PHARMACOLOGICAL POTENTIAL OF ENDOTHELIN RECEPTORS AGONISTS AND ANTAGONISTS

The endothelin (ET) family of peptides are very potent endogenous vasoconstrictors and pressor agents, secreted by various cells and tissues in the human. The ET family consists of 3 structurally similar isopeptides: ET-l,-2, and -3. The genes that encode these peptides are cloned and are found to be on chromosomes 6, 1 and 20, respectively (14). Of the three isoforms, endothelin ET-1 is the predominant isoform produced by the vascular endothelium. ET-l is synthesized in the human vasculature and is the most potent vasoconstrictor substance known (32). ET-2 has similar vasoconstrictor potency to ET-l and appears to be synthesized predominantly in the kidney and intestines (13). ET-3 is the least potent vasoconstrictor. It is detectable in the central nervous system, lungs, kidneys, pancreas and spleen (12). All the three endogenous isoforms of ET in humans mediate their actions via two ET receptor subtypes: ETA and ETB. These ETs play an important role in human physiology and in the pathophysiology of vasospastic conditions, renal failure, chronic heart failure and asthma (10). Chemical compounds that interact with ET receptors, natural and/or synthetic agonists and antagonists, respectively, represent important group of substances with weighty pharmacological potential (23). Because ETs were thought to be important in cardiovascular homeostasis, many investigators focused on the physiological and pathophysiological significance of ET. Accordingly, ET receptor antagonists have been developed rapidly, mostly for the treatment of cardiovascular diseases (11). The current review will focus on the recent developments in the endothelin field, with special emphasis on the ET-1 antagonists and their clinical use.


Introduction
The endothelin (ET) family of peptides are very potent endogenous vasoconstrictors and pressor agents, secreted by various cells and tissues in the human.The ET family consists of 3 structurally similar isopeptides: ET-l,-2, and -3.The genes that encode these peptides are cloned and are found to be on chromosomes 6, 1 and 20, respectively (14).Of the three isoforms, endothelin ET-1 is the predominant isoform produced by the vascular endothelium.ET-l is synthesized in the human vasculature and is the most potent vasoconstrictor substance known (32).ET-2 has similar vasoconstrictor potency to ET-l and appears to be synthesized predominantly in the kidney and intestines (13).ET-3 is the least potent vasoconstrictor.It is detectable in the central nervous system, lungs, kidneys, pancreas and spleen (12).All the three endogenous isoforms of ET in humans mediate their actions via two ET receptor subtypes: ET A and ET B .These ETs play an important role in human physiology and in the pathophysiology of vasospastic conditions, renal failure, chronic heart failure and asthma (10).Chemical compounds that interact with ET receptors, natural and/or synthetic agonists and antagonists, respectively, represent important group of substances with weighty pharmacological potential (23).Because ETs were thought to be important in cardiovascular homeostasis, many investigators focused on the physiological and pathophysiological significance of ET.Accordingly, ET receptor antagonists have been developed rapidly, mostly for the treatment of cardiovascular diseases (11).The current review will focus on the recent developments in the endothelin field, with special emphasis on the ET-1 antagonists and their clinical use.

Endothelins
ETs represent a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear C-terminal tail.These peptides were originally described on the basis of their potent vasoconstrictor activity (27).The three peptides are encoded by three different genes.Biologically active endothelins are produced from pre-pro-polypeptides through two steps of proteolytic processing.The approximately 200-residue prepro-endothelins are first processed by a furin-like processing protease(s) into biologically inactive intermediates termed big endothelins (big ETs) (25).These are further proteolytically cleaved between Trp 21 and Val/Ile 22 to produce active endothelins.This proteolytic conversion is catalyzed by specific endothelin-converting enzymes (ECEs).Two isozymes of ECE, ECE-1 and ECE-2, have been molecularly identified.Both these enzymes are membrane proteins with highly conserved Zn 2+ metalloprotease motifs.
The ECE-1 cleaves big endothelins at a neutral pH, while ECE-2 functions in an acidic pH range.This implies that these enzymes function in different subcellular locations: ECE-1 may act in early components of the secretory path-way, presumably in the Golgi apparatus, as well as on the cell surface.In contrast, ECE-2 probably functions in highly acidified compartments of the secretory pathway, including a portion of the trans-Golgi apparatus.These isozymes also appear to have different functions during embryonic development (9).Targeted disruption of the ECE-1 gene in mice revealed that ECE-1 is the major enzyme involved in the activation of big ET-1 and big ET-3 at specific developmental stages.Importantly, it appears that loss of ECE-1 protein cannot be compensated for by ECE-2.

