Eleven new analogues of arginine vasopressin (AVP) modified in position 2 by 3,3-diphenyl-l-alanine or its d-enantiomer (Dip or d-Dip) were synthesized and pharmacologically evaluated for their pressor, antidiuretic and in vitro uterotonic activities. Both the Dip and d-Dip modifications at position 2 of AVP are sufficient to completely change the pharmacological profile of the peptides. They preserve or increase antidiuretic activity, cause its prolongation, transform uterotonic property in antagonistic one and cancel the effect on blood pressure. Four of the new peptides ([Mpa 1,d-Dip 2]AVP, [Mpa 1,d-Dip 2,Val 4]AVP, [Mpa 1,d-Dip 2,d-Arg 8]VP, [Mpa 1,d-Dip 2,Val 4,d-Arg 8]VP) are exceptionally potent antidiuretic agents with significantly prolonged activities.
Arginine vasopressin (AVP), a neurohypophyseal hormone and neuromodulator, is a cyclic nonapeptide with a disulfide bridge between Cys residues at positions 1 and 6 [1]. This results in a six-amino-acid cyclic part and a C-terminal amidated three-residue tail.
Main physiological roles played by AVP are the regulation of water balance, the control of blood pressure, and the secretion of adrenocorticotropin hormone (ACTH) [1], [2], [3]. Moreover, AVP also exhibits to some extent typical oxytocin (OT, a closely related neurohypophyseal peptide) activities such as the galactogogic and the uterotonic effects [4]. Apart from these well-known functions, AVP is also involved in glycogenolysis in the liver [5], in the steroid and catecholamine secretion by the adrenals [6], release of insulin and glucagon by the pancreas [7], and in the secretion of atrial natriuretic factor from the heart [8]. Vasopressin exerts its biological functions upon binding to four different 7-transmembrane G-protein-coupled receptors (GPCRs) termed: V 1a, V 1b, V 2 and OT receptors [1], [2], [3]. V 1a receptors, present in many tissues including brain, mediate the vasopressor actions of AVP [1]. V 1b receptors present in the pituitary, pancreas and adrenals mediate the ACTH-release from the anterior pituitary gland [3], steroid secretion from the adrenal medulla [6] and the insulin and glucagon release from the pancreas [7]. OT receptors control milk ejection and uterine smooth muscle contractions. These three receptor subtypes function via the phosphatidylinositol pathway. The antidiuretic activity of AVP is evoked by V 2 receptors present in the renal tubule and particularly the collecting ducts. They are linked to adenylate cyclase signaling [3].
Vasopressin plays an important role in water metabolism and impairment of its synthesis, secretion or metabolism induces some clinical disorders such as inappropriate antidiuretic hormone secretion syndrome (SIADH) or diabetes insipidus. Elevated AVP secretion leads to renal water retention and extracellular fluid expansion that is compensated for by enhanced urinary Na+ excretion. The combination of water retention and Na+ excretion leads to hyponatremia. Criteria for SIADH were first described by Bartter and Schwartz [9] and are still applicable today. These are: hypotonic hyponatremia, urine osmolality higher than appropriate for the concomitant plasma osmolality, increased natriuresis, absence of edema or volume deletion, and normal renal and adrenal functions [10]. On the other hand, diabetes insipidus is a heterogeneous condition characterized by polyuria and polydipsia caused by lack of vasopressin secretion (physiological suppression following excessive water intake) or kidney resistance to its action [11].
Since 1954, when du Vigneaud et al. characterized and synthesized AVP [12], many analogues of this hormone have been obtained and pharmacologically evaluated. A synthetic vasopressin analogue, V 2 agonist, 1-deamino-8-d-arginine vasopressin (dDAVP, commercial names Minirin, Minrin, Desmopressin, Adiuretin-SD) has been a standard drug for the treatment of diabetes insipidus for over 30 years [13]. On the other hand, V 2 antagonists have potential therapeutic value in the treatment of SIADH [14]. The OT antagonists have been useful for suppressing premature labor [15]. The design of analogues that selectively interact with appropriate AVP receptors is still a matter of interest.
Parallel to the development of selective peptide agonist and antagonists, the research of non-peptide agonists and antagonists was carried out. As a result, a number of interesting compounds of varied structure have been synthesized and tested for their activity. At present there are available non-peptide antagonists for all 4 types of neurohypophyseal hormone receptors [16], [17], e.g. orally active V 2 receptor antagonists (Vaptans), notably, Tolvaptan, Lixivaptan and Satavaptan [16] and the mixed V 1a/V 2 receptor antagonist Conivaptan (YM087) [18]. To date, only Conivaptan (also known as Vaprisol) has been approved by the US FDA for clinical use (by i.v. administration) for the treatment of euvolemic and hypervolemic hyponatremia in hospitalized patients [16]. Especially interesting is the recent development of orally active specific V 1b receptor antagonist (SSR149415) which opened new possibilities for the study and treatment of stress and depression [19].
