An l -RNA-based aquaretic agent that inhibits vasopressin in vivo

Abstract

A class of diuretic͞aquaretic agents based on mirror-image oligonucleotides (so-called Spiegelmers) has been identified. These molecules directly bind and inhibit the neuropeptide vasopressin (AVP). AVP is the major regulatory component of body fluid homeostasis mediated through binding to the renal V2 receptor. Elevated plasma levels of AVP are implicated in several pathological conditions, mainly cardiovascular diseases. In congestive heart failure, AVP is part of a neuroendocrine imbalance that is responsible for progressive worsening of the disease. Employing in vitro selection techniques, RNA aptamers that bind to the unnatural D-configuration of AVP were isolated. The best aptamer displayed an affinity to D-AVP of Ϸ560 pM at 37°C. The corresponding Spiegelmer, a 38-mer mirror-image oligonucleotide (L-RNA) termed NOX-F37, inhibits vasopressin-dependent activation of V1a as well as V2 receptors with IC50 values of 6.1 nM and 1 nM, respectively. NOX-F37 administered to healthy rats effectively neutralized AVP and increased diuresis dose-dependently for 24 h. The mode of action was strictly aquaretic, i.e., the increase in urine volume was not accompanied by an increase in electrolytes. These results clearly prove the in vivo efficacy of NOX-F37 and points out its potential as a drug in the treatment of diseases that are associated with body fluid overload.aptamer ͉ congestive heart failure ͉ diureses ͉ water homeostasis ͉ Spiegelmer T he cyclic nonapeptide vasopressin (AVP) is a neurohormone that is synthesized in the hypothalamus and stored in the hypophysis from where it is released into the circulation (1). AVP is also known as antidiuretic hormone because its main peripheral function is the maintenance of volume and osmolality of body fluids by regulating the free water reabsorption in the kidneys. Under physiological concentrations, AVP acts vasoconstrictive. AVP's actions are mediated through specific G proteincoupled receptors (2). The V 2 receptor, mainly expressed on cells of the basolateral membrane of the collecting duct in the kidneys, mediates the antidiuretic effect via enhanced cAMP levels (3). As a result, aquaporin channels are inserted into the cytoplasma membranes, and the transcription of aquaporin mRNA is induced. The V 1a receptor, mainly expressed on vascular smooth muscle cells and on cells of the myocardium, exerts vasoconstrictive effects through the second messengers inositoltrisphosphate and diacylglycerol. Subsequently, intracellular Ca 2ϩ is mobilized from the endoplasmic reticulum, and Ca 2ϩ channels as well as Na ϩ ͞H ϩ exchangers are activated (4). In addition to vasoactive effects, binding of AVP to the V 1a receptor also stimulates cell growth and proliferation in vascular smooth muscle cells through activation of the mitogen-activated protein kinase pathway (5).AVP release into the circulation is tightly regulated by osmoreceptors in the hypothalamus and baroreceptors in the heart and large arteries. Whereas osmoreceptors respond to small changes (1%) in osmolality, barorec...

Targeting complement component 5a promotes vascular integrity and limits airway remodeling

“…Spiegelmers act like conventional aptamers by forming a 3D structure that binds with high affinity to the target of choice. A number of Spiegelmers have been generated by in vitro selection (13)(14)(15), and three Spiegelmers are currently being tested in clinical trials. The purpose of this study was to evaluate whether increased vascular thrombin was available to compensate for or to replace as a C5 convertase in a relevant in vivo model and to determine whether interrupting C5a activity through administration of the C5a inhibitor NOX-D19 would preserve microvascular function and airway architecture.…”

Spiegelmer Inhibition of CCL2/MCP-1 Ameliorates Lupus Nephritis in MRL-(Fas)lpr Mice

“…11 Hence, Spiegelmers show excellent biostability without any further chemical modifications, which renders then very well suited for in vitro and in vivo applications. [12][13][14][15] We therefore hypothesized that Spiegelmer-based blockade of CCL2 would be suitable for the treatment of lupus nephritis and other disease manifestations of systemic lupus erythematosus. Here we report the identification of the Spiegelmer mNOX-E36 that specifically inhibits murine CCL2 (mCCL2) in vitro in the absence of type I IFN induction in dendritic cells, recently described for natural and synthetic RNA.…”

Polyetheylenimine-Polyplexes of Spiegelmer NOX-A50 Directed against Intracellular High Mobility Group Protein A1 (HMGA1) Reduce Tumor Growth in Vivo

“…Spiegelmer antagonists to a number of extracellular targets have been described (11,14,24,35). Two Spiegelmers have proven to be safe and well tolerated in Phase I clinical studies 4 providing evidence that the Spiegelmer technology is suitable to generate human medicines.…”

“…By further reasons of analogy, it is most likely that besides HMGA1b, the alternative splice product HMGA1a which also comprises the 21-amino acid fragment chosen for Spiegelmer identification, is recognized. Only few such fragment approaches for aptamers have been published to date (24,37,38).…”

“…In Vitro Selection and Pulldown Assays-Automated and manual in vitro selection experiments were performed against the biotinylated 21-amino acid mirror-image peptide, representing a central sequence of HMGA1a/b in selection buffer resembling intracellular ion conditions and pH (25 mM TrisHCl, pH 7.0, 140 mM KCl, 12 mM NaCl, 0.8 mM MgCl 2 , and 0.1% Tween 20) at 37°C (13,23,24). Pulldown assays were carried out as described (13,23,24).…”