W. Y. Chan

Characterization of a novel, linear radioiodinated vasopressin antagonist: an excellent radioligand for vasopressin V1a receptors.

We report on the pharmacological properties of a potent and selective linear vasopressin (AVP) V1a receptor antagonist HO-Phenylacetyl1-D-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-NH2 (HO-LVA). Iodinated on the phenolic substituent at position 1, [125I]-HO-LVA displayed the highest affinity for rat liver V1a receptors (8 pM) ever reported. Furthermore, affinities of HO-LVA and I-HO-LVA for V1b, V2 and oxytocin (OT) receptors was 400- to 1,000-fold lower than for V1a receptors, rendering it a highly selective ligand. Both HO-LVA and its iodinated derivative are V1 antagonists, they potently inhibited AVP-induced inositol-phosphate accumulation in WRK1 cells, and also, although with a much lower potency, the AVP-induced ACTH release from freshly prepared pituitary cells. Using autoradiography [125I]-HO-LVA appeared to be the first radioligand to successfully identify and localize the presence of V1a receptors in rat liver and blood vessel walls. Moreover, several new brain regions expressing V1a receptors could be identified, in addition to those brain regions that were previously identified with other radiolabelled AVP analogues.

Discovery and design of novel and selective vasopressin and oxytocin agonists and antagonists: the role of bioassays.

Synthetic oxytocin and vasopressin agonists and antagonists have become important tools for research and were instrumental in the identification of the four known receptor subtypes, V1a, V2, V1b (V3) and oxytocin, of these peptide hormones. However, the relative lack of receptor selectivity, particularly of the antagonists, has limited their usefulness as experimental probes and their potential as therapeutic agents. We now present some findings from our continuing studies aimed at the design of more selective oxytocin and vasopressin agonists and antagonists and a structure‐activity relationship update on our recently discovered novel hypotensive vasopressin peptides. Bioassays have been, and continue to be, of critical importance in leading to the discovery of the novel agonists, antagonists and hypotensive peptides reported here. This paper highlights three main aspects of these studies. (1) Replacement of the tyrosine2 and/or phenylalanine3 residues in the V2 agonist deamino,[Val4,D‐Arg8]arginine‐vasopressin (dVDAVP)by thienylalanine resulted in selective V2 agonists with strikingly high potencies. However, the peptide solutions were unstable and lost activity over time. These highly potent V2 agonists, which are devoid of vasopressor activity, are promising leads for improving drugs for treating diabetes insipidus, enuresis and coagulation disorders. (2) Diaminopropionic acid and diaminobutyric acid substitution at position‐5 in oxytocin and in V1a antagonists yielded, respectively, the first specific antagonist for the oxytocin receptor, desGly‐NH2,d(CH2)5[D‐Trp2, Thr4, Dap5]OVT and the first specific antagonist for the vasopressin V1a receptor, d(CH2)5[Tyr(Me)2, Dab5]AVP. The availability of single receptor subtype‐specific or selective antagonists will enhance our ability to delineate receptor functions. Utilising these new receptor specific probes, we were able to show thatthe uterotonic action of vasopressin is mediated principally by oxytocin and not by V1a receptors. (3) Replacement of the phenylalanine8 residue in the V1a/V2/oxytocin antagonist, d(CH2)5[D‐Tyr(Et)2, Val4]AVP, with arginine3 yielded the novel, selective, hypotensive vasopressin peptide, d(CH2)5[D‐Tyr(Et)2, Arg3, Val4]AVP (Peptide I). Bioassay characterisations of Peptide I show that its vasodepressor action is independent of the peripheral autonomic, bradykinin, nitric oxide and prostaglandin systems and is not mediated by the known classical oxytocin and vasopressin receptors. These findings suggest the existence of a new vasopressin receptor subtype that may be relevant to the vasodilating action of vasopressin in regional vascular beds. Iodinatable hypotensive peptides have been synthesised and could be developed as markers for the putative new receptor. Ongoing structure‐activity relationship studies on Peptide I have led to more potent and selective hypotensive peptides for use as new research tools and as leads for the development of a new class of antihypertensive agents.

