val-D-Pro-Asp-Trp)

Compounds (3) and (5_), which have activity as endothelin antagonists, were synthesized for use as controls (see, EP 0436189 A1 to BANYU PHARMACEUTICAL CO., LTD. (October 7, 1991 )).

EXAMPLE 1 Synthesis of cyclo(D-Tyr-Phe-D-His-Gly-D-Trp) (1) A. Preparation of Boc-D-Trp(For)-oxime resin. One percent crosslinked polystyrene substituted with the oxime linker (2.01 g) was preswollen in 30 ml DCM for 30 min in a 60 ml reaction vessel. Boc-D-Trp(For)-OH (0.13 g, 0.4 mmol) and DCC (0.082 g, 0.4 mmol) were dissolved in 10 ml DCM and stirred for 30 min at room temperature. The solution was removed and the resin was washed as follows: 2 x 10 ml DMF/DCM (1 :1 ), 2 x 10 ml DCM, 2 x 10 ml ethanol/DCM (1 :1 ), and 2 x 10 ml DCM. The remaining free oxime groups were capped with 3.0 ml (14.8 mmol) trimethyl acetic anhydride and 1.0 ml (5.74 mmol) DIEA in 1 1 ml DCM at room temperature overnight, after which the resin was washed with 4 x 20 ml DCM, 2 x 20 ml ethanol and then dried. The yield was 2.17 g.

Substitution was determined using the picric acid test (Steward et al. (1984) in Solid Phase Peptide Synthesis, Freeman, W.H., Pierce, NY, pages 105-122). The test was performed in triplicate in three separate glass filter funnels. About 20 mg of resin was introduced into each glass filter funnel and washed with 2 x 1 ml DCM, followed by 1 min cleavage with 1 ml 25% TFA in DCM and 20 min cleavage with 1 ml 25% TFA in DCM. The filter was washed with 2 x 1 ml DCM, neutralized with 1 ml 5% DIEA in DCM, washed with 2 x 1 ml DCM, neutralized with 1 ml 5% DIEA in DCM, and washed. with 3 x 1 ml DCM. Picric acid (3 x 1 ml 0.1 M (2.29 g/100 ml DCM)) was adsorbed to the material in the filter funnels, washed with 6 x 1 ml DCM, desorbed with 3 x 1 ml 5% DIEA, and the resulting solution was collected. The material in the funnels was washed with 3 x 1 ml DCM and the filtrate combined with the collected solution. The resulting solution was diluted to 100 ml with 95% ethanol and the absorbance(OD) at 358 nm (e = 14,500) was measured.

The amount of substitution (nmol/g) = (OD x volume (ml))/(e x weight (g)) = 0.1 195 nmol/g for the Boc-D-Trp(for)-oxime resin.

B. Preparation of D-Tyr(BrZ)-Phe-D-His(Bom)-Gly-D-Trp(For) oxime resin.

The D-Trp(For)-oxime resin was washed with 2 x 10 ml DCM, followed by 1 min cleavage with 1 x I0 ml 25% TFA in DCM and 30 min cleavage with 1 x 10 ml 25% TFA in DCM. The resulting material was washed with Z. x 10 ml DCM, 1 x 10 ml isopropanol, 2 x 10 ml DCM, 1 x 10 ml isopropanol, 2 x 10 ml DCM, neutralized with 3 x 10 ml 5% DIEA in DCM, and washed with 4 x 10 ml DCM. The material was then coupled for 60 min with 3 eq Boc-aa-OH, in which aa is the next amino acid in the chain. In this instance, Gly. Prior to coupling, the Boc-aa-OH had been preactivated by treatment for 30 min with 3 eq DIC in 10 ml DCM. The resin was washed with 2 x 10 ml DMF/DCM (1 : 1 ), 2 x 10 ml DCM, 2 x 10 ml ethanol/DCM (1 :1 ), and 2 x 10 ml DCM. About 5 mg of the resin was collected for a ninhydrin test. The remainder was acetylated for 60 min with 3 eq trimethylacetic anhydride/2.2 eq DIEA in 10 ml DCM and washed with 4 x 10 ml DCM. To couple the third residue, D-His in this instance, the above proceedure was repeated, except that the last neutralization step and subsequent washings were eliminated. Five eq, rather than 3 eq of protected and activated amino acid were added to the resin. The base (1.2 eq DIEA) was added to the resin after the protected and activated amino acid were added. No HOBt was added. The last two amino acid residues, L-Phe and D-Tyr in this instance, were sequentially coupled to the peptide, with appropriate protecting groups, where necessary, using the above procedure.

