Synthesis of new vasotocin analogues: effects on renal water and ion excretion in rats.

Vasopressin and nonmammalian hormone vasotocin are known to increase the water permeability of mammalian collecting ducts, frog skin and the urinary bladder. Neurohypophysial nonapeptides have also been shown to interfere with the regulation of renal ion transport. The subject of this study was a search for vasopressin and vasotocin analogues with selective effects on renal water, sodium and potassium excretion. During this study, we synthesised the following peptides: 13 vasotocin analogues modified at positions 4 (Thr or Arg), 7 (Gly or Leu) and 8 (D-Arg, Lys or Glu); 4 vasopressin analogues modified at positions 4 and 8; and 9 peptides shortened or extended at the C-terminal or with substitutions for Gly-NH2. Most of these peptides had mercaptopropionic acid (Mpa) instead of Cys in position 1. The effects of these nonapeptides on renal water, sodium and potassium transport were evaluated in in vivo experiments using Wistar rats. Some nonapeptides possessed antidiuretic, natriuretic and kaliuretic activities ([Mpa(1)]-arginine vasotocin, [Mpa(1), homoArg(8)]-vasotocin, [Mpa(1), Thr(4)]-arginine vasotocin and [Mpa(1), Arg(4)]-arginine vasopressin). Substitutions at positions 4 and 8 increased the selectivity of peptide actions. The antidiuretic [D-Arg(8)]-vasotocin analogues had no effects on sodium excretion. [Mpa(1), Arg(4)]-arginine vasotocin was antidiuretic and kaliuretic but not natriuretic. [Mpa(1), Glu(8)]-oxytocin had weak natriuretic activity without any effects on water and potassium transport. In accordance with the data obtained, synthesised vasotocin analogues could be good candidates for pharmaceuticals selectively regulating renal sodium and potassium transport, which is of clinical importance.

[Selective effect of vasotocin analogues with amino acid substitutions in 4, 7, 8 positions on the water and cations excretion by the rat kidney].

Analogues ofarginine vasotocin with replacement of amino acids in 4, 7 and 8 positions of the hormone were synthesized. After water loading the injection of 1 x 10(-12) mol per 100 g BW 1-deamino-8-homoarginine vasotocin, 1-deamino-4-threonine-8-arginine vasotocin and 1-deamino-4-threonine-8-D-arginine vasotocin increased solute-free water reabsorption, but did not affect cations excretion (Na, K, Ca, Mg). Presence of glycine in 9th position and proline in 7th position ofa vasotocin molecule is essential for hormone interaction with a V2-receptor. In rats, injection of 5 x 10(-11) mol 1-deamino-8-homoarginine vasotocin or 1-deamino-4-threonine-8-arginine vasotocin dramatically increased urinary sodium and potassium excretion and enforced osmotically free water reabsorption but very little affected urinary magnesium and calcium excretion. The injection in the same dose of other synthesized vasotocin analogues did not affect the cations excretion. The issue of physiological mechanisms of kidney's selective response related to water, one- and bivalent cations excretion after injection of vasotocin analogues is discussed. Inhibition of cations transport is associated with activation of any V-receptor (but not V2-receptor). This effect takes place only in presence of L-arginine instead of D-arginine in the analogue.

Synthesis of oxytocin analogues with replacement of sulfur by carbon gives potent antagonists with increased stability.

[Chemical reaction: See text] The neuropeptide oxytocin 1 controls mammary and uterine smooth muscle contraction. Atosiban 2, an oxytocin antagonist, is used for prevention of preterm labor and premature birth. However, the metabolic lifetimes of such peptide drugs are short because of in vivo degradation. Facile production of oxytocin analogues with varying ring sizes wherein sulfur is replaced by carbon (methylene or methine) could be achieved by standard solid-phase peptide synthesis using olefin-bearing amino acids followed by on-resin ring-closing metathesis (RCM). These were tested for agonistic and antagonistic uteronic activity using myometrial strips taken from nonpregnant female rats. Peptide 8 showed agonistic activity in vitro (EC50= 1.4 x 10(3) +/- 4.4 x 10(2) nM) as compared to 1 (EC50= 7.0 +/- 2.1 nM). Atosiban analogues 17 (pA2= 7.8 +/- 0.1) and 18 (pA2= 8.0 +/- 0.1) showed substantial activity compared to the parent oxytocin antagonist 2 (pA2= 9.9 +/- 0.3). Carba analogue 35 (pA2= 6.1 +/- 0.1) had an agonistic activity over 2 orders of magnitude less than its parent 3 (8.8 +/- 0.5). A comparison of biological stabilities of 1,6-carba analogues of both an agonist 8 and antagonist 18 versus parent peptides 1 and 2 was conducted. The half-lives of peptides 8 and 18 in rat placental tissue were shown (Table 2) to be greatly improved versus their parents oxytocin 1 and atosiban 2, respectively. These results suggest that peptides 8 and 18 and analogues thereof may be important leads into the development of a long-lasting, commercially available therapeutic for initiation of parturition and treatment of preterm labor.

