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

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.


| 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 Less well known is that nitrite enzymatically reacts with deoxyhaemoglobin to generate nitric oxide in the RBC, which serves as an "erythrocrine," that is endocrine carrier and exporter of NO . in the circulation under low oxygen conditions. 3 Platelets are disc-shaped cell fragments, 1/14th the volume of erythrocytes, that change shape and aggregate at wound sites to ini-stimuli such as collagen, 4 adenosine diphosphate (ADP), epinephrine, thromboxane A2 (TxA2) and thrombin. In addition to sealing vascular breaches, platelets are also implicated in the pathogenesis of hypertension, hypercholesterolaemia, cigarette smoking, 5 diabetes and cancer 6 and their activation predisposes to thrombotic vascular complications, leading to ischaemia and the development of coronary, cerebrovascular and peripheral artery disease. 7 Excessive activation, therefore, is tightly regulated, mostly through the action of inhibitory factors such as NO, released continually by eNOS (endothelial nitric oxide synthase) in the endothelium (except under hypoxic conditions when its activity is impaired), that limits both platelet adhesion to the vascular wall and platelet aggregation by activation of cGMP/ PKG pathway, which in turn leads to reduction in concentration of Ca 2+ with vasodilation and increased blood flow. 8,9 Nonetheless, a common feature of endothelium dysfunction is reduced NO bioavailability, which induces abnormal platelet activation leading to thrombus formation and vessel occlusion.
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 NO 15 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 plateletrich plasma (PRP) with those of diethylenetriamine NONOate (DETA-NONOate), which spontaneously releases nitric oxide in aqueous solution. A diagram, which depicts NO release from RRx-001, DETA-NONOate and eNOS in the endothelium, is shown in

| RBC aggregation
Red blood cells were separated from the blood by centrifugation at

| Coagulation studies
Coagulation studies were performed on plasma separated at 4°C from citrated whole blood.

| Platelet aggregation and ATP release
Whole blood was centrifuged at 1100 g for 10 minutes.

| Statistical analysis
All data are means ± SD. Statistical analysis was analysed by Graph-Pad Prism version 6 (GraphPad software Inc., San Diego, CA, USA).
Correlation was analysed by Pearson's method. ANOVA with Tukey's multiple comparison were used to compare with acceptable P-value, 0.05. F I G U R E 1 A Diagram, which Depicts Four Methods of NO Release, Leading to Increased Blood Flow: (a) nitric oxide is postulated to be released from RRx-001 (formula shown below) directly via a Neflike reaction; (b) RRx-001 binds to the betaCys93 residue on haemoglobin, which accelerates the formation of nitric oxide from deoxyhemoglobin in RBCs; (c) spontaneous release of nitric oxide from a NONOate in aqueous media; (d) production of NO via eNOS in the endothelium 3 | RESULTS

| RRx-001 had no effect on RBC rouleaux formation or aggregation
In red blood cells exposed to RRx-001, no rouleaux formation or increased aggregation was observed compared with vehicle or untreated RBCs, as shown in Figure 2.

| RRx-001 modified ADP, collagen and epinephrine-induced platelet aggregation but not coagulation parameters
At concentrations of 10 mg/mL, RRx-001 did not affect coagulation parameters (APTT, PTT and fibrinogen were all within normal range) and, similarly, thrombocyte count was within the normal range (278 ± 54 × 10 3 /mm 3 ). However, RRx-001 non-significantly attenuated or modified platelet adhesion/aggregation induced by collagen, epinephrine or ADP, as shown in Figure 3.

DETA-NONOate are reversed by the addition of CPTIO, a nitric oxide scavenger
Platelet aggregation was studied in platelet-rich plasma (PRP). In PRP, DETA-NONOate and RRx-001 both exhibited a dose-dependent inhibition of platelet aggregation induced by 8 μmol/L ADP; however, the effect of DETA-NONOate on platelet aggregation was much more pronounced that RRx-001. However, the antiplatelet effects of both DETA-NONOate and RRx-001 were reversed by CPTIO, an NO scavenger, which suggests an NO-mediated action. (Figure 4).

DETA-NONOate are dependent on haematocrit
In platelet aggregation measured by impedance (electrical resistance), the magnitude of effect for RRx-001 and DETA-NONOate varied inversely with each other depending on the haematocrit. As RRx-001 stimulates red blood cells to reduce nitrite to NO and since haemoglobin scavenges nitric oxide, the effect of haematocrit on platelet inhibition was tested. The impedance aggregometry method was used because RBCs interfered with the light transmission method. The inhibitory effect of DETA-NONOate on platelet aggregation decreased in the presence of RBCs with a 20% haematocrit attenuating the effect of DETA-NONOate to a greater extent than a 1% haematocrit, presumably due to NO destruction by haemoglobin in RBCs. By contrast, the inhibitory effect of RRx-001 on platelet aggregation increased in the presence of RBCs with a 20% haematocrit > than 1% haematocrit, likely due to stimulation of the nitrite reductase function of deoxyhaemoglobin, which converts nitrite to NO. However, irrespective of the haematocrits, the antiplatelet effects of both DETA-NONOate and RRx-001 were reversed by CPTIO. (Figure 5).

| The addition of nitrite enhanced the inhibitory action of RRx-001 on platelet activation
The potential effect of nitrite addition (0.1-10 μmol/L) on the RRx-001-mediated regulation of platelet activity, given that RRx-001-F I G U R E 2 A, Blood Smear for Aggregation Studies Obtained from Heparinized Human Blood Untreated or Treated with Vehicle, RRx-001 (10 mg/kg) or Dextran 500 kDa (10 mg/kg in Sterile Saline, Positive Control). From left to right vehicle-treated blood, RRx-001-treated blood and Dextran-treated blood. Clumping and rouleaux formation is only seen with Dextran, the positive control; B, RBC aggregation index of human blood untreated, or treated with Vehicle, RRx-001 (10 mg/kg) or Dextran 500 kDa (10 mg/kg in sterile saline, positive control). RBC aggregation index was unchanged between untreated blood, vehicle-treated blood and RRx-001 treated blood. Only Dextran, the positive control, showed a significant (P < 0.05) increase in RBC clumping and aggregation (far right panel) bound deoxyhaemoglobin reduces nitrite to NO, was examined. In  between RRx-001 and the CVA event in this one patient is improbable (although not impossible).
Currently, RRx-001 is dosed as a single agent and in combination with standard chemotherapy and radiation; its mechanism of anticancer action is thought to be related to pan-epigenetic inhibition 21,22 and the repolarization of tumour associated macrophages and neutrophils 23 ; however, the synergistic activity between nitrite and RRx-001 on platelet aggregation suggests that co-administration with nitrite, which would lead to overproduction of NO . and other genotoxic reactive nitrogen species under the hypoxic conditions presumably endemic only to tumours, may not only result in enhanced cytotoxicity but also improved safety, as patients with cancer are prone to increased platelet activation and thromboembolic events.
In conclusion, these data, which confirm previous observations that RRx-001 catalyses the conversion of nitrite to nitric oxide from deoxyhaemoglobin 24 suggest that RRx-001, apart from its anticancer effects and unlike all other known NO-donating agents, reduces platelet aggregation and protects against thromboembolism as haemoglobin and haematocrit levels increase. Moreover, also unlike other known NO-donating agents, because RRx-001 mediates increased nitric oxide production under hypoxic conditions, NO is released locally on demand where it is most needed when eNOS is impaired.

CONF LICT OF I NTEREST
The authors disclose that EpicentRx, Inc. funds research of RRx-001.