C. Cyclization of the Peptide on Oxime Resin.

The product of step B (1.1 g, 0.132 mmol) was placed in a solid phase reaction vessel with 15 ml DCM for 30 min. The Boc group was cleaved, as described in the synthesis protocol (Example 1.B.), after which 68 μ\ (0.12 mmol) of acetic acid in 15 ml DCM was added to the resin and the resulting mixture was shaken at room temperature for 15 hours. The solution was then filtered and collected. The resin was washed with 4 x10 ml DCM, 2 x 10 ml DCM, 2 x10 ml isopropanol and 2 x 10 ml DCM. The filtrate and washes were combined and evaporated. The concentrate was added dropwise to 50 ml dry and cold ether. The colorless precipitate was collected by centrifugation, the solution decanted and the residue washed with 2 x 30 ml ether. The crude peptide was then purified by HPLC useing a gradient composed of 0.1 % TFA in water and 0.1 % TFA in CH₃CN.

D. Removal of side chain protective groups

To cleave the formyl group from Trp, the crude cyclic pepide was stirred in 20% piperidine in DMF at room temperature for 1 hr. The solvent was evaporated and the residue was dried. The Tyr, Asp and His side chain protective groups, Brz, Bzl, and Bom, respectively, were removed by catalytic hydrogenation.

Catalytic hydrogenation was typically accomplished by dissolving the peptide in3 ml HFIP and diluting with 3 ml TFE. Pd/C catalyst was added and the mixture was hydrogenolyzed at room temperature for 15 hours. The catalyst was removed by filtration over cellite and washed with 3 x 1 ml TFE and 5 x 2 ml methanol. The solution was concentrated to about 1 ml and added dropwise to 35 ml ice cold dry ether. The precipitated peptide was collected by centrifugation, the solution decanted and the residue was washed with 2 x 30 ml ether. The product was dried, diluted with 1 ml HFIP and water and then lyophilzed. The final product, cyclo(D-Tyr-Phe-D-His-Gly-D-Trp) was purified by HPLC as decribed in step C above. For a typical procedure, the overall yield was 2.3%.

FAB-MS: m/z 691 (Theoretical m/z: 690.79)

Amino acid analysis: Tyr (1.08), Phe (0.99), His (1 .00), Gly

(0.92) Trp not determined.

EXAMPLE 2 Synthesis of cyclo(D-Tyr-Phe-D-His-β-AIa-D-Trp) (2)

Using the method described in Example 1 , cyclo(D-Tyr-Phe-D-His-β- Ala-D-Trp) was prepared.

FAB-MS: m/z 705 (Theoretical m/z: 704.81 ) Amino acid analysis: Tyr (0.96), Phe (0.99), His (1.05), Trp and β-Ala not determined.

EXAMPLE 3 Synthesis of cyclo(D-Glu-Ala-D-Val-Leu-D-Trp) (3)

Using the method described in Example 1 , cyclo(D-Glu-Ala-D- Val- Leu-D-Trp) was synthesized. FAB-MS: m/z 599 (Theoretical m/z: 598.72)

Amino acid analysis: Glu (1.02), Ala (1.01 ), Val (0.95), Leu (1.01 ), Trp not determined.