Improved radiotracing of oxytocin receptor-expressing tumours using the new [111In]-DOTA-Lys8-deamino-vasotocin analogue.

Oxytocin receptors (OTR) have been described in a number of tumours of different origin, and represent a new target for specific radiolabelled oxytocin (OT) analogues in cancer diagnosis and therapy. By linking the DOTA chelating agent to position 8 of the deamino derivative of Lys(8)-vasotocin (dLVT), we obtained a new compound (DOTA-dLVT) with the following characteristics: (1) it forms a monomeric and stable compound that binds to OTR with an affinity comparable to that of the endogenous OT ligand; (2) it is characterised by a very good selectivity profile for the human OTR, with a low affinity binding to the closely related V1a, V1b and V2 vasopressin receptor subtypes; (3) it induces rapid and persistent receptor internalisation and (4) when radiolabelled, [(111)In]-DOTA-dLVT is efficiently and selectively taken up by OTR-positive tumours grown in mice. These features makes radiolabelled DOTA-dLVT a very good candidate for the radiotargeting of OTR-expressing tumours.

111In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-lys(8)-vasotocin: a new powerful radioligand for oxytocin receptor-expressing tumors.

We developed a radioactive ligand for tumors expressing oxytocin receptors (OTRs) by linking the chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) to Lys(8)-vasotocin (LVT), an analogue of oxytocin with high affinity for OTRs. The new reagent (DOTA-LVT) retained high affinity for human OTRs, as proved by in vitro affinity binding to cells endogenously expressing OTRs, such as MCF7 breast carcinoma and MOG-U-V-W glioblastoma cells lines, as well as to transiently transfected COS7 cells. In in vivo experiments, DOTA-LVT carrying (111)In showed specific binding activity to OTR-positive TS/A mouse mammary tumors. The present study opens new perspectives for imaging and, possibly, therapy of OTR-positive human tumors such as breast and endometrial carcinomas, neuroblastomas, and glioblastomas.

A new tritiated oxytocin receptor radioligand--synthesis and application for localization of central oxytocin receptors.

A new tritiated oxytocin antagonist radioligand was synthesized by introducing a tritiated propionic acid residue into the free amino group of ornithine in position 8 of the parent peptide [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(O-methyl)-tyrosine, 4-threonine, 8-ornithine, 9-tyrosylamide]vasotocin (OTA), that was previously described. The tritiated compound [3H][1-(beta-mercapto-beta,beta-cyclo-pentamethylene propionic acid), 2-(O-methyl)-tyrosine, 4-threonine, 8-(N6-propionyl)-ornithine, 9-tyrosylamide]vasotocin ([3H]PrOTA) was obtained in good yield with high specific activity (100 Ci/mmol). [3H]PrOTA exhibited the same affinity (Kd = 0.8 nM) and selectivity for the myometrial oxytocin receptor as the iodinated antagonist [125I]OTA. Autoradiographic localization of oxytocin receptors in the rat brain showed specific binding sites for [3H]PrOTA within regions of the limbic system, the neocortex, and hypothalamus, which is consistent with the binding pattern obtained with [125I]OTA. The high specific activity in combination with the long half-life of tritium and its low radiotoxicity as compared to iodine-125 makes the new tritiated antagonist a valuable tool for pharmacological studies.

A fluorescent analogue of hydrin 1: a new probe for vasotocin receptors.

