Platelet inhibitory effects of the Phase 3 anticancer and normal tissue cytoprotective agent, RRx-001.

Bryan Oronsky,Neil Oronsky,Pedro Cabrales

doi:10.1111/jcmm.13791

Abstract

The platelet inhibitory effects of the Phase 3 anticancer agent and nitric oxide (NO) donor, RRx‐001, (1‐bromoacetyl‐3,3‐dinitroazetidine) were examined ex vivo and compared with the diazeniumdiolate NO donor, diethylenetriamine NONOate (DETA‐NONOate), which spontaneously releases nitric oxide in aqueous solution. In the absence of red blood cells and in a dose‐dependent manner, DETA‐NONOate strongly inhibited platelet aggregation induced by several stimuli (ADP, epinephrine and collagen) whereas RRx‐001 only slightly inhibited platelet aggregation under the same conditions in a dose‐dependent manner; these antiaggregant effects were blocked when both DETA‐NONOate and RRx‐001 were co‐incubated with carboxy‐PTIO (CPTIO 0.01‐100 micromol), a widely accepted NO scavenger. However, in the presence of red blood cells from healthy human donors, RRx‐001, which binds covalently to haemoglobin (Hb) and catalyses the production of NO from endogenous nitrite, more strongly inhibited the aggregation of platelets than DETA‐NONOate in a dose‐dependent manner likely because haemoglobin avidly scavenges nitric oxide and reduces its half‐life; the RRx‐001‐mediated platelet inhibitory effect was increased in the presence of nitrite. The results of this study suggest that RRx‐001‐bound Hb (within RBCs) plays an important role in the bioconversion of NO2− to NO ., which makes RRx‐001 a more physiologically relevant inhibitor of platelet aggregation than other nitric oxide donors, whose effects are attenuated in the presence of red blood cells. Therefore, RRx‐001‐mediated platelet inhibition is a potentially useful therapeutic property, especially in hypercoagulable cancer patients that are at an increased risk of thrombotic complications.

Highlights

It is well known that haemoglobin (Hb) in circulating red blood cells (RBCs) acts as a high‐affinity depot or storage pool for nitric oxide (NO)[1] and that, cell‐free Hb, an even more avid scavenger of NO, contributes to the pathology of haemolytic anaemias like sickle cell disease, malaria and transfusion of older stored blood.[2]
The main objective of this pilot study was to investigate ex vivo the effects of RRx‐001 and DETA‐NONOate on platelet aggregation induced by adenosine diphosphate (ADP), epinephrine and collagen in whole blood and platelet‐ rich plasma (PRP)
The antiplatelet effects of RRx‐001 and DETA‐NONOate varied depending on the dose and presence or absence of RBCs

Summary

| INTRODUCTION

It is well known that haemoglobin (Hb) in circulating red blood cells (RBCs) acts as a high‐affinity depot or storage pool for nitric oxide (NO)[1] and that, cell‐free Hb, an even more avid scavenger of NO, contributes to the pathology of haemolytic anaemias like sickle cell disease, malaria and transfusion of older stored blood.[2]. RRx‐001, a new class of aerospace‐derived anticancer and normal tissue cytoprotective agent,[10] binds selectively and irreversibly to an accessible and highly conserved thiol on haemoglobin called beta (β) cysteine 93; this binding of RRx‐001 to Hb β cysteine 93 significantly accelerates the rate of reduction in endogenous nitrite, which RBCs carry in bulk,[11] to nitric oxide.[12,13] In addition, to a much lesser extent, one of the nitro groups on RRx‐001 is postulated to be the site of a Nef‐like reaction, a standard reaction of organic nitro derivatives,[14] wherein nitric oxide is non‐enzymatically released This RRx‐001‐mediated hyperinduction of NO15 under hypoxic conditions and lack of hypotensive side‐effects as well as headache, facial flushing, etc.[16] differentiates it from other nitric oxide donors such as organic nitrates, S‐nitrosothiols, diazeniumdiolates‐NONOates, furoxans, zeolites, NO hybrid drugs and hydroxyurea[17] that act systemically, resulting in potential toxicities.[18] The primary objective of this study was to investigate the potential antiaggregant and antithrombotic effects of RRx‐001, as an NO donor, and to compare these ex vivo effects in whole blood and platelet‐ rich plasma (PRP) with those of diethylenetriamine NONOate (DETA‐NONOate), which spontaneously releases nitric oxide in aqueous solution.

| METHODS

| RESULTS

Findings

| DISCUSSION