4-Leucine-Oxytocin: Natriuretic, Diuretic, and Antivasopressin Polypeptide

During water diuresis in anesthetized rats, 4-leucine-oxytocin increased the urine output and the rates of sodium and chloride excretion. The potassium excretion rate was only slightly increased. During vasopressin-suppressed water diuresis, 4-leucine-oxytocin produced similar effects on urine and electrolyte excretions. In addition, it inhibited the vasopressin-induced free-water reabsorption, and it could reverse reabsorption to freewater clearance.

The role of oxytocin receptors and vasopressin V1a receptors in uterine contractions in rats : Implications for tocolytic therapy with oxytocin antagonists

OBJECTIVE The objective of the study was to determine in the rat model whether the uterotonic action of vasopressin is mediated by the vasopressin V1a receptor in the uterus, by the oxytocin receptor, or by both. The purpose is to assess whether the anti-V1a activity of oxytocin antagonists is a desirable pharmacologic property in tocolytic therapy for preterm labor. STUDY DESIGN Dose-response characteristics of the uterotonic action of oxytocin and arginine vasopressin were compared and analyzed by the in vitro cumulative dose-response curve technique. A nonselective oxytocin-V1a receptor antagonist, a selective oxytocin receptor antagonist, and a selective V1a receptor antagonist were selected for this study. Their relative effectiveness in inhibiting the uterine contractile responses induced by oxytocin and by arginine vasopressin in the isolated rat uterus was examined. RESULTS The uterotonic dose-response curves for oxytocin and arginine vasopressin were parallel and had the same maximal response. The nonselective oxytocin/V1a receptor antagonist and the selective oxytocin receptor antagonist were equally potent in inhibiting the uterine contractions induced by either oxytocin or arginine vasopressin, whereas the selective V1a receptor antagonist had no antiuterotonic activity. Inhibition by the selective oxytocin antagonist caused a similar parallel shift to the right of the dose-response curves for oxytocin and arginine vasopressin. CONCLUSIONS The parallel dose-response curves for oxytocin and arginine vasopressin suggest that the uterotonic action of vasopressin is also mediated by the oxytocin receptor. Arginine vasopressin binds to both oxytocin and V1a receptors in the uterus, but the activation of V1a receptors appears not to be a mechanism involved in the uterine-stimulating action of vasopressin. The anti-V1a activity of oxytocin antagonists does not contribute to tocolytic efficacy and may represent an undesirable side effect. By blocking the vascular V1a receptors, it may compromise the patients ability to maintain arterial blood pressure during hemorrhage.

Discovery of novel selective hypotensive vasopressin peptides that exhibit little or no functional interactions with known oxytocin/vasopressin receptors

1 Arginine‐vasopressin (VP) has both vasoconstricting and vasodilating action. We report here the discovery of four novel selective hypotensive VP analogues: d(CH2)5[D‐Tyr(Et)2,Arg3,Val4]AVP; d(CH2)5[D‐Tyr(Et)2,Lys3,Val4]AVP and their iodinatable Tyr‐NH29 analogues. 2 Bioassays in rats for activities characteristic of neurohypophysial peptides showed that the four VP peptides possessed little or no V1a, V2 or oxytocin (OT) receptor agonistic or antagonistic activities. 3 In anaesthetized rats, these peptides (0.05–0.10 mg kg−1 i.v.) elicited a marked fall in arterial blood pressure. 4 Blockade of cholinoceptors, adrenoceptors and bradykinin B2 receptors, and inhibition of prostaglandin synthesis had little effect on their vasodepressor action. 5 Classical V1a, V2 and OT receptor antagonists did not block the vasodepressor response. 6 L‐NAME, 0.2 mg kg−1 min−1, markedly suppressed the hypotensive response to ACh but not the vasodepressor response to the hypotensive VP peptides. However, the duration of the vasodepressor response was shortened. Very high doses of L‐NAME attenuated both the vasodepressor response and the duration of action. 7 These findings indicate that the vasodepressor action of these VP peptides is independent of the peripheral autonomic, bradykinin and PG systems and is not mediated by the known classical OT/VP receptors. NO does not appear to have an important role in their vasodepressor action. 8 The discovery of these novel VP peptides could lead to the development of new tools for the investigation of the complex cardiovascular actions of VP and the introduction of a new class of hypotensive agents. The two iodinatable hypotensive VP peptides could be radiolabelled as potential markers for the localization of the receptor system involved.