Hydrin 1 is the biosynthetic precursor of vasotocin in Xenopus laevis. We have synthesized deamino and fluorescein analogues of hydrin 1 and characterized their physiological action in the urinary bladder of the toad, Bufo marinus. 1-Deamino-hydrin 1 (d-hydrin) was more potent than vasotocin in stimulating osmotic water flow across intact bladders and more potent than vasotocin in displacing tritium-labeled vasopressin [( 3H]AVP) from cell membranes. 1-Deamino-[11-lysine (fluorescein)]-hydrin 1 (flu-hydrin) was found to be the most potent fluorescent vasotocin receptor probe synthesized to date. Flu-hydrin increased osmotic water flow across bladders with a half-maximal effective dose (ED50) value of 6 x 10(-10) M and displaced [3H]AVP from membranes with a half-maximal concentration (IC50) value of 3 x 10(-9) M. The hydrosmotic response to flu-hydrin was blocked by 1-deamino-[4-lysine (p-azido-benzoyl)]arginine vasotocin [d4Lys(N3)-AVT]. Epifluorescence light microscopic studies showed vesicular uptake of flu-hydrin at the basolateral membrane of toad bladder epithelial cells, and this uptake was blocked by d4Lys(N3)AVT. This study shows that d-hydrin can serve as a foundation molecule to which reporter groups, such as fluorescent residues, can be attached with better preservation of hydrosmotic activity than is possible with similar modifications of vasotocin.

Synthesis and characterization of fluorescein- and rhodamine-labeled probes for vasotocin receptors.

Fluorescent analogues of vasotocin, [1-(beta-mercaptopropionic acid), 9-(p-aminofluoresceinylphenylalanine)]arginine vasotocin [[MPA1, (p-NH2flu)Phe9]AVT] and [1-(beta'-mercaptopropionic acid), 9-(p-amino rhodaminylphenylalanine)]AVT [[MPA1, (p-NH2rhod)-Phe9]AVT], were synthesized by the solid-phase method. These compounds yielded half-maximal hydrosmotic responses (half-maximal values) in the toad urinary bladder at 2 X 10(-9) M. Epifluorescence microscopy showed punctate basal localization of analogues on the majority of bladder epithelial cells within 20 min. The cellular localization was prevented by vasotocin. These fluorescent analogues may serve as useful probes for vasotocin receptors in toad bladder and in tissues from other species that use vasotocin as their antidiuretic-pressor hormone.

Photoaffinity, biotinyl, and iodo analogues as probes for vasotocin receptors.

Vasotocin (AVT) analogues with tyrosine or phenylalanine in position 9, i.e., [9-tyrosine]AVT, [2-phenylalanine,-9-tyrosine]AVT, and 1-desamino[9-(p-aminophenylalanine)]AVT, were synthesized by the solid-phase method. These compounds showed a high biological activity in the hydroosmotic toad bladder assay. Using the chemically reactive functional groups on tyrosine and p-aminophenylalanine in position 9, we prepared iodinated, photoreactive, and affinity ligands, i.e., [2-phenylalanine,9-(iodotyrosine)]AVT, 1-desamino[9-(p-azidophenylalanine)]AVT, and 1-desamino[9-(biotinylphenylalanine)]AVT, respectively. Half-maximal hydroosmotic responses (ED-50 values) were obtained with 2.5 X 10(-9) M for the iodinated analogue, with 0.9 X 10(-10) M for the photoaffinity analogue, and with 1.2 X 10(-8) M for the biotinyl analogue. The hydroosmotic activity of the biotinyl analogue was reversed following addition of avidin, whereas the photoaffinity analogue induced a persistent response following UV irradiation that was not reversed upon repeated and prolonged periods of washout. These analogues of vasotocin are the most potent that have been synthesized to date, and they should serve as useful probes in the isolation and characterization of vasotocin receptors in toad bladders and tissues from other species that use vasotocin as an antidiuretic/pressor principle. The photoaffinity and biotinyl analogues had a rat antidiuretic activity of 66 and 40 units/mg, respectively. These compounds are, therefore, also suitable for the isolation of V-2 vasopressin receptors from mammalian tissues.

Synthesis and biological activities of a photoaffinity probe for vasotocin receptors.