EXAMPLE 4 Synthesis of Cyclo(D-Tyr-Phe-Gly-D-His-Gly-D-Trp) (4) Using the method described in Example 1 , cyclo(D-Tyr-Phe-Gly-D-

His-Gly-D-Trp) was synthesized with a 1 .5% overall yield. FAB-MS: m/z 748 (Theoretical m/z: 747.85) Amino acid analysis: Tyr (1.02), Phe (1 .05), Gly (1 .76), His (1.18), Trp not determined. EXAMPLE 5 Synthesis of cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (5)

Using the method described in Example 1 , cyclo(D-Asp-Pro-D-Val- Leu-D-Trp) was synthesized and resulted in a good overall yield. FAB-MS: m/z 61 1 (Theoretical m/z: 610.74)

Amino acid analysis: Asp (1.06), Pro (1.07), Val (0.91 ), Leu (0.97), Trp not determined.

EXAMPLE 6 Synthesis of cyclo(D-Asp-Ala-D-Ala-Ala-D-Trp) (6) Using the method described in Example 1 , cyclo(D-Asp-Ala-D-Ala-

Ala-D-Trp) was synthesized.

FAB-MS: m/z 515 (Theoretical m/z: 514.56) Amino acid analysis: Asp (1.01 ), Ala (2.99), Trp not determined. EXAMPLE 7

Synthesis of cyclo(D-Tyr-Ala-D-Ala-Ala-D-Trp) (7)

Using the method described in Example 1 , cyclo(D-Tyr-Als-D-Ala- Ala-D-Trp) (7) was synthesized to produce a 5.5% overall yield. FAB-MS: m/z 563 (Theoretical m/z: 562.65) Amino acid analysis: Tyr (1.02), Ala (2.98), Trp not determined.

EXAMPLE 8 Synthesis of cyclo(D-Leu-L-Val-D-Pro-L-Asp-L-Trp) (8) A. Preparation of Boc-Trp-oxime resin One percent crosslinked polystyrene substituted with the oxime linker (2.67 g = 2.0 mmol) was preswollen in 60 ml DCM for 30 min. in a 60 ml reaction vessel. Boc-Trp-OH (1.216 g, 4.0 mmol) and DCC (0.825 g, 4.0 mmol) were dissolved in 60 ml DCM and stirred for about 15.5 hrs. at room temperature; The solution was removed and the resin was washed as follows: 2 x 40 ml DMF/DCM (1 :1 ), 2 x 40 m DCM, 2 x 40 ml ethanol/DCM (1 :1 ), and 2 x 40 ml DCM. The remaining free oxime groups were capped with 4.0 ml (19.71 mmol) trimethyl acetic anhydride and 1.3 ml (7.4 mmol) DIEA in 1 1 ml DCM at room temperature overnight, after which the resin was washed with 4 x 40 ml DCM, 2 x 25 ml ethanol and then dried. The yield was 3.3 g.

Substitution was determined using the picric acid test [Steward and Young (1984) in Solid Phase Peptide Synthesis. Freeman, W.H., Pierce, NY, pp. 105-122]. The test was performed in duplicate in three separate glass filter funnels. About 20 mg of resin (accurately weighed) was introduced into each glass filter funnel and washed with 2 x 1 ml DCM, followed by 1 min. cleavage with 1 ml 25% THF in DCM and 20 min. cleavage with 1 ml 25% TFA in DCM. The filter was washed with 2 x 1 ml DCM, neutralized with 1 ml 5% DIEA in DCM for 3 min., washed with 2 x 1 ml DCM, neutralized again with 1 ml 5% DIEA in DCM for 3 min., and washed with 3 x 1 ml DCM. Picric acid (3 x 1 ml 0.1 M (2.29 g/100 mi DCM) was absorbed to the material in the filter funnels, washed with 6 x 1 ml DCM, desorbed with 3 x 1 m. 5% DIEA, and the resulting solution was collected. The material in the funnels was washed with 3 x 1 ml DCM and the filtrate combined with the collected solution. The resulting solution was diluted to 100 ml with 95% ethanol and the absorbance(OD) at 358 nm ε = 14,500) was measured.

The amount of substitution (nmol/g) = (OD x volume (ml)/ε x weight (g)) = 0.4075 nmol/g for the Boc-Trp-oxime resin. B. Preparation of (D-Leu-Val-D-Pro-Asp-Trp) oxime resin.