An investigation of position 3 in arginine vasopressin with aliphatic, aromatic, conformationally-restricted, polar and charged amino acids.

We report the solid‐phase synthesis and some pharmacological properties of 23 new analogs of arginine vasopressin (AVP) which have the Phe3 residue replaced by a broad variety of amino acids. Peptides 1–9 have at position 3: (1) the mixed aromatic/aliphatic amino acid thienylalanine (Thi) and the aliphatic amino acids; (2) cyclohexylalanine (Cha); (3) norleucine (Nle); (4) Leu; (5) norvaline (Nva); (6) Val; (7) alpha‐aminobutyric acid (Abu); (8) Ala; (9) Gly. Peptides 10–23 have at position 3: the aromatic amino acids, (10) homophenylalanine (Hphe); (11) Tyr; (12) Trp; (13) 2‐naphthylalanine (2‐Nal); the conformationally‐restricted amino acids (14) Pro; (15) 2‐aminotetraline‐2‐carboxylic acid (Atc); the polar amino acids (16) Ser; (17) Thr; (18) Gln; and the charged amino acids (19) Asp; (20) Glu; (21) Arg; (22) Lys; (23) Orn. All 23 new peptides were evaluated for agonistic and, where appropriate, antagonistic activities in in vivo antidiuretic (V2‐receptor) and vasopressor (V1a‐receptor) assays and in in vitro (no Mg2+) oxytocic assays. The corresponding potencies (units/mg) in these assays for AVP are: 323±16; 369±6 and 13.9±0.5. Peptides 1–9 exhibit the following potencies (units/mg) in these three assays: (1) 379±14; 360±9; 36.2±1.9; (2) 294±21; 73.4±2.7; 0.33±0.02; (3) 249±28; 84.6±4.3; 4.72±0.16; (4) 229±19; 21.4±0.6; 2.1±0.2; (5) 134±5; 31.2±0.9; 28.4±0.2; (6) 114±9; 45.3±2.3; 11.3±1.6; (7) 86.7±2.5; 4.29±0.13; 0.45±0.03; (8) 15.5±1.5; 0.16±0.01; ∼0.02; (9) 3.76±0.03; <0.02; in vitro oxytocic agonism was not detected. These data show that the aliphatic amino acids Cha, Nle, Leu, Nva and Val are well‐tolerated at position 3 in AVP with retention of surprisingly high levels of antidiuretic activity. Peptides 2–9 exhibit significant gains in both antidiuretic/vasopressor (A/P) and antidiuretic/oxytocic (A/O) selectivities relative to AVP. [Thi3]AVP appears to be a more potent antidiuretic and oxytocic agonist than AVP and is equipotent with AVP as a vasopressor agonist. The antidiuretic potencies of peptides 10–23 exhibit drastic losses relative to AVP. They range from a low of 0.018±0.001 units/mg for the Lys3 analog (peptide 22) to a high of 24.6±4.6 units/mg for the Hphe3 analog (peptide 10). Their vasopressor potencies are also drastically reduced. These range from a low of <0.002 units/mg for peptide 22 to a high of 8.99±0.44 units/mg for the Atc3 analog (peptide 15). Peptides 10–23 exhibit negligible or undetectable in vitro oxytocic agonism. The findings on peptides 10–23 show that position 3 in AVP is highly intolerant of changes with aromatic, conformationally‐restricted, polar and charged amino acids. Furthermore, these findings are in striking contrast to our recent discovery that position 3 in the potent V2/V1a/OT antagonist d(CH2)5d‐Tyr(Et)2VAVP tolerates a broad latitude of structural change at position 3 with many of the same amino acids, to give excellent retention of antagonistic potencies. The data on peptides 1–4 offer promising clues to the design of more potent and selective AVP V2 agonists. Copyright