This study reports the synthesis and biological activities of 1-desamino, 7-lysine-(4-azidobenzoyl), 8-arginine vasotocin (d7-N3-AVT). This compound was found to be biologically active in the rat antidiuretic assay (20 U/mg), to behave as an antagonist of vasopressin in the rat pressor assay (pA2 = 6.6), and to yield a half-maximal hydroosmotic response in the isolated toad urinary bladder at a bath concentration of 2.4 X 10(-8) M. When toad bladders were exposed to d7-N3-AVT in the presence of long wavelength UV light, the hydroosmotic response persisted in spite of prolonged and repeated periods of washout. By contrast, the hydroosmotic response in control bladders after stimulation with d7-N3-AVT in the absence of UV irradiation was fully reversed within 15 min of washout. A membrane preparation derived from bladders that had been photolabeled with d7-N3-AVT and washed for 1 h specifically bound 325 fmol [3H]vasopressin/mg protein. Matched bladders exposed to the analog in the absence of UV irradiation and washed for 1 h specifically bound 591 fmol [3H]vasopressin per mg of protein. These studies indicate that d7-N3-AVT binds covalently to hydroosmotic receptors of toad urinary bladder and forms a complex that is functional in triggering an increase in the permeability to water of the epithelium. This analog may prove useful in the isolation and purification of vasotocin receptors in lower vertebrates.

Synthesis and biological activities of a photoaffinity probe for vasotocin and oxytocin receptors.

The present study describes the synthesis and biological activities of the photoreactive vasotocin analog 1-deamino[8-lysine(N epsilon-4-azidobenzoyl)] vasotocin ([Mpa1, Lys(N epsilon-4-azidobenzoyl)8]vasotocin). The analog was obtained by introducing the photoreactive aryl azido group at the epsilon-amino group of Lys8 in [Mpa1, Lys8]-vasotocin, which was synthesized by the solid phase method. In the isolated toad urinary bladder the photoaffinity analog of vasotocin retained hydroosmotic activity in the absence of u.v.-light. After irradiation the osmotic water flow across the bladder wall increased. Moreover, the water permeability remained high during repeated periods of washout, suggesting that the analog formed covalent complexes with vasotocin receptors in the toad bladder. In the rat uterotonic assay the photoreactive vasotocin analog was without photoactivation a mild agonist. These studies suggest that the photoaffinity analog of vasotocin might be useful for the isolation of vasotocin receptors in low vertebrates and oxytocin receptors in mammals.

[Tritium-labeled reactive analogs of [1,6-alpha-aminosuberic acid, 8-arginine] vasopressin (deamino-dicarba-[8-arginine] vasopressin. synthesis and properties]. / Tritium-markierte reaktive Analoga des [1,6-alpha-Aminosuberinsäure, 8-Arginin]-Vasopressins (Desamino-dicarba-[8-Arginin]vasopressin). Synthese und Eigenschaften.

The synthesis of three reactive analogues of [1,6-alpha-aminosuberic acid, 8-arginine]-vasopressin ([Asu1,6,Arg8]vasopressin) is described. Two peptide hormone analogues contain in the p-position of Phe2 the azido or 3-(3-methyl-3-diazirinyl)propanoylamino residue, which can be converted by photoactivation into nitrenes and carbenes, respectively. The third derivative contains the chemically reactive (bromoacetyl)amino group in the same position. The analogues are prepared via the precursor [Asu1,6,Phe(pNH2)2,Arg8]vasopressin, which is obtained by peptide synthesis in solution. Modifications of the p-amino group of Phe(pNH2) give the reactive analogues. The analogue containing p-azidophenylalanine in Pos. 2 shows a similar high binding affinity for the antidiuretic receptor in bovine kidney as vasopressin. By iodination of the Phe(pNH2) residue, followed by catalytic dehalogenation, the 2-(p-amino-phenylalanine) analogue is labelled with tritium at a specific radioactivity of 16 Ci/mmol. It can be converted into the tritium-labelled 2-(p-azidophenylalanine) analogue without loss of binding affinity for vasopressin receptors.

Solid phase synthesis and some hormonal activities of 8-L-homoarginine-vasotocin and 1-deamino-8-L-homoarginine-vasotocin.

8-L-Homoarginine-vasotocin and its 1-deamino derivative were synthesized by the solid phase method. Both compounds possessed high activities in the uterus contraction and fowl depressor assays and showed higher pressor activities than the corresponding vasopressins. Homoarginine-vasotocin, unexpectedly, was a more potent antidiuretic agent than its deamino analogue.