Endothelin receptors
ET A and ET B have been isolated and cloned from human tissues (2).Both subtypes belong to the seven-transmembrane-domain-spanning, G-protein-coupled receptor superfamily (6).ET receptors are widely expressed in all tissues, consistent with the physiological role of ET-1, the most abundant isoform, as a ubiquitous endothelium-derived vasoactive peptide contributing to the maintenance of normal vascular tone.ET receptors are also localized to non-vascular structures, such as epithelial cells, and expressed in the central nervous system.ET-1 stimulates proliferation in a number of different cell types, including smooth muscle cells (mainly via the ET A subtype) and astrocytes (via ET B ).In human blood vessels, ET A receptors are present mainly on vascular smooth muscle cells, and are chiefly responsible for contraction whereas ET B receptors mediate vasodilatation.The mechanisms of ET A activation by endothelins and signal transfer mechanism of vascular smooth muscle contraction is schematically shown in Fig 1 [according to Potaczek and Sanak (26)].ET B receptors are also localized in the single layer of endothelial cells lining the vessel wall.Activation of endothelial ET B receptors may lead to the release of endothelium-derived relaxing factors (nitric oxide and prostanoids).
ET-1 has dual vasoactive effects, mediating vasoconstriction via ET A receptor activation of vascular smooth muscle cells and vasorelaxation via ET B1 receptor activation of endothelial cells.ET B -receptor is also present in vascular smooth musle like ET A -receptor.The receptor on endothelium has been called ET B1 , while the vascular smooth muscle subtype is termed ET B2.Agonists acting on ET B1 cause, as already mentioned, vasodilation.In contrast, ET B2 -mediated responses are vasoconstrictor, like responses mediated by ET A .Vasodilation is, at least partly, caused by stimulation of nitric oxide (NO) synthesis and PGI 2 synthesis,, however, the precise signal tranduction is unknown (21).
Non-vascular ET B receptors in organs including the kidney may beneficially clear ET-1 from the circulation (6).All these receptors have become important targets for drugs developed to inhibit vasoconstrictor actions (19).
It is widely accepted that the vascular, cardiac, and renal adverse effects of ET-1 are mediated by ET A , while activation of ET B receptors leads to beneficial effects such as: attenuating the vascular and cardiac hypertrophic effects of ET-1 as well as the vasodilatory action of this peptide (1).

Endothelin receptors agonists
Natural ET agonists are all endogenous ET isoforms and ET receptors to be classified according to their rank order of potency for the individual ETs (5).Other natural ET agonists are sarafotoxins, toxic peptides from the venom of mole viper Atractaspis engaddensis (burrowing asp), which may pose a serious threat to humans.At present, four sarafotoxins (STX) are known: S6a, S6b, S6c, and S6d (3,30).A new member of the endothelin/sarafotoxin family of vasoconstrictor peptides, bibrotoxin (BTX), was isolated from the venom of the burrowing asp Atractaspis bibroni (4).The primary structure of all known endothelins, sarafotoxins and bibrotoxin are summarized in Tab. 1.They are 21-amino acid peptides with high degree of similarity whose structure consists of two rings formed by intra-chain disulphide bonds.The venom from the snake A. engaddensis has a very high lethal potency, with an i.v.LD 50 of 0.06-0.075mg.kg -1 body weight in mice.The onset of action of the venom is rapid (30-45 min) and death results from seemingly neurotoxic effects.However, even at high concentrations, the venom does not block contractions of skeletal muscles that are directly or indirectly stimulated.The changes observed in the ECG are similar to those recorded in human victims (18) and are the result of an A-V block that is caused by an apparent direct action of the venom on the heart (34).Concerning ETs already a low dose of ET-1 has a lethal effect in experimental animals by inducing disturbances in the cardiovascular system (16).