Properties of a new radioiodinated antagonist for human vasopressin V2 and V1a receptors

A vasopressin receptor antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-o-ethyl-D-tyrosine, 4-valine, 9-tyrosylamide] arginine vasopressin (d(CH2)5[o-ethyl-D-Tyr2,Val4,Tyr-NH9(2)]AVP), has been prepared. This antagonist is a potent antiantidiuretic, antivasopressor and antioxytocic peptide with pA2 values of 7.69-7.94 and affinities of 1.12-11.0 nM. When radioiodinated at the phenyl moiety of the tyrosylamide residue at position 9, this peptide was demonstrated to bind to vasopressin V2 and V1a receptors with a dissociation constant of 0.22-0.75 nM. This ligand is a good tool for further studies on human vasopressin V2 receptor localization and characterization, when used in combination with a selective vasopressin V1a ligand.

Long-acting oxytocin antagonists: effects of 2-D-stereoisomer substitution on antagonistic potency and duration of action.

Abstract Recently we reported the discovery of a series of 2-O-alkyltyrosine- (or 2-p-alkylphenylalanine), 4-threonine-, and 8-ornithine-substituted analogs of [1-penicillamine]oxytocin ([Pen1]OT) which possess prolonged anti-OT activity. In this study, we attempt to improve the potency and the duration of action of this series of OT antagonists by exploring the effects of D-stereoisomer substitution in the 2 position. We compare the in vitro anti-OT potency, expressed in pA 2 values, and the duration of in vivo inhibitory action, expressed in recovery t 1/2, of [Pen1]OT, [Pen1,Orn8]OT, [Pen1,Thr4]OT, [Pen1Tyr(Ome)2, Th4, Orn8]OT, [Pen1,Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,D-Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,Phe2,Thr4]OT, [Pen1,Phe(Me)2,Thr4,Orn8]OT, [Pen1,D-Phe(Me)2,Thr4,Orn8]OT, [Pen1,Phe(Et)2,Thr4,Orn8]OT, and [Pen1,D-Phe(Et)2,Thr4,Orn8]OT. The results show that modifications of the amino acid in position 2 by alkylation of the aromatic ring and use of D-stereoisomerism produce nonparallel effects on the in vitro potency and duration of action of OT antagonists. Time-action curve determinations show that long-acting OT antagonists exhibit delayed peak inhibitory action. Long action is not coupled with high potency in all cases. This dissociation between potency and duration of action gives support to our hypothesis that the potency and duration of action of these peptides may each have different conformational Structure requirements.

Discovery and design of novel vasopressin hypotensive peptide agonists.

This presentation will trace the serendipitous discovery of novel vasopressin (VP) hypotensive agonists d(CH2)5[D-Tyr(Et)2,X3]VAVP (where X = Arg, Lys). These peptides were uncovered as part of an ongoing program aimed at the design of potent and selective VP antidiuretic (V2 receptor) antagonists. We will also present highlights of our subsequent preliminary studies seeking (i) to design high affinity radioiodinatable ligands for the localization and characterization of the putative VP vasodilatory (V1c?) receptor; (ii) to identify the structural features of selective and non-selective cyclic and linear VP and oxytocin (OT) antagonists of the V2 receptor, the vascular (V1a) receptor and of the uterine (OT) receptor required for hypotensive agonism and; (iii) to enhance hypotensive potency. These novel VP hypotensive agonists could serve as valuable research tools in studies on the roles of VP in blood pressure regulation and may also lead to the development of a new class of therapeutically useful antihypertensives.