Biological activity of peptides is determined by their structure and conformation. Conformational restriction is therefore a well-established strategy to change their pharmacological profile. Peptide flexibility can be restricted by a local constraint imposed, e.g. by introducing amino acids with limited conformational freedom that has an impact on specific orientations of the other amino acid side chains and peptide backbone.
It is generally accepted that the conformation of the N-terminal part of neurohypophyseal hormone analogues is crucial for their pharmacological activity [20], [21]. This has also been supported by our already 12-years research focused on the impact of steric restriction and bulky substituent in the N-terminal part of AVP molecule on biological properties of the resulting analogues. We demonstrated that the arginine vasopressin analogues modified at position 2 with either l or d β-(1-naphthyl)-alanine were moderately potent and selective oxytocin antagonists in vitro [22], while [d-Arg 8]VP substituted at position 3 with β-(2-naphthyl)-alanine turned out to be a potent and selective antagonist of the pressor response to AVP [23]. We have also shown that introduction of either 1-aminocyclohexane-1-carboxylic acid (Acc, also known as Ac 6 c) or 1-aminocyclopentane-1-carboxylic acid (Apc, also known as Ac 5 c) into position 2 of AVP and some of its analogues resulted in compounds having high antidiuretic potency, low and graded pressor activity, and either no activity or low oxytocin antagonizing activity in the uterotonic in vitro tests [24], [25], [26]. Recently, we described some pharmacological activities of three analogues having bulky 3,3-diphenyl-l-alanine (Dip) in position 2 [27]. The new peptides had strikingly different biological properties in comparison with those of the parent hormone. Two of them, [Dip 2]AVP and [Mpa 1,Dip 2]AVP, displayed no pressor activity while their antidiuretic potency was preserved and prolonged (a steep dose–response curve). Both were moderately potent blockers of the oxytocin uterotonic activity.
In this study we further checked the influence of the bulky Dip isomers (l- and d, see Fig.1) at position 2 of the AVP and some of its analogues on the pharmacological properties. The synthesized analogues I–XI are listed in Table 1.
Thin-layer chromatography (TLC) was carried out on silica plates (Merck), and spots were visualized with iodine. The solvent system used was butan-1-ol/acetic acid/water/ethyl acetate (1:1:1:1, v/v). High-performance liquid chromatography was carried out on a Waters (analytical and preparative) chromatograph equipped with a UV detector (λ = 226 nm). The purity of the peptides was determined on Vydac C 18 column (5 μm, 4.6 × 250 mm) or Hypersil ODS C 18 column (5 μm, 4.6 × 250 mm) or Hypersil BDS C 18 column
Wistar rats were used in all experiments. Handling of the experimental animals was done under supervision of the Ethics Committee of the Academy of Sciences according to § 23 of the Law of the Czech Republic No. 246/1992.
The uterotonic test was carried out in vitro on the strips of rat uterus in the absence of magnesium ions [35], [36]. Rats in induced estrus by the injection of estrogen 48 h before the experiments were used. After decapitation, the uterine horns were excised, longitudinally
The 11 new analogues of AVP (I–XI) were synthesized by Boc strategy, purified by HPLC and characterized. The purity of all the analogues was higher than 98%. Physicochemical properties of the new peptides and their pharmacological data are presented in Table 1, Table 2, respectively.
The activities of the analogues were determined in the in vitro rat uterotonic test in the absence of magnesium ions, in the rat pressor test, and in the antidiuretic assay using conscious rats as described in the
We have previously demonstrated significant impact of steric restrictions and bulkiness of the substituents in the N-terminal part of the AVP and OT molecules on pharmacological properties of the resulting analogues. Our studies included among others the modification of AVP and some of its analogues with Acc and Apc, which resulted in compounds having high antidiuretic potency, decreased pressor activity and either no activity or low oxytocin antagonizing activity in the uterotonic in vitro
In summary, our studies have shown once more that the modification of position 2 which changes conformation of the N-terminal part of the analogue has a dramatic impact on the pharmacological profile of the compound (in this case it resulted in a high and prolonged V 2 agonistic activity). This finding supports our previous results showing that single substitution with some bulky residues may result in quite potent antagonists of OT receptors [44]. Such substitution is clearly disadvantageous
Partial funding for this work was provided by grant 0230/B/H03/2008/35 and by research project no. Z4055905 of the Academy of Sciences of the Czech Republic.