The Trp-oxime resin (1 g, 0.406 mmol) was washed with 2 x 10 ml DCM, followed by 1 min cleavage with 1 x 10 ml 25% TFA in DCM. The resulting material was washed with 2 x 10 ml DCM, 1 x 10 ml isopropanol, 2 x 10 ml DCM, 1 x 10 ml isopropanol, 2 x 10 ml DCM, neutralized with 3 x 10 nl 5% DIEA in DCM, and washed with 4 x 10 ml DCM. The material was then coupled for 60 min. with 3 eq of Asp (OBzl) which was preactivated by treatment for 30 min. with 3 eq DIC in 10 ml DCM. The resin was washed with 2 x 10 ml DMF/DCM (1 : 1 ), 2 x 10 ml DCM, 2 x 10 ml ethanol/DCM (1 :1 ), and 2 x 10 ml DCM. About 5 mg of the resin was collected for a ninhydrin test. The remainder was acylated for 60 min. with 3 eq trimethylacetic anhydride/2.2 eq DIEA in 10 ml DCM and washed with 4 x 10 ml DCM.

To couple the third residue, D-Pro, the above procedure was repeated, except that the neutralization step and subsequent washings were eliminated. Five eq, rather than 3 eq of preactivated protected amino acid were added to the resin. The base (1.2 eq DIEA) was added to the resin after the protected and activated amino acids were added. No HOBt was added. L-Val and D-Leu residues were sequentially coupled to the peptide using the above procedure. C. Cyclization of the Peptide on Oxime Resin.

One half of the product of step B (0.631 g, 0.256 mmol) was placed in a solid phase reaction vessel with 20 ml DCM for 30 min. The Boc group was cleaved as described in Example 1 .B after which 146.6μl of acetic acid in 20 ml DCM was added to the resin and the resulting mixture was shaken at room temperature for 21 hrs.

The solution was then filtered and collected. The resin was washed 4 x 10 ml DCM, 2 x 10 ml DCM, 2 x 10 ml isopropanol and 2 x 10 ml DCM. The filtrate and washes were combined and evaporated. The crude peptide was then purified on Silica gel column using 25:5 CH₂CI₂/MeOH.

D. Removal of side chain protective groups The side chain Bzl protective group on Asp was moved by catalytic hydrogenation. The peptide was dissolved in 3 ml MeOH. Pd/C catalyst was added and the mixture was hydrogenolyzed at room temperature for 2 hrs. The catalyst was removed by filtration, washed with 5 x 2 ml nriethanol. The solution was concentrated to dryness. The final product, cyclo(D-Leu-Val-D-Pro-Asp-Trp) was purified by HPLC as described in step C above and the overall yield was 24.1 %.

EXAMPLE 9 The synthetic peptides exhibited endothelin antagonistic activity A. Assays

1. Endothelin Binding Inhibition Test #1. Ventricles from 4-day old rats were removed and rinsed in PBS. The tissue was minced with scissors, suspended in a solution of 0.1 % collagenase in Dulbecco's Modified Eagles Medium (MEDIA) and incubated for 15 min at 37°C in a shaking water bath. The tissue was then triturated, the dispersed cells removed and added to MEDIA containing 10% Fetal calf serum (HEART MEDIA). Fresh collagenase solution was added to the tissue and the above process was repeated three times. The dispersed cells were pooled, washed three times in heart media, placed in a T-75 tissue culture dish and incubated for one hour at 37°C in an atmosphere of 5% CO₂. The flask was tapped gently several times and the media, containing mainly cardiocytes, removed and centrifuged at 30,000 X g. The resulting pellet was resuspended in ultra pure water containing aprotinin (100 KlU/ml) and was homogenized using a Dounce homogenizer fitted with a loose fitting pestle. The cell/membrane suspension was frozen and thawed once and then recentrifuged at 30,000 X g for 10 minute. The resultant membranes were resuspended in 30 mM HEPES buffer, pH 7.4, containing aprotinin (100 KlU/ml) to give a protein concentration of 5 mg/ml and stored at -70°C until use.

Two μ\ of this membrane suspension were added to 98 μ\ of binding buffer (30 mM HEPES buffer, pH 7.4 containing 150 mM NaCI, 5 mM MgCI₂, 0:5% Bacitracin, 0.1 % BSA). Fifty /vl of (A) endothelin-1 (to measure non specific binding: to give a final concentration 80 nM), (B) binding buffer (to measure total binding), or (C) a test compound (final concentration 1 nM to 100 μM) was added to the membrane suspension.

Each mixture was shaken and incubated at 25 °C for 60 min. and 50 μ\

¹²⁵l-endothelin-1 (3,000 cpm) was added. Each mixture was shaken, incubated at 4°C for 16 hours and centrifuged at 4°C for 25 min at

2,500 X g. The supernatant, containing unbound radioactivity, was decanted and the pellets counted on a Genesys multiwell gamma counter.

The degree of inhibition of binding (D) was calculated according to the following equation: (C) - (A)

% D = 100 X 100

(B) - (A)

Each test was performed in triplicate.

2. Endothelin Binding Inhibition Binding Test #2: Inhibition of binding to ET_B receptors

COS-7 cells were transfected with DNA encoding the ET_B receptor.

Transfected cells that expressed the human ET_B receptor were grown to confluence in T-175 flasks. Cells from multiple flasks were collected by scraping, pooled and centrifuged for 10 min. at 190 X g. The cells were resuspended in phosphate buffered saline (PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. The suspension was centrifuged at 4°C at 57,800 X g for 15 min, the pellet was resuspended in 5 ml of buffer A (5mM HEPES buffer, pH 7.4 containing aprotinin (100 KlU/ml)) and then frozen and thawed once. Five ml of buffer B (5 mM HEPES Buffer, pH 7.4 containing 10 mM MnCI₂ and 0.001 % deoxyribonuclease Type 1 ) was added, the suspension mixed by inversion, incubated at 37°C for 30 min. and centrifuged at 57,800 X g as described above. The pellet was washed twice with buffer A and then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containing aprotinin (100 KlU/ml) to give a final protein concentration of 2 mg/ml. The binding assay was performed and the degree of inhibition of binding was calculated as described above for Test #1 , except that the mixture was diluted to a final concentration of 1 μg protein/100 μ\ of binding buffer. 3. Endothelin Binding inhibition Binding Test #3: Inhibition of binding to ET_A receptors

TE 671 cells (ATCC Accession No. HTB 139) were transfected with DNA encoding ET_A receptors. The resulting transfected cell lines, which express ET_A receptors on the cell surfaces, were grown to confluence in T-175 flasks. Cells from multiple flasks were collected by scraping, pooled and centrifuged for 10 min at 190 X g. The cells were resuspended in phosphate buffered saline (PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. The suspension was centrifuged at 4°C at 57,800 X g for 15 min, the pellet was resuspended in 5 ml of buffer A (5mM HEPES buffer, pH 7.4 containing aprotinin (100 KlU/ml)) and then frozen and thawed once. 5 ml of Buffer B (5 mM HEPES Buffer, pH 7.4 containing 10 mM MnCI₂ and 0.001 % deoxyribonuclease Type 1 ) was added, the suspension mixed by inversion and then incubated at 37°C for 30 minutes. The mixture was centrifuged at 57,800 X g as described above, the pellet washed twice with buffer A and then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containing aprotinin (100 KlU/ml) to give a final protein concentration of 2 mg/ml and stored at -70°C until use.

The membrane suspension was diluted with binding buffer (30 mM HEPES buffer, pH 7.4 containing 150 mM NaCI, 5mM MgCI₂, 0.5% Bacitracin, 0.1 % BSA) to a concentration of 6 μg/100 μ\. To this suspension 50μi of (A) endothelin-1 (for non specific binding: to give a final concentration 80 nM), (B) binding buffer (for total binding), or (C) a test compound (final concentration 1 nM to 100 μM) were added. . Mixtures were shaken and incubated at 25° C for 60 minutes prior to the addition of 50 μl ¹²⁵I-ET-1 (3,000 cpm). Mixtures were shaken, incubated at 4° C for 16 hours and centrifuged at 4° C for 25 min at 2,500 X g.

The supernatant, containing unbound radioactivity, was decanted and the pellet counted on a Genesys multiwell gamma counter. The degree of inhibition of binding (D) was calculated as described above. 4. Test for activity against endothelin-induced contraction of isolated rat thoracic arterial rings

Compounds to be tested were prepared as 100 μM stocks. If necessary to effect dissolution, the compounds are first dissolved in a minimum amount of DMSO and diluted with 150 mM NaCI. Because DMSO can cause relaxation of the aortic ring, control solutions containing varying concentrations of DMSO were tested.

The thoracic portion of the adult rat aorta was excised, the endothelium abraded by gentle rubbing and then cut into 3 mm ring segments. Segments were suspended under a 2 g preload in a 10 ml organ bath filled with Krebs'- Henseleit solution saturated with a gas mixture of 95% O₂ and 5% CO₂ (1 18 mM NaCI, 4,7 mM KCI; 1 .2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 2.5 mM CaCI₂, 10 mM D- glucose) gassed with 95% O₂/5% CO₂. Changes in tension were measured isometrically and recorded using a Grass Polygraph coupled to a force transducer.

Endothelin was added to the organ bath in a cumulatively increasing manner, and the effects of the test compounds on the concentration-response curve for endothelin-1 were examined.

Compounds were added 15 min prior to the addition of endothelin-1 . 5. A second assay for identifying compounds that have antagonistic activity against ET_B receptors

Since endothelin-1 stimulates the release of prostacyclin from cultured bovine aortic endothelial cells, the cyclic peptides are screened for their ability to inhibit endothelin-1 induced prostacyclin release from such endothelial cells by measuring 6-keto PGF_1σ substantially as described by (Filep et al. (1991 ) Biochem. Biophvs. Res. Commun. 177 171-176. Bovine aortic cells are obtained from collagenase-treated bovine aorta, seeded into culture plates, grown in Medium 199 supplemented with heat inactivated 15% fetal calf serum, and L- glutamine (2 mM), penicillin, streptomycin and fungizone, and subcultured at least four times. The cells are then seeded in six-well plates in the same medium. Eight hours before the assay, after the cells reach confluence, the medium is replaced. The cells are then incubated with a) medium alone, b) medium containing endothelin-1 (10 nM), c) cyclic peptide alone, and d) cyclic peptide + endothelin-1 (10 nM). After a 15 min incubation, the medium is removed from each well and the concentrations of 6-keto PGF_1<7 are measured by a direct immunoassay. Prostacyclin production is calculated as the difference between the amount of 6-keto PGF_1α released by the cells challenged with the endothelin-1 minus the amount released by identically treated unchallenged cells. Cyclic peptides which stimulate 6-keto PGF_1σ release possess agonist activity and those which inhibit endothelin-1 6-keto PGF₁₍₇ release possess antagonist activity. B. Assay Results

1. The test results from binding assay # 1 are set forth in Table 1 : TABLE 1

2. The test results from binding assay # 2 are set forth in Table 2:

TABLE 2

3. Inhibition of Endothelin-1 induced contraction

Compounds (6) cyclo(D-Asp-Ala-D-Ala-Ala-D-Trp) and (7) cyclo(D- Tyr-Ala-D-Ala-Ala-D-Trp) showed no agonistic activity in the contraction assay.

Compound (6) cyclo(D-Asp-Ala-D-Ala-Ala-D-Trp) at a concentration of 10 μM caused 75% inhibition of the contraction induced by 100 nM endothelin-1. Compound (7) cyclo(D-Tyr-Ala-D-Ala-Ala-D-Trp) at a concentration of 10 μM caused a 75% inhibition of the contraction induced by 100 nM endothelin-1 .

Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims

WE CLAIM:

1. A substantially pure peptide formula (I): cyclo(X¹-X²-X³-X⁴-D-Trp) (I) or pharmaceutically acceptable salts or esters thereof, wherein: X¹ is D-Tyr or D-Asp; X² is Phe, Ala or Pro; X³ is D-His, D-Ala, D-Val, Gly, β-Ala; and X⁴ is D-His, L-His, Ala, β-Ala, Aib, Gly, D-His-gly or D-Leu, provided that the peptide is not cyclo(D-Asp-Ala-Ala-D-Trp).

2. A peptide of claim 1 selected from group consisting of cyclo(D-Tyr-Phe-D-His-Gly-D-Trp); cyclo(D-Tyr-Phe-D-His-β-Ala-D-Trp); cyclo(D-Tyr-Phe-Gly-D-His-Gly-D-Trp); cyclo(D-Tyr-Phe-β-Ala-D-His-D-Trp); cyclo(D-Tyr-Ala-D-Ala-Ala-D-Trp); cyclo(D-Asp-Ala-D-His-Ala-D-Trp); cyclo(D-Asp-Ala-D-His-Leu-D-Trp); cyclo(D-Asp-Ala-D-Val-Aib-D-Trp); cyclo(D-Asp-Pro-D-Ala-Aib-D-Trp); and cyclo(D-Tyr-Phe-Gly-D-His-Gly-D-

Trp).

3. The peptide of claim 1 , which is cyclo(D-Tyr-Ala-D-Ala-Ala-D-

Trp) or cyclo(D-Tyr-Phe-D-His-β-Ala-D-Trp).

4. A substantially pure peptide of formula II: cyclo(X -L-Phe-X³-X⁴-X⁵) (II) or pharmaceutically acceptable salts or thereof, wherein: X¹ is D-Tyr, D- Asp or D-Glu; X³ is His, D-His, β-Ala-D-His or gly-D-His; X⁴ is Gly, D-His or β-Ala; and X⁵ is D-Trp or N-Me-D-Trp.

5. A substantially pure peptide of claim 4, which is selected from the group consisting of cyclo(X-L-Phe-His-Gly-D-Trp); cyclo(X-L-Phe-D- His-β-Ala-D-Trp); cyclo(X-L-Phe-Gly-D-His-Gly-D-Trp); cyclo(X-L-Phe-β-Ala- D-His-Gly-D-Trp); cyclo(X-L-Phe-D-His-Gly-N-Me-D-Trp); cyclo(X-L-Phe-β- Ala-D-His-Gly-D-Trp); and cyclo(D-Asp-Ala-D-Val-Aib-σ-Me-D-Trp), wherein X is D-Tyr, D-Asp or D-Glu and N-Me-Trp is N-CH₃-D-Trp.

6. A substantially pure peptide of formula (III) cyclo(X¹-X²-X³-X -L-Trp) (III) or pharmaceutically acceptable salts or pharmaceutically acceptable esters thereof, wherein: X¹ is D-Ala, Aib, Gly, D-Val, D-Leu, D-Ile, D-Nva, D-Nle or D-Alle; X² is D-Val, D-Leu, D-Ile, D-Ala, D-GIn, Gly, Aib, D-Nva, D-Nle or D-Alle; X³ is L-Pro, Gly, Aib, L-Val, L-Leu, L-Nva, L-Nle, L-Alle or L-Hyp; and X⁴ is D-Asp, D-Glu, D-Ser, D-Thr, D-Tyr, D-Cys(O₃H) or D- Pen(O₃H).

7. The substantially pure peptide of claim 6 or pharmaceutically acceptable salts or esters thereof, wherein: X¹ is D-Ala, Aib or Gly; X² is D-Val, D-Leu, D-Ile, D-Ala or D-GIn; and X⁴ is D-Asp, D-Glu or D-Ser.