Synthesis and structure–activity investigation of novel vasopressin hypotensive peptide agonists

We report the solid phase synthesis and vasodepressor potencies of the novel hypotensive peptide [1(‐β‐mercapto‐β,β‐pentamethylene propionic acid)‐2‐O‐ethyl‐D‐tyrosine, 3‐arginine, 4‐valine] arginine vasopressin, d(CH2)5[D‐Tyr(Et)2, Arg3, Val4]AVP (A), its related Lys3 (B), Tyr‐NH29 (C), [Lys3, Tyr‐NH29] (D) analogs and in a preliminary structure–activity study of positions 2–4 and 7–9, 24 analogs (1–24) of A–C. Peptides 1–6, 9–14 have the following single substituents at positions 2, 3, 4, 8 and 9 in (A): 1, D‐Tyr(Me)2; 2, L‐Tyr(Et)2; 3, Orn3; 4, N‐Me‐Arg3; 5, Glu3; 6, Arg4; 9, D‐Arg8; 10, Eda9; 11, Arg‐NH29; 12, Ala‐NH29; 13, desGly9; 14, desGly‐NH29. Peptides 15 and 16 are analogs of B which possess the following single modifications: 15, Arg‐NH29; 16, desGly9. Peptides 7 and 8 are analogs of (C) with the following single modification: 7, Gln4; 8, Lys8. Peptides 17–24 are analogs of A possessing the following multiple modifications: 17, [Sar7, Eda9]; 18, [Arg7, Eda9]; 19, [Arg7, Eda9←Tyr10]; 20, [Arg4, Arg‐NH29]; 21, [Ile4, desGly9]; 22, [Arg4, desGly9]; 23, [Arg7, desGly9]; 24, [Arg7, Lys8, desGly9]. All 24 new peptides were evaluated for agonistic and antagonistic activities in in vivo antidiuretic (V2‐receptor), vasopressor (V1a‐receptor) and in in vitro (no Mg2+) oxytocic (OT‐receptor) assays and like the parent peptides (A–D) (Chan et al. Br. J. Pharmacol. 1998; 125: 803–811) were found to exhibit no or negligible activities in these assays. Vasodepressor potencies were determined in anesthetized male rats with baseline mean arterial blood pressure maintained at 110–120 mmHg. The effective dose (ED), in μg 100 g−1 i.v., required to produce a vasodepressor response of 5 cm2, area under the vasodepressor response curve (AUC) during the 5‐min period following the injection of the test peptide, was determined. Therefore, the EDs measure the relative vasodepressor potencies of the hypotensive peptides. The following ED values were obtained for A–D and for peptides 1–24: A, 4.66; B, 5.75; C, 10.56; D, 11.60; 1, ∼20; 2, ∼30; 3, 6.78; 4, non‐detectable (ND); 5, ND; 6, ∼32; 7, ND; 8, 8.67; 9, ND; 10, 2.43; 11, 3.54; 12, 10.57; 13, 4.81; 14, ND; 15, 4.47; 16, 9.78; 17, 5.72; 18, 1.10; 19, 1.05; 20, 10.41; 21, 9.13; 22, ∼33; 23, 3.01; 24, 1.71. A is clearly the most potent of the four original hypotensive peptides A–D. These data provide insights to which modification of A enhance, retain or abolish hypotensive potencies. Six of the new hypotensive peptides are significantly more potent than A. These are peptides 10, 11, 18, 19, 23 and 24. Peptide 19, a radioiodinatable ligand, is ten times more potent than C or D. The Gln4 modification of C and the N‐Me‐Arg3, Glu3, D‐Arg8 and desGly‐NH29 modifications of A abolished hypotensive potency. By contrast, the Eda9, Arg‐NH29, [Sar7, Eda9], [Arg7, Eda9←Tyr10], [Arg7, desGly9], [Arg7, Lys8, desGly9] modifications of A all led to enhancements of hypotensive potency. This initial structure–activity exploration provides useful clues to the design of (a) more potent vasodepressor peptides and (b) high affinity radioiodinatable ligands for the putative AVP vasodilating receptor. Some of the peptides here may be of value as pharmacological tools for studies on the complex cardiovascular actions of AVP and may lead to the development of a new class of anti‐hypertensive agents. Copyright