ISOLATION AND CHARACTERIZATION OF THE RAT LIVER AVP RECEPTOR USING [125I][d(CH2)5''SARCOSINE7]AVP1

1. A vasopressin (AVP) binding protein was purified from rat liver membranes by an improved method using [125I][d(CH2)5'Sarcosine7]AVP, a selective V1 AVP radioligand and a combination of CHAPS solubilization, gel filtration, lectin affinity and FPLC ion exchange chromatography. 2. The purified protein exhibited a maximum binding activity of 2480 pmol/mg protein with a KD of 4.5 nmol/L, which corresponds to a purification of approximately 26,700-fold. The molecular weight of this protein was 70,000 Da. 3. The binding of [125I][d(CH2)5'Sarcosine7]AVP to the solubilized membranes was dependent on the protein concentration, and was inhibited by the unlabelled peptides [d(CH2)5'Sarcosine7]AVP, AVP, and to a lesser degree by peptides with high V2 receptor affinity, such as 1-desamino-D-AVP and [d(CH2)5'D-Ileu2-Ileu4]AVP. 4. In addition, an AVP anti-idiotypic monoclonal antibody bound to both the partially purified and purified lectin affinity AVP binding protein in a concentration-dependent manner. These results indicate that the purified protein displays similar characteristics to the liver membrane-bound AVP V1 receptor.     

CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO A RAT LIVER VASOPRESSIN RECEPTOR

1. Balb/c mice were immunized against a vasopressin binding protein purified from rat liver. The hybrids produced from two cell fusions were screened against this receptor. Three hybrids were selected, cloned and expanded in serum-free media. The monoclonal antibodies (MoAb) secreted by these three hybrids were of the subclass IgM and were able to immunoprecipitate [125I]-labelled purified receptor. 2. All three MoAb bound to the purified solubilized receptor, crude liver and kidney membranes in a concentration-dependent manner. However, the binding of MoAb to the membranes did not inhibit the binding of [125I]-[d(CH2)5,Sar7]AVP, a selective V1 receptor radioligand, to the liver membrane-bound receptor. 3. These results suggest that the three MoAb recognize epitopes on the V1 receptor which are not denatured by solubilization, but are common to both rat liver and kidney membranes.     

Investigational vasopressin receptor modulators in the pipeline

The vasopressin system is complex and interacts with the central nervous, cardiovascular, renal, and hematological systems. Vasopressin plays an important role in the control of blood osmolarity and vascular tone, but is also involved in many other physiological events, which are mediated mainly via three types of vasopressin receptor: V₁R, V₂R, and V₃R. V₁R primarily mediate the vascular, and V₂R the aquaretic, effects of vasopressin. Vasopressin may also interact with other receptors, like adrenergic and angiotensin-II receptors, or with distinct biological pathways, including those of nitric oxide and the K_ATP channel. There are numerous clinical situations where vasopressin receptor modulators (agonists or antagonists) could be used. Currently, vasopressin and terlipressin are most commonly used to stimulate V₁R in vasodilatory shock and cardiac arrest, while desmopressin, a synthetic analogue of vasopressin, acts on V₂R; but new molecules are becoming available in the treatment of inappropriate antidiuretic hormone (ADH) secretion.     

加压素及其类似物研究进展

加压素用于尿崩症、食管静脉曲张出血等疾病治疗已经在临床上应用多年。本文简述了加压素及加压素受体的结构、分布及生理功能,介绍了对加压素结构修饰而产生的一系列类似物及其不同的生物活性,加压素及其类似物在各药典收载及质量控制情况,以及加压素及其类似物质量标准中对杂质的要求,并对加压素在临床上的应用情况和前景进行了展望。     

Recent advances in the treatment of hyponatremia

Hyponatremia is a common and potentially serious electrolyte disorder, most often caused by excessive arginine vasopressin (AVP) secretion. Conventional management of hyponatremia is based on graded steps starting from water restriction in mild cases to administration of saline in symptomatic cases, which may not be effective. A major new approach in the treatment of hyponatremia is the development of AVP receptor antagonists that directly inhibit the effect of increased AVP which results in the excretion of electrolyte free water. This review summarizes pathophysiology, conventional treatment and future directions in the treatment of hyponatremia.     