Endothelin receptors antagonists
Antagonists are classified as ET A -selective, ET B -selective, or non-selective mixed antagonists that display similar affinities for both receptor subtypes.However, only ET A -selective or ET A /ET B antagonists are currently being evaluated in clinical trials.Nevertheless, ET B -selective antagonists are also known.
Generally more potent non-peptide antagonists for the ET A receptors, such as PD156707, SB234551, L754142, A127722, and TBC11251 are known and some of them are characterized in Tab. 2.
A limited number of peptide (BQ788) and non-peptide (A192621) ET B antagonists have also been developed.Some of them are summarized in Tab. 2. They are generally less potent than ET A antagonists and display lower selectivity (usually only 1 to 2 orders of magnitude) for the ET B receptor.Radioligands highly selective for either ET A ( 125 I-PD151242, 125 I-PD164333, and 3 H-BQ123) or ET B receptors ( 125 I-BQ3020 and 125 I-IRL1620) have further consolidated classification into only these two types, with no strong molecular or pharmacological evidence to support the existence of further receptors in mammals.

Endothelin receptors antagonists in clinical development
While peptide antagonists are useful as research tools, their therapeutic use is limited by the fact that they can only be administered i.v. and have a short duration of action.Being peptides, they are degraded in the gastrointestinal tract by proteolytic enzymes.
The limitations of peptide antagonists have been partly overcome by non-peptide antagonists (5).ET receptors antagonists in clinical trial are summarized in Tab. 3. Manipulation of the activity of ET-1, especially using ET receptor antagonist, has allowed the further elucidation of the role of this neurohormonal system and development of novel therapeutic strategies in the treatment of heart failure (28).To date, published clinical studies of these agents have involved relatively small numbers of patients with severe heart failure, followed for a relatively short time period, and have mainly examined surrogate endpoints.Large-scale trials that address to significant clinical outcomes are ongoing and their results forthcoming.A key question that remains is whether selective ET A or dual ET A /ET B receptor blockade will be more effective (1).
The same refers to utilization of ET antagonists in other pathological conditions, such as pulmonary arterial hypertension (PAH), subarrachnoid hemorrhage or hepatorenal syndrome.Bosentan (Tracleer) (Fig. 2) was the first ET antagonist approved by the Food and Drug Administration for the treatment of pulmonary arterial hypertension (8) and recently tezosentan (Veletri) has been designated for the same indication (17) and is under investigation for acute heart failure.The development of bosentan, a novel, well-tolerated, orally active endothelin antagonist, has significantly changed the therapeutic approach to PAH.Recent clinical trials have demonstrated that treatment with bosentan produces favourable effects on cardiopulmonary haemodynamics (22).Also tezosentan, dual ET A /ET B receptor antagonist that has demonstrated efficacy in improving cardiac index and reducing pulmonary capillary wedge pressure in patients with acute, decompensated heart failure, was introduced into clinical practice recently (31).

Conclusions
The ET system plays an important role in the pathophysiology of a variety of cardiovascular diseases including congestive heart failure, essential and pulmonary hypertension, renal failure, and cerebrovascular disease.The biological effects of ET-1 on its target organs are mediated by two receptor types: ET A and ET B .It is widely accepted that the vascular, cardiac, and renal adverse effects of ET-1 are mediated by ET A , while activation of ET B receptors leads to beneficial effects such as: attenuating the vascular and cardiac hypertrophic effects of ET-1 as well as they mediate the vasodilatory action of this peptide (activation of ET B1 -receptor subtype).In the last decade, a plenty of peptide and non-peptide ET-1 antagonists have been developed.Several clinical studies have revealed that ET-1 antagonists are clinically beneficial therapeutic agents for the treatment of several cardiovascular diseases, leading to the approval of bosentan (ET A /ET B antagonist) for the treatment of pulmonary hypertension.

Tab. 3: Classification of ET receptor antagonists in clinical development.
ET receptors antagonists in II and III phase of clinical trials.CHF, congestive heart failure; PH, primary pulmonary hypertension; PC, prostate cancer; PORH, portal hypertension, SAH, subarachnoid hemorrhage; HRS, hepatorenal syndrome.

Fig. 2 :
Fig. 2: Chemical structure of bosentan (Tracleer), the first endothelin receptor antagonist approved by the Food and Drug Administration for the treatment of pulmonary arterial hypertension.