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RRx-001 is a shape shifting small molecule with Fast Track designation for the prevention/amelioration of chemoradiation-induced severe oral mucositis (SOM) in newly diagnosed Head and Neck cancer. It has been intentionally developed or “engineered” as a chimeric single molecular entity that targets multiple redox-based mechanisms. Like an antibody drug conjugate (ADC), RRx-001 contains, at one end a “targeting” moiety, which binds to the NLRP3 inflammasome and inhibits it as well as Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of Nrf2, and, at the other end, a conformationally constrained, dinitro containing 4 membered ring, which fragments under conditions of hypoxia and reduction to release therapeutically active metabolites i.e., the payload. This “payload”, which is delivered specifically to hypoperfused and inflamed areas, includes nitric oxide, nitric oxide related species and carbon-centered radicals. As observed with ADCs, RRx-001 contains a backbone amide “linker” attached to a binding site, which correlates with the Fab region of an antibody, and to the dinitroazetidine payload, which is microenvironmentally activated. However, unlike ADCs, whose large size impacts their pharmacokinetic properties, RRx-001 is a nonpolar small molecule that easily crosses cell membranes and the blood brain barrier (BBB) and distributes systemically. This short review is organized around the de novo design and in vivo pro-oxidant/pro-inflammatory and antioxidant/anti-inflammatory activity of RRx-001, which, in turn, depends on the reduced to oxidized glutathione ratio and the oxygenation status of tissues.

BackgroundBromonitrozidine (RRx-001) is a minimally toxic, NLRP3 inhibitor that has been observed, in experimental systems, to also downregulate CD47, repolarize tumor associated macrophages (TAMs) and normalize aberrant tumor perfusion. This phase 1 pilot study was undertaken to determine the safety and feasibility of RRx-001 and nivolumab in patients with advanced cancer and no standard options.MethodsThis single arm, single site, open-label pilot study (NCT02518958) called PRIMETIME was designed to evaluate the safety profile of RRx-001 and nivolumab in patients with advanced malignancies and no other standard therapeutic options. A 3 + 3 trial design was used to establish safety of the combination at each dose level and guide the decision to escalate dose. RRx-001 is infused once weekly while nivolumab is given at 3mg/kg once every 2 weeks. The RRx-001 starting dose was 2 mg IV weekly with 4 dose level escalations up to 16 mg IV weekly. From January 2015 to November 2015, twelve patients received treatment for only 4 cycles (total 12 weeks) with the combination due to unavailability of nivolumab, which was not supplied to the Sponsor. Treatment-emergent (all cause, TEAEs) and treatment-related (TRAEs) adverse events that occurred within 16 weeks of the first dose of RRx-001 and nivolumab were characterized according to CTCAE v4.03.ResultsTwelve patients received ≥1 dose of RRx-001 and nivolumab. One discontinuation occurred due to pneumonitis and one to voluntary withdrawal after a post-procedural infection. There were no DLTs. The main adverse event related to RRx-001 was infusion reaction (33.3%). The main adverse event related to the combination was pseudoprogression manifested by larger tumors in patients that were symptomatically improved (25%). The most common immune-related treatment-emergent AEs were pneumonitis (8.3%), and hypothyroidism (8.3%). The objective response rate at 12 weeks was 25% and the disease control rate (DCR) consisting of ≥SD was 67% by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. 25% of the patients progressed on the combination.ConclusionsThe combination of RRx-001 and nivolumab was safe and well-tolerated with preliminary evidence of anti-cancer activity. Further clinical trials with RRx-001 and nivolumab are warranted.Clinical trial registrationClinicalTrials.gov identifier, NCT02518958.

For the past thirty years, hemoglobin-based oxygen carriers (HBOCs) have been under development as a red blood cell substitute. Side-effects such as vasoconstriction, oxidative injury, and cardiac toxicity have prevented clinical approval of HBOCs. Recently, high molecular weight (MW) polymerized human hemoglobin (PolyhHb) has shown positive results in rats. Studies have demonstrated that high MW PolyhHb increased O2 delivery, with minimal effects on blood pressure, without vasoconstriction, and devoid of toxicity. In this study, we used guinea pigs to evaluate the efficacy and safety of high MW PolyhHb, since like humans guinea pigs cannot produce endogenous ascorbic acid, which limits the capacity of both species to deal with oxidative stress. Hence, this study evaluated the efficacy and safety of resuscitation from severe hemorrhagic shock with high MW PolyhHb, fresh blood, and blood stored for 2 weeks. Animals were randomly assigned to each experimental group, and hemorrhage was induced by the withdrawal of 40% of the blood volume (BV, estimated as 7.5% of body weight) from the carotid artery catheter. Hypovolemic shock was maintained for 50 min. Resuscitation was implemented by infusing 25% of the animal’s BV with the different treatments. Hemodynamics, blood gases, total hemoglobin, and lactate were not different before hemorrhage and during shock between groups. The hematocrit was lower for the PolyhHb group compared to the fresh and stored blood groups after resuscitation. Resuscitation with stored blood had lower blood pressure compared to fresh blood at 2 h. There was no difference in mean arterial pressure between groups at 24 h. Resuscitation with PolyhHb was not different from fresh blood for most parameters. Resuscitation with PolyhHb did not show any remarkable change in liver injury, inflammation, or cardiac damage. Resuscitation with stored blood showed changes in liver function and inflammation, but no kidney injury or systemic inflammation. Resuscitation with stored blood after 24 h displayed sympathetic hyper-activation and signs of cardiac injury. These results suggest that PolyhHb is an effective resuscitation alternative to blood. The decreased toxicities in terms of cardiac injury markers, vital organ function, and inflammation following PolyhHb resuscitation in guinea pigs indicate a favorable safety profile. These results are promising and support future studies with this new generation of PolyhHb as alternative to blood when blood is unavailable.

Extracorporeal circulation (ECC) procedures, such as cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO), take over the function of one or more organs, providing clinicians time to treat underlying pathophysiological conditions. ECMO and CPB carry significant mortality rates for patients, despite prior decades of research focused on the resulting failure of critical organs. Since the focus of these procedures is to support blood flow and provide oxygen-rich blood to tissues, a shift in research toward the effects of ECMO and CPB on the microcirculation is warranted. Along with provoking systemic responses, both procedures disrupt the integrity of red blood cells, causing release of hemoglobin (Hb) from excessive foreign surface contact and mechanical stresses. The effects of hemolysis are especially pronounced in the microcