[1-deamino-4-cyclohexylalanine] arginine vasopressin: a potent and specific agonist for vasopressin V1b receptors.

To date, there are no vasopressin (VP) agonists that exhibit a high affinity and selectivity for the VP V1b receptor with respect to the V1a, V2, and oxytocin receptors. In this study, we describe the synthesis and pharmacological properties of [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP). Binding experiments performed on various membrane preparations revealed that d[Cha(4)]AVP exhibits a nanomolar affinity for V1b receptors from various mammalian species (rat, bovine, human). It exhibits high V1b/V1a and V1b/oxytocin selectivity for rat, human, and bovine receptors. Furthermore, it exhibits high V1b/V2 specificity for both bovine and human vasopressin receptors. Functional studies performed on biological models that naturally express V1b receptors indicate that d[Cha4]AVP is an agonist. Like VP, it stimulated basal and corticotropin-releasing factor-stimulated ACTH secretion and basal catecholamine release from rat anterior pituitary and bovine chromaffin cells, respectively. In vivo experiments performed in rat revealed that d[Cha4]AVP was able to stimulate both ACTH and corticosterone secretion and exhibits negligible vasopressor activity. It retains about 30% of the antidiuretic activity of VP. This long-sought selective VP V1b receptor ligand with nanomolar affinity will allow a better understanding of V1b-mediated VP physiological effects and is a promising new tool for V1b receptor structure-function studies.

Design of oxytocin antagonists, which are more selective than atosiban.

We report the solid phase synthesis of four pairs of L- and D-thienylalanine (Thi/D-Thi) position two modified analogues of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly(NH2)9,d (CH2)5[Tyr(Me)2,Thr4]OVT) (A); the Tyr-(NH2)9 analogue of (A), d(CH2)5[Tyr(Me)2,Thr4,Tyr-(NH2)9]OVT (B); the Eda9 analogue (where Eda = ethylenediamine) of (A), d(CH2)5[Tyr(Me)2, Thr4, Eda9]OVT (C); and the retro Tyr10 modified analogue of (C), d(CH2)5[Tyr(Me)2, Thr4, Eda9<--Tyr10]OVT (D). The eight new analogues of A-D are (1) desGly(NH2),d(CH2)5[Thi2,Thr4]OVT, (2) desGly(NH2),d(CH2)5[D-Thi2,Thr4]OVT, (3) d(CH2)5[Thi2, Thr4,Tyr-(NH2)9]OVT, (4) d(CH2)5[D-Thi2,Thr4,Tyr-(NH2)9]OVT (5) d(CH2)5[Thi2,Thr4Eda9]OVT, (6) d(CH2)5[D-Thi2,Thr4,Eda9]OVT, (7) d(CH2) [Thi2,Thr4,Eda9<--Tyr10]OVT, (8) d(CH2),[D-Thi2,Thr4,Eda9<--Tyr10]OVT. We also report the synthesis of (C). Peptides 1-8 and C were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in in vivo vasopressor (V1a receptor) assays and in in vivo antidiuretic (V2 receptor) assays. None of the eight peptides nor C exhibit oxytocic or vasopressor agonism. Peptides 1-8 are extremely weak V2 agonists (antidiuretic activities range from < 0.0005 to 0.20 U/mg). Peptide C is a weak mixed V2 agonist/antagonist. Peptides 1-8 and C exhibit potent in intro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.76 to 8.05). Peptides 1-8 are all OT antagonists in vivo (estimated in vivo anti-OT pA2 values range from 6.54-7.19). With anti-V1a pA2 values of approximately 5-5.80, peptides 1-8 exhibit marked reductions in anti-V1a potencies relative to those of the parent peptides A-D (anti-V1a pA2 range from 6.48 to 7.10) and to l-deamino[D-Tyr(Et)2, Thr4]OVT (Atosiban, trade name Tractocile) (anti-V1a pA2-6.14). Atosiban has recently been approved in Europe for clinical use for the prevention of premature labour (Pharm. J. 264(7-100): 871). Peptides 1-8 exhibit striking gains in in vitro anti-OT/anti-V1a selectivities with respect to the parent peptides A, B, C and D and to Atosiban. Peptides 1-8 exhibit anti-OT (in vitro)/anti-V1a selectivities of 450, 525, 550, 450, approximately 1080, 116, 355, 227 respectively. The corresponding values for A-D and Atosiban are 30, 4.2, 4.3, 2.6 and 37. With the exception of peptide 6, the remaining seven peptides exhibit 3-18-fold gains in anti-OT (in vivo)/anti-V1a selectivity with respect to Atosiban, peptides 1-8 exhibit anti-OT (in vivo)/anti-V1a selectivities of 22, approximately 82, approximately 82, 147, approximately 83, 11, 31 and 42. By comparison, Atosiban exhibits an anti-OT (in vivo)/anti-V1a selectivity = 8. With an estimated in vivo anti-OT pA2 value = 7.19+/-0.06, peptide 4 is equipotent with Atosiban (pA2 = 7.05+/-0.05). However, with its significantly reduced anti-vasopressor potency, pA2 = approximately 5, it is approximately 18 times more selective for OT receptors with respect to VP V1a receptors than Atosiban. Since we have shown that V1a antagonism could be an unwanted side-effect in tocolytics, peptide 4 and some of the OT antagonists reported here have advantages over Atosiban and thus may be suitable candidates for evaluation as potential tocolytic agents for the treatment of preterm labour.

Novel selective hypotensive vasopressin peptides: cardiovascular and structure-activity-relationship studies.

Recently, we discovered a series of peripheral acting selective hypotensive vasopressin peptides. Whether these peptides may interact with receptors outside the vasopressin receptor family and affect cardiac function could not be excluded. Accordingly, we tested the effects of these hypotensive vasopressin peptides on blood pressure and heart rate in intact rats and on the heart rate, ventricular contractile force and coronary flow of isolated perfused rat hearts. We found that the hypotensive vasopressin peptides did not modify cardiac function, either in vivo or in vitro. The vasodepressor potency was reduced when assayed in rats with vasopressin-maintained baseline blood pressure, suggesting that vasopressin and the hypotensive peptide compete for a common vasodilating vasopressin receptor in the vasculature. We have now synthesized more potent and radioiodinatable hypotensive peptides that could serve as lead compounds for the development of a radiomarker for the putative vasodilating vasopressin receptor.

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 that the uterotonic action of vasopressin is mediated principally by oxytocin and not by V1a receptors. (3) Replacement of the phenylalanine3 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.

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(-beta-mercapto-beta,beta-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-NH(9)2 (C), [Lys3, Tyr-NH(9)2 (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-NH(9)2; 12, Ala-NH(9)2; 13, desGly9; 14, desGly-NH(9)2. Peptides 15 and 16 are analogs of B which possess the following single modifications: 15, Arg-NH(9)2; 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-NH(9)2]; 21, [Ile4, desGly9]; 22, [Arg4, desGly9]l; 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 microg 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, approximately 20; 2, approximately 30; 3, 6.78; 4, non-detectable (ND); 5, ND; 6, approximately 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, approximately 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-NH(9)2 modifications of A abolished hypotensive potency. By contrast, the Eda9, Arg-NH(9)2, [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.

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; approximately 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.

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.

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-NH2(9) 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.

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.

Position three in vasopressin antagonist tolerates conformationally restricted and aromatic amino acid substitutions: a striking contrast with vasopressin agonists.

We report the solid-phase synthesis and some pharmacological properties of 12 position three modified analogues (peptides 1-12) of the potent non-selective antagonist of the antidiuretic (V2-receptor), vasopressor (V1a-receptor) responses to arginine vasopressin (AVP) and of the uterine contracting (OT-receptor) responses to oxytocin (OT), [1(-beta mercapto-beta,beta-pentamethylenepropionic acid)-2-O-ethyl-D-tyrosine 4-valine] arginine vasopressin [d(CH2)5D-Tyr(Et)2VAVP] (A) and two analogues of (B) (peptides 13,14), the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid3 (Tic3) analogue of (A). Peptides 1-12 have the following substituents at position three in (A): (1) Pro; (2) Oic; (3) Atc; (4) D-Atc; (6) D-Phe; (7) Ile; (8) Leu; (9) Tyr; (10) Trp; (11) Hphe; (12) [HO]Tic; Peptide (13) is the Tyr-NH2(9) analogue of (B): Peptide (14) is the D-Cys(6) analogue of (B). All 14 new peptides were evaluated for agonistic and antagonistic activities in in vivo V2 and V1a assays and in vitro (no Mg2+)n oxytocic assays. With the exception of the D-Phe3 peptide (No. 6), which exhibits very weak V2 agonism (approximately 0.0017 U/mg), none of the remaining 13 peptides exhibit any agonistic activities in these assays. In striking contrast to their deleterious effects on agonistic activities in AVP, the Pro3, Oic3, Tyr3 and Hphe3 substitutions in (A) are very well tolerated, leading to excellent retention of V2, V1a and OT antagonistic potencies. All are more potent as V2 antagonists than the Ile3 and Leu3 analogues of (A). The Tyr-NH2(9) and D-Cys(6) substitutions in (B) are also well tolerated. The anti-V2 pA2 values of peptides 1-5 and 7-14 are as follows (1) 7.77 +/- 0.03; (2) 7.41 +/- 0.05; (3) 6.86 +/- 0.02; (4) 5.66 +/- 0.09; (5) approximately 5.2; (7) 7.25 +/- 0.08; (8) 6.82 +/- 0.06; (9) 7.58 +/- 0.05; (10) 7.61 +/- 0.08; (11) 7.59 +/- 0.07; (12) 7.20 +/- 0.05; (13) 7.57 +/- 0.1; (14) 7.52 +/- 0.06. All analogues antagonize the vasopressor responses to AVP, with anti-V1a pA2 values ranging from 5.62 to 7.64, and the in vitro responses to OT, with anti-OT pA2 values ranging from 5.79 to 7.94. With an anti-V2 potency of 7.77 +/- 0.03, the Pro3 analogue of (A) is surprisingly equipotent with (A), (anti-V2 pA2 = 7.81 +/- 0.07). These findings clearly indicate that position three in AVP V2/V1a antagonists, in contrast to position three in AVP agonists, is much more amenable to structural modification than had heretofore been anticipated. Furthermore, the surprising retention of V2 antagonism exhibited by the Pro3, Oic3, Tyr3, Trp3 and Hphe3 analogues of (A), together with the excellent retention of V2 antagonism by the Tyr-NH2(9) and D-Cys6 analogues of (B) are promising new leads to the design of potent and possibly orally active V2 antagonists for use as pharmacological tools and/or as radioiodinatable ligands and for development as potential therapeutic agents for the treatment of the hyponatremia caused by the syndrome of the inappropriate secretion of the antidiuretic hormone (SIADH).

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 patient's ability to maintain arterial blood pressure during hemorrhage.

Isosteric substitution of Asn5 in antagonists of oxytocin and vasopressin leads to highly selective and potent oxytocin and V1a receptor antagonists: new approaches for the design of potential tocolytics for preterm labor.

Substitution of Asn5 in oxytocin (OT) or vasopressin (VP) invariably leads to a dramatic loss of the biological activities of the peptides. Because of this observation, few structure-activity-relationship studies of OT and VP peptides have involved modifications in the 5 position. It is now recognized that peptide agonists and antagonists may use different structural and conformational features in their interactions with the receptors. Our prior studies showed that OT and VP antagonists, unlike the agonists, tolerate amino acid substitutions in the 5 position. This opens new approaches for the design of antagonists. We describe the effects of isosteric replacement of Asn5 by diaminopropionic acid (Dap) or diaminobutyric acid (Dab) in three OT and VP antagonists: (1) the V1a (vasopressor receptor) antagonist d(CH2)5[Tyr(Me)2]AVP; (2) the OT (uterine OT receptor) antagonist d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH29] OVT and (3) three selective OT antagonists, desGly-NH2,d(CH2)5[D-Tyr2, Thr4]OVT, desGly-NH2, d(CH2)5[D-Phe2, Thr4]OVT and desGly-NH2, d(CH2)5- [D-Trp2, Thr4]OVT. The Dap5 and Dab5 substitutions were tolerated remarkably well, with the less isosteric Dap5 substitution leading to a greater retention of anti-OT potency than the Dab5 substitution. Furthermore, the Dap5 and Dab5 and OT and VP antagonist analogues were surprisingly shown to be much more selective than their respective parent compounds. The Dab5 analogue of (1) was devoid of anti-OT activity. The three Dap5 analogues of (3) were devoid of anti-V1a activities. These appear to be the first single-receptor-type-selective OT and VP antagonists discovered to date. These findings could provide new leads for the development of single-receptor-type-selective receptor probes for the localization and characterization of OT and VP receptors and potential selective tocolytics for the treatment of premature labor.

Design and synthesis of highly selective in vitro and in vivo uterine receptor antagonists of oxytocin: comparisons with Atosiban.

We report the solid phase synthesis and some pharmacological properties of seven position two analogues (peptides 1-7) of one of our lead oxytocin antagonists, des-9-glycinamide[1-(beta-mercapto-beta,beta-pentamethylenepropionic+ ++ acid),2-O-methyltyrosine,4-threonine]ornithinevasotocin(desGly+ ++-NH2, d(CH2)5-[Tyr(Me)2,Thr4]OVT) (A). Peptides 1-7 have the following substituents at position two (1) D-Tyr(Me); (2) L-Tyr(Et); (3) D-Tyr(Et); (4) L-Tyr; (5) D-Tyr; (6) D-Phe and (7) D-Trp. These were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in vivo vasopressor (V1a-receptor) assays and in vivo antidiuretic (V2-receptor) assays. None of the seven peptides exhibits oxytocic or vasopressor agonism. Peptides 1, 2, 4, 6 and 7 are extremely weak V2 agonists (V2 activities range from 0.001 to 0.02 U/mg). Peptides 3 and 5 exhibit weak V2 antagonism (pA2 < 6.0 and < 5.5, respectively). Peptides 1-7 exhibit potent in vitro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.66 to 8.03). Peptides 1 and 4-7 exhibit potent in vivo OT antagonism. Estimated in vivo anti-OT pA2 values range from 7.06 to 7.79 (peptides 2 and 3 were not tested). With anti-V1a pA2 values of 5.17-6.25 all seven peptides exhibit reduced anti-V1a potencies relative to the parent peptide (A) (anti-V1a pA2 = 6.46). Four of these peptides (4-7) exhibit striking gains in in vitro and in vivo anti-OT/anti-V1a selectivities compared to (A) which has an in vitro selectivity of 30 and an in vivo selectivity of 18. The D-Tyr2 (5), D-Trp2 (7), D-Phe2 (6) and L-Tyr2 (4) analogues of (A) exhibit anti-OT (in vitro)/anti-V1a selectivities = 240, 390, 404 and 540, respectively. The L-Tyr2 (4), D-Trp2 (7), D-Phe2 (6) and D-Tyr2 (5) analogues exhibited anti-OT (in vivo)/anti-V1a selectivities of 72, 80, 88 and 95, respectively. Peptides 4-7 appear to be the most selective peptide OT antagonists reported to date. In this regard it may be noted that they appear to be as or more potent and much more selective than the closely related OT antagonist 1-deamino[D-Tyr(Et)2,Thr4]OVT (Atosiban) which is currently undergoing clinical trial as a potential therapeutic agent for the prevention of premature labor. Atosiban (peptide 8) was resynthesized and pharmacologically evaluated in our laboratories. Atosiban exhibits the following antagonistic potencies. Anti-OT (in vitro, no Mg2+) pA2 = 7.71; anti-OT in vivo pA2 = 7.05; anti-V1a pA2 = 6.14 and anti-V2 pA2 approximately 5.9. Its anti-OT (in vivo)/anti-V1a selectivity is 8. Some of these antagonists may be suitable candidates for evaluation as potential tocolytic agents for use in the treatment of pre-term labor. They could also serve as useful new pharmacological tools for studies on the physiological roles of oxytocin. Finally, the findings presented here provide useful clues for the design of more potent and more selective OT antagonists.

Effects of a D-Cys6/L-Cys6 interchange in nonselective and selective vasopressin and oxytocin antagonists.

We report the solid-phase synthesis of the D-Cys6 analogues of arginine-vasopressin (AVP), peptide 1, of the selective AVP vasopressor (V1a receptor) antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-methyltyrosine]arginine-vasopressin (d(CH2)5[Tyr(Me)2]-AVP, (A)), peptide 2, of the three nonselective antidiuretic/vasopressor (V2/V1a receptor) AVP antagonists d(CH2)5[Tyr(Et)2]VAVP (B), d(CH2)5[D-Tyr(Et)2]VAVP (C), and d(CH2)5[D-Phe2]VAVP (D) (where V = Val4), peptides 3-5, of the nonselective oxytocin (OT) antagonists d(CH2)5-[Tyr(Me)2]OVT (E) and d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT (F) (where OVT = ornithine-vasotocin), peptides 6 and 7, and of the selective OT antagonists desGly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT (G) and d(CH2)5]D-Trp2,Thr4]OVT (H), peptides 8 and 9. We also present the repeat syntheses of the previously reported d(CH2)5[D-Trp2]AVT (peptide 10) and its D-Cys6 analogue (peptide 11) (where AVT = arginine-vasotocin). Peptides 1-11 were assayed for agonistic and antagonistic activities in in vivo V1a, V2, and oxytocic assays and in in vitro oxytocic assays without and with 0.5 mM Mg2+. With V2 and V1a agonistic potencies of 0.82 and 0.41 units/mg, [D-Cys6]AVP has retained less than 0.3% of the V2 and V1a potencies of AVP. It exhibits no oxytocic activity and is an in vitro OT antagonist. pA2 = 6.67 (no Mg2+); pA2 = 5.24 (0.5 mM Mg2+). By contrast, with one or two exceptions, a D-Cys6/L-Cys6 interchange in antagonists 2-9, although resulting in reductions of antagonistic potencies in all assays for virtually all peptides 2-9 relative to A-H, has been well tolerated. For peptides 2-5, the anti-V2 and anti-V1a pA2 values range from approximately 5.54 to 7.33 and from 7.19 to 8.06, respectively; the range of in vitro anti-OT pA2 values (no Mg2+) is 7.35-7.87; with 0.5 mM Mg2+, the range is 7.24-8.21. Peptides 2 and 4 have in vivo anti-OT pA2s = 6.60 and 7.16, respectively. For peptides 6-9, the range of in vitro anti-OT pA2 values (no Mg2+) is 7.65-7.96; with 0.5 mM Mg2+, the range is 7.41-7.65, and the in vivo anti-OT pA2 values range from 6.85 to 7.33. With an in vivo anti-OT pA2 = 7.33, peptide 6 is equipotent with its parent E. The in vivo anti-OT potencies of peptides 7-9 are significantly reduced relative to those of F-H. The in vitro anti-OT (0.5 mM Mg2+) pA2 values of 10 and 11 are 7.54 and 7.50, both significantly lower than those previously reported.(ABSTRACT TRUNCATED AT 400 WORDS)

An exploration of the effects of L- and D-tetrahydroisoquinoline-3-carboxylic acid substitutions at positions 2, 3 and 7 in cyclic and linear antagonists of vasopressin and oxytocin and at position 3 in arginine vasopressin.

We have investigated the effects of mono-substitutions with the conformationally restricted amino acid, 1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid (Tic) at position 3 in arginine vasopressin (AVP), at positions 2, 3 and 7 in potent non-selective cyclic AVP V2/V1a antagonists, in potent and selective cyclic and linear AVP V1a antagonists, in a potent and selective oxytocin antagonist and in a new potent linear oxytocin antagonist Phaa-D-Tyr(Me)-Ile-Val-Asn-Orn-Pro-Orn-NH2 (10). We report here the solid-phase synthesis of peptide 10 together with the following Tic-substituted peptides: 1. [Tic3]AVP: 2. dICH2)5[D-TIc2]VAVP: 3, d(CH2)5[D-Tyr(Et)2Tic3]VAVP: 4, d(CH2)5[Tic2Ala-NH2(9)]AVP: 5. d(CH2)5[Tyr]Me)2.Tic3,Ala-NH2(9)]AVP: 6. d(CH2)5 [Tyr(Me)2,Tic7]AVP: 7, Phaa-D-Tyr(Me)-Phe-Gln-Asn-Lys-Tic-Arg-NH2: 8, desGly-NH2,d[CH2]5[Tic2,Thr4]OVT: 9. desGly-NH2d(CH2)5[Tyr(Me)2Thr4, Tic7[OVT; 11, Phaa-D-Tic-Ile-Val-Asn-Orn-Pro-Orn-NH2, using previously described methods. The protected precursors were synthesized by the solid-phase method, cleaved, purified and deblocked with sodium in liquid ammonia to give the free peptides 1-11 which were purified by methods previously described. Peptides 1-11 were examined for agonistic and antagonistic potency in oxytocic (in vitro, without Mg2+) and AVP antidiuretic (V2-receptor) and vasopressor (V1a-receptor) assays. Tic3 substitution in AVP led to drastic losses of V2, V1a and oxytocic agonistic activities in peptide 1, L- and D-Tic2 substitutions led to drastic losses of anti-V2/anti-V1a and anti-oxytocic potencies in peptides 2, 4, 8 and 11 (peptide 2 retained substantial anti-oxytocic potency; pA2 = 7.25 +/- 0.025). Whereas Tic3 substitution in the selective V1a antagonist d(CH2)5[Tyr(Me)2,Ala-NH2(9)]AVP(C) led to a drastic reduction in anti-V1a potency (from anti-V1a pA2 8.75 to 6.37 for peptide 5, remarkably, Tic3 substitution in the V2/V1a antagonist d(CH2)5(D-Tyr(Et)2]VAVP(B) led to full retention of anti-V2 potency and a 95% reduction in anti-V1a potency. With an anti-V2 pA2 = 7.69 +/- 0.05 and anti-V1a pA2 = 6.95 +/- 0.03. d(CH2)5[D-Tyr(Et)2, Tic3]VAVP exhibits a 13-fold gain in anti-V2/anti-V1a selectivity compared to (B). Tic7 substitutions are very well tolerated in peptides 6, 7 and 9 with excellent retention of the characteristic potencies of the parent peptides. The findings on the effects of Tic3 substitutions reported here may provide promising leads to the design of more selective and possibly orally active V2 antagonists for use as pharmacological tools and as therapeutic clinical agents for the treatment of the syndrome of the inappropriate secretion of antidiuretic hormone (SIADH).

Potent V2/V1a vasopressin antagonists with C-terminal ethylenediamine-linked retro-amino acids.

We report the solid-phase synthesis and antagonistic potencies of 25 analogues (1-25) of [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-ethyl-D-tyrosine,4-valine]arginine-vasopressin (d(CH2)5D-Tyr(Et)2-VAVP) (A) and of the related Ile4 (D) and [D-Phe2,Ile4] (E) analogues, potent antagonists of the antidiuretic (V2-receptor) and of the vasopressor (V1a-receptor) responses to arginine-vasopressin (AVP). Six of these peptides (1, 13, 17, 19, 21, and 23) have the Pro-Arg-Gly-NH2 tripeptide side chain fully or partially replaced or extended by ethylenediamine (Eda). The remaining 19 peptides have L- or D-amino acids retrolinked to these six C-terminal Eda peptides. Peptides 1, 13, 17, and 19 all have the ring structure of (A). Their side-chain structures are as follows: 1, Eda; 13, Pro-Eda; 17, Pro-Arg-Eda; 19, Arg-Gly-Eda. Peptide 21 is the Pro-Arg-Eda analogue of D; peptide 23 is the Pro-Arg-Gly-Eda analogue of E. Peptide 2 is the retro-Arg analogue of 1. Its side-chain structure is Eda<--Arg. Peptides 3-6 are analogues of 2 which have the D-Tyr-(Et)2 residue replaced by L-Tyr(Et)2 (3), D-Phe2 (4), D-Ile2 (5), or D-Leu2 (6), respectively. Peptides 7-12 are analogues of 2 which have the C-terminal retro-Arg replaced in retrofashion by D-Arg (7), Gly (8), Orn (9), D-Orn (10), D-Lys (11), or Arg-Arg (12). Peptides 14-16 have D-Orn (14), D-Lys (15), and D-Arg (16) retrosubstituted to peptide 13. Peptides 18, 20, and 22 are the retro-Arg-substituted analogues of 17, 19, and 21, respectively. Peptides 24 and 25 have Val and D-Val in retrolinkage with 23, respectively. All 25 peptides were examined for agonistic and antagonistic potencies in AVP V2/V1a assays. With the exception of peptides 5 and 6, all exhibit potent anti-V1a antagonism, with anti-V1a pA2 values in the range 7.64-8.33.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and some pharmacological properties of potent and selective antagonists of the vasopressor (V1-receptor) response to arginine-vasopressin.

We report the solid-phase synthesis of eight position-9-modified analogues of the potent V1-receptor antagonist of arginine-vasopressin, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-methyltyrosine]arginine-vasopressin (d(CH2)5Tyr(Me)AVP) (1-8) and five position-9-modified analogues of the closely related beta,beta-dimethyl less potent V1 antagonist, [1-deaminopenicillamine,2-O-methyltyrosine]arginine-vasopressin (dPTyr(Me)AVP) (9-13). In d(CH2)5Tyr(Me)AVP the C-terminal Gly-NH2 was replaced by (1) ethylenediamine (Eda), (2) methylamine (NHMe), (3) Ala-NH2, (4) Val-NH2, (5) Arg-NH2, (6) Thr-NH2, (7) Gly-Eda, (8) Gly-N-butylamide (Gly-NH-Bu); in dPTyr(Me)AVP the C-terminal Gly-NH2 was replaced by (9) Ala-NH2, (10) Val-NH2, (11) Thr-NH2, (12) Arg-NH2, and (13) Tyr-NH2. All 13 analogues were tested for agonistic and antagonistic activities in in vivo rat vasopressor (V1-receptor) and rat antidiuretic (V2-receptor) assays. They exhibit no evident vasopressor agonism. All modifications in both antagonists were well-tolerated with excellent retention of V1 antagonism and striking enhancements in anti-V1/anti-V2 selectivity. With anti-V1 pA2 values of 8.75, 8.73, 8.86, and 8.78, four of the analogues of d-(CH2)5Tyr(Me)AVP (1-3 and 6) are equipotent with d(CH2)5Tyr(Me)AVP (anti-V1 pA2 = 8.62) but retain virtually none of the V2 agonism of d(CH2)5Tyr(Me)AVP. They are in fact weak V2 antagonists and strong V1 antagonists with greatly enhanced selectivity for V1 receptors relative to that of d(CH2)5Tyr(Me)AVP. With anti-V1 pA2 values respectively of 8.16, 8.05, 8.04, 8.52, and 8.25, all five analogues (9-13) of dPTyr(Me)AVP are at least as potent V1 antagonists as dPTyr(Me)AVP (pA2 = 7.96) and three of these (9, 12, 13) actually show enhanced V1 antagonism over that of dPTyr(Me)AVP. In fact, the Arg-NH2(9) analogue (12) is almost equipotent with d(CH2)5Tyr(Me)AVP. These new V1 antagonists are potentially useful as pharmacological tools for studies on the cardiovascular roles of AVP. Furthermore the analogues of dPTyr(Me)AVP may be useful in studies on the role(s) of AVP in the V1b-receptor-mediated release of ACTH from corticotrophs.

Design of potent and selective linear antagonists of vasopressor (V1-receptor) responses to vasopressin.

We report the solid-phase synthesis of 21 linear analogues of A and D, two nonselective antagonists of the vasopressor (V1) and antidiuretic (V2) responses to arginine vasopressin (AVP). A is Aaa-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (where Aaa = adamantylacetyl at position 1). D is the des-Arg9 analogue of A. Nine new analogues of A (1-9) and 12 new analogues of D (10-21) were obtained. The following substitutions either alone or in combination were incorporated in A and/or in D: phenylacetic acid (Phaa) and tert-butylacetic acid (t-Baa) at position 1; D-Tyr2, D-Tyr(Me)2; Gln4; Arg6, Lys6, Orn6, MeAla7. The nine new analogues of A are (1) [Arg6], (2) [Lys6], (3) [Orn6], (4) [Phaa1,Lys6], (5) [Phaa1,Orn6], (6) [D-Tyr2], (7) [D-Tyr2,Arg6], (8) [Phaa1,D-Tyr2], (9) [Phaa1,D-Tyr2,Arg6]. The 12 new analogues of D are (10) [Arg6], (11) [Lys6], (12) [Orn6], (13) [Phaa1,Lys6], (14) [Phaa1,Gln4,Lys6], (15) [Phaa,D-Tyr(Me)2,Lys6], (16) [Phaa,D-Tyr(Me)2,Gln4,Lys6], (17) [Phaa1,D-Tyr2,Gln4,Lys6], (18) [t-Baa1,Lys6], (19) [t-Baa1,Gln4,Lys6], (20) [Arg6,MeAla7], (21) [t-Baa1,Arg6,MeAla7]. All 21 peptides were examined for agonistic and antagonistic potencies in AVP V2 and V1 assays in rats. With the exception of 6, the eight remaining new analogues of A are equipotent or more potent than A as V1 antagonists. Peptides 2-9 are less potent than A as V2 antagonists. Three, 4, 5, and 9, exhibit significant gains in anti-V1/anti-V2 selectivities (selectivity ratio = 41, 14, and infinite, respectively), compared to A (anti-V1, pA2 = 7.75 +/- 0.07; selectivity ratio = 0.44). Peptide 9 is unique in both series. It is a highly potent V1 antagonist (anti-V1 pA2 = 8.62 +/- 0.11 and is the first linear peptide to exhibit substantial antidiuretic agonism (4.1 +/- 0.2 units/mg). With the exception of 12, the remaining 11 analogues of D are 8-40 times more potent than D as V1 antagonists. Eight of these peptides exhibit significant gains in anti-V1/anti-V2 selectivities compared to D (anti-V1 pA2 = 7.43 +/- 0.06; selectivity ratio = 1.6).(ABSTRACT TRUNCATED AT 400 WORDS)

Invertebrate neuropeptides resembling vasotocin and some analogues: synthesis and pharmacological properties.

The solid phase synthesis of three invertebrate vasopressin-oxytocin homologs: AVP-like factor, F1(1), ([Leu2, Thr4] AVT)2 isolated from subesophageal and thoracic ganglia of Locusta migratoria3, Arg-conopressin-S4. ([Ile2, Arg4] AVT), Lys-conopressin-G4 ([Phe2, Arg4] LVT), both isolated from the venom of fish-hunting marine snails of the genus Conus and six of their analogues is reported. These analogues are: [Arg4] AVT, [Ile2] AVT, [Leu2] AVT, [Phe2, Arg4] AVT, [Arg4] LVT and [Ile2, Arg4] LVT. All peptides were tested for antidiuretic and vasopressor activities.