8. The peptides of claim 6, wherein X² is D-GIn.

9. The peptides of claim 6, wherein X² is D-Val, D-Leu, D-Ile or D- Ala.

10. A substantially pure peptide of formula (IV): cyclo(X¹-X²-X³-X⁴-L-Trp) (IV) or pharmaceutically acceptable salts and esters thereof, wherein: X¹ is D-Leu, D-Val, D-Ile, D-Ala, Gly, Aib, D-Nva, D-Nle or D-Alle; X² is L-Val, L-lle, L-Leu, L-Ala, L-GIn, L-Gly, Aib, L-Nva, L-Nlle or L-Alle; X³ is D-Pro, D-Hyp, D-Ala, D-Val, D-Ile, Gly, Aib, D-Nva, D-Nle or D-Alle; and X⁴ is L- Asp, L-Glu, L-Tyr, L-Ser, L-Thr, L-Cys(O₃H) or L-Pen(O₃H).

1 1 . The substantially pure peptide of claim 10 or pharmaceutically acceptable salts or esters thereof, wherein: X¹ is D-Leu, D-Val, D-Ile or D-Ala; X² is L-Val, L-lle, L-Leu or L-Ala; X³ is D-Pro, D-Ala, D-Val or D-Ile; and X⁴ is L-Asp, L-Glu, L-Tyr or L-Ser

12. A peptide of any of claims 1 -1 1 , wherein said peptide is an endothelin antagonist that inhibits the interaction of endothelin with an endothelin receptor with an IC₅₀ of about 100 μM or less.

13. A pharmaceutical composition for the treatment of endothelin- mediated disorders formulated for administration in single doses that comprise an effective amount of one or more cyclic peptides of any of claims 1 -12 or pharmaceutically acceptable salts or esters of the cyclic peptides of any of claims 1 -12 in a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for the treatment of hypertension, pulmonary hypertension, cardiovascular disease, bronchoconstriction, asthma, inflammatory diseases, ophthalmologic disease, gastroenteric disease, renal failure, endotoxin shock, anaphylactic shock, or hemorrhagic shock, menstrual disorders, obstetric conditions, erythropoietin-mediated vasoconstriction, and wounds, formulated for administration in single dosages that comprise an effective amount of one or more cyclic peptides of any of claims 1 -12 or pharmaceutically acceptable salts or esters of the peptides of claims 1 -12 in a pharmaceutically acceptable carrier, wherein the amount is effective for ameliorating the symptoms of hypertension, pulmonary hypertension, cardiovascular disease, bronchoconstriction, asthma, inflammatory diseases, ophthalmologic disease, gastroenteric disease, renal failure, endotoxin shock, anaphylactic shock, hemorrhagic shock, menstrual disorders, obstetric conditions, erythropoietin-mediated vasoconstriction, or wounds.

15. A pharmaceutical composition for the treatment of hypertension, formulated for administration in single dosages that comprises an effective amount of one or more cyclic peptides of any of claims 1 -12 or pharmaceutically acceptable salts or esters of the cyclic peptides of any of claims 1-12 in a pharmaceutically acceptable carrier, wherein the amount is effective for reducing blood pressure.

16. A pharmaceutical composition for the treatment of endotoxin shock formulated for administration in single dosages that comprise an effective amount of one or more cyclic petides of any of claims 1-12 or pharmaceutically acceptable salts or esters of the cyclic peptides of any of claims 1 -12 in a pharmaceutically acceptable carrier, wherein the concentration is effective for ameliorating the symptoms of endotoxin shock.

17. A method for the treatment of hypertension, pulmonary hypertension, cardiovascular disease, bronchoconstriction, asthma, inflammatory diseases, ophthalmologic disease, gastroenteric disease, renal failure, endotoxin shock, anaphylactic shock, or hemorrhagic shock, menstrual disorders, obstetric conditions, erythropoietin-mediated vasoconstriction, or wounds comprising administering an effective amount of the pharmaceutical composition of any of claims 13-16 to an individual experiencing hypertension, pulmonary hypertension, cardiovascular disease, bronchoconstriction, asthma, inflammatory diseases, ophthalmologic disease, gastroenteric disease, renal failure, endotoxin shock, anaphylactic shock, or hemorrhagic shock, menstrual disorders, obstetric conditions, erythropoietin-mediated vasoconstriction, or wounds.

18. Use of the cyclic peptides or pharmaceutically acceptable salts or esters of the cyclic peptides of any of claims 1-12 formulated as a medicament for the treatment of endothelin-mediated disorders.