Vasopressin V1a and V1b receptor modulators: a patent review (2012 – 2014)

Introduction: The vasopressin V1a and V1b receptors are involved in many crucial physiological, reproductive, behavioral and social functions. Consequently, they are also involved in several pathological conditions, thus the ligands capable of selective stimulation/inhibition of these receptors may present therapeutic benefit in a variety of diseases.

Areas covered: In this review, the author focuses on the vasopressin V1a and V1b receptors, their biological functions and agonists and antagonists patented in the years 2012 – 2014. This paper is divided according to both the target receptor and the applicant and describes the compounds from the patents along with their biological activity.

Expert opinion: In the recent years, pharmaceutical companies have discovered and patented new compounds which act through vasopressin V1a and/or V1b receptors, both peptide and non-peptide. Among the V1bR antagonists published in the last years, the oxindole derivatives appear to be the most promising drug candidates.     

MONOCLONAL ANTIBODIES TO ARGININE VASOPRESSIN RECEPTOR BIND TO LIVER, KIDNEY AND PITUITARY MEMBRANES

1. A vasopressin binding protein purified from rat liver membranes was used to immunize Balb/c mice and, subsequently, for the screening of hybrids raised in two different cell fusions. 2. Three hybrids were obtained which secreted monoclonal antibodies (MoAb) that bound to the purified solubilized receptor as detected by an enzyme-linked immunosorbent assay technique. All three MoAb immunoprecipitated the purified receptor. 3. In addition, the MoAb bound in a concentration-dependent manner to crude liver, kidney and anterior pituitary membranes, tissues known to contain arginine vasopressin (AVP) receptors but not to cardiac ventricle membranes which lack AVP receptors. 4. However, the binding of [¹²⁵I]-[d(CH₂)₅, Sar⁷]AVP (a specific radiolabelled V₁ antagonist) to the membrane-bound receptor was not inhibited by these antibodies. 5. These results suggest that MoAb recognize epitopes which are common to rat liver, kidney and anterior pituitary membranes but are not at the ligand binding site.     

EFFECTS OF AN ORALLY ACTIVE VASOPRESSIN V1 RECEPTOR ANTAGONIST

1. This paper reports on the in vitro and in vivo characteristics of a non-peptide vasopressin V₁ receptor antagonist 1-{1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-quinolinone (OPC-21268). 2. OPC-21268 caused a concentration-dependent displacement of the selective V₁ receptor antagonist radioligand, [¹²⁵I]-[d(CH₂)₅, sarcosine⁷]AVP from vasopressin V₁ receptors in rat liver and kidney membranes, inhibitory concentration of 50% (IC₅₀) 4 ± 10-⁸, 0.3 mol/L liver and 1.5 ± 10-⁸, 0.2 mol/L kidney. OPC-21268 had little effect on the selective V₂ antagonist radioligand [³H]desGly-NH₂⁹-d(CH₂)₅[d-Ileu², Ileu⁴]AVP binding to V₂ receptors in renal membranes (IC₅₀ > 10-⁴ mol/L). 3. After oral administration to rats, OPC-21268 was an effective V₁ antagonist to both liver and kidney V₁ receptors, in a dose-dependent manner. 4. These studies confirm that OPC-21268 is a potent non-peptide, orally effective V₁ vasopressin receptor antagonist.     

Effects of Viscous Hyaluronate–Sodium Solutions on the Nasal Absorption of Vasopressin and An Analogue

The effects of viscous solutions of hyaluronate-sodium of various average molecular weights (MW) on the nasal absorption of vasopressin (AVP) and its analogue, l-deamino-8-D-arginine vasopressin (l-d-8-DAVP), were examined in rats. Solutions of hyaluronate with MW greater than 3 × 10⁵ daltons enhanced the nasal absorption of AVP; solutions of MW 5.5 × 10⁴ daltons were not effective. The enhancing effects on the nasal absorption of AVP and l-d-8-DAVP were dependent on the concentration in the range of 0–1.5% (w/v) hyaluronate (MW 1.4 × 10⁶ daltons). The nasal absorption of AVP was increased with this solution at lower pH. Bioavailabilities after nasal administration of AVP and l-d-8-DAVP in hyaluronate solutions (MW 1.4 × 10⁶ and 2 × 10⁶ daltons) increased more than 2- and 1.6-fold as compared to nasal administration of AVP and 1-d-8-DAVP in buffer solutions (pH 7.0), respectively. Hyaluronate solution (MW 1.4 × 10⁶ daltons) did not affect the ciliary beat frequency of rabbit nasal mucosal membranes in vitro. Therefore, hyaluronate solution may be useful as a vehicle for nasal delivery of AVP and l-d-8-DAVP.     

Lixivaptan: a novel vasopressin receptor antagonist

Arginine vasopressin, also known as antidiuretic hormone, is a neuropeptide that functions in the maintenance of body water homeostasis. Inappropriate secretion of vasopressin has been implicated in the pathophysiology of multiple diseases, including polycystic kidney disease, syndrome of inappropriate antidiuretic hormone (SIADH) secretion, and the hyponatremia commonly associated with cirrhosis and congestive heart failure. Vasopressin receptor antagonists are novel agents that block the physiologic actions of vasopressin. Lixivaptan is a vasopressin receptor antagonist with high V2 receptor affinity and is now undergoing Phase III clinical trials. Studies so far have demonstrated that lixivaptan is efficacious in the correction of hyponatremia in SIADH, heart failure and liver cirrhosis with ascites, and few adverse effects have been noted. Thus, lixivaptan remains a promising therapeutic modality for the treatment of multiple diseases and prevention of the associated morbidity and mortality associated with hyponatremia.     

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 V_1a, 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 V_1a, 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.

     

Pharmacological chaperones: a new twist on receptor folding

Protein misfolding is at the root of several genetic human diseases. These diseases do not stem from mutations within the active domain of the proteins, but from mutations that disrupt their three-dimensional conformation, which leads to their intracellular retention by the quality control apparatus of the cell. Facilitating the escape of the mutant proteins from the quality control system by lowering the temperature of the cells or by adding chemicals that assist folding (chemical chaperones) can result in proteins that are fully functional despite their mutation. The discovery that ligands with pharmacological selectivity (pharmacological chaperones) can rescue the proper targeting and function of misfolded proteins, including receptors, might help to develop new treatments for ‘conformational diseases’.     

Endogenous vasopressin damages duodenal mucosa during haemorrhagic shock in rats

The role of endogenous vasopressin was studied in the development of mucosal erosions induced by haemorrhagic shock in the duodenum of the rat. Ischaemia-reperfusion provoked duodenal haemorrhagic lesions and elevated circulating and intramucosal vasopressin level. This mucosal injury was significantly attenuated by a vasopressin pressor receptor antagonist. Moreover, in the vasopressin-deficient Brattleboro homozygous rat, mucosal injury induced by haemorrhagic shock was also reduced. By contrast, when the vasopressin agonist, lysin-vasopressin, was administered, significant aggravation of ischaemia-reperfusion-induced duodenal mucosal injury was seen. These findings indicate the aggressive role of endogenous vasopressin, via its pressor receptors, in the generation of duodenal mucosal stress erosions in haemorrhagic shock. This paper was presented at the Symposium on ‘Cell injury and protection in the gastrointestinal tract: from basic science to clinical perspectives’, October 8–11, 1995, Pécs, Hungary.     

HUMAN INTERNAL MAMMARY ARTERY RESPONSES TO NON-PEPTIDE VASOPRESSIN ANTAGONISTS

1. OPC-21268 and OPC-31260 are newly developed orally active non-peptide vasopressin (AVP) V₁ and V₂ receptor antagonists, respectively. The effects of the two compounds on human vessels have not been studied. 2. The effects of the two compounds on AVP-induced contraction of human internal mammary arteries (IMA) were investigated. Their effects were compared with the peptide V₁ and V₂ antagonists d(CH₂)_sSar⁷AVP (SAVP) and d(CH₂)₅D-Ileu²11eu⁴AVP (Ileu² Ileu⁴AVP), respectively. 3. The V₁ antagonist OPC-21268 failed to antagonize AVP-induced contraction at low concentrations and potentiated the contraction at higher concentration (3 × 10^?-⁷ mol/L, P<0.05). It also caused a mild direct contractile effect on IMA. In contrast, the peptide V₁ antagonist SAVP potently inhibited the AVP-induced contraction, indicating that functionally constrictor V₁ receptors exist in IMA. Both the nonpeptide and peptide V₂ antagonists OPC-31260 (3X10^?-⁶ mol/L) and Ileu²Ileu⁴AVP significantly antagonized the AVP-induced contraction (P<0.01). 4. The AVP-induced contraction was reversed by high concentrations of OPC-31260 (10^?-⁶ mol/L-3×10^?-⁵ mol/L) but not by OPC-21268 (up to 3X 10^?-⁵ mol/L). 5. These studies indicate that, in human IMA, OPC-21268 is a partial VI receptor agonist with no V₁ receptor antagonist activity, while OPC-31260 is a V1 receptor antagonist. The results also indicate that Ileu² Ileu⁴AVP may be a V_l receptor antagonist in humans.     

Mechanism of the CNS-mediated pressor response to intracerebroventricular injection of noradrenaline in unanaesthetized rats

Catecholamines administered intracerebroventricularly (i.c.v.) have cardiovascular effects mediated by the CNS. Although hypotension constitutes the more prominent response, an increase in blood pressure has also been reported after the intracerebroventricular injection of these amines. Anaesthesia interferes with pressor responses mediated by the CNS to a larger extent than with depressor mechanisms and constitutes one of the major factors influencing the pattern of response to the amines. The depressor response observed after the intracerebroventricular administration of noradrenaline is reversed into increases in blood pressure in awake animals. In the present experiment, the action of intracerebroventricularly injected noradrenaline was compared in anaesthetized and conscious rats. The results indicated that the pressor response in awake rats was not mediated by the sympathetic nervous system and involved the release of a pituitary humoral-factor, most probably vasopressin, whereas the depressor response observed in anaesthetized animals was not dependent on pituitary mediation. The involvement of histaminergic mechanisms in the CNS in the control of the pressor response to intracerebroventricularly administered noradrenaline in the rat is proposed.     

INTERACTION BETWEEN EPIDERMAL GROWTH FACTOR AND ARGININE VASOPRESSIN IN RENAL MEDULLARY MEMBRANES

1. Epidermal growth factor is a potent mitogen that causes natriuresis, diuresis and inhibition of arginine vasopressin-induced water reabsorption. 2. The aim of this study was to determine any interaction between epidermal growth factor and the V1 (vascular) and/or V2 (antidiuretic) arginine vasopressin receptor subtypes. 3. Radioligand binding displacement assays demonstrated that although arginine vasopressin related peptides displaced both radioligands from renal medullary membranes at low concentrations epidermal growth factor displaced neither. 4. Arginine vasopressin V2 receptor second messenger cyclic adenosine monophosphate (CAMP) production was inhibited by epidermal growth factor (IC₅₀ 2 ± 10^?7 mol/L) as was sodium fluoride cAMP production but only at much higher concentrations. 5. Therefore the diuretic effect of epidermal growth factor is not via direct antagonism of arginine vasopressin receptors but seems mediated via inhibition of the V2 second messenger system.     

Inhibition by Diphenylhydantoin of Vasopressin Release from Isolated Rat Neurohypophyses*

Abstract: Diphenylhydantoin (40 μg/ml) inhibited vasopressin release from isolated hemilobes of rat neurohypophyses, both during resting conditions and after electrical stimulation.     

Tolvaptan for Volume Management in Heart Failure

Volume management in acute decompensated and chronic heart failure (HF) remains a significant challenge. Although progress has been made in the development of mortality-reducing neurohormonal regimens in the reduced ejection fraction population, no clinical trial has yet demonstrated anything more than symptomatic relief or biomarker reduction with pharmacotherapeutic volume-based interventions made in the acutely decompensated individual or those with evolving outpatient congestion. As the number of patients with HF continues to grow, in addition to HF-related hospitalizations, identifying therapies that have the potential to aid in diuresis more safely and efficaciously is paramount to decreasing inpatient length of stay and preventing unnecessary admissions. More recently, a significant amount of research has been dedicated to the use of vasopressin antagonists, specifically tolvaptan, as adjunctive therapy to loop and thiazide diuretics. Although these agents do not seem to have a pervasive role in fluid management in the acute decompensated and chronic HF populations, they are effective tools to have available for specific clinical situations. This review summarizes the literature surrounding the use of tolvaptan for volume management in congestive HF, as well as offering practical guidance for use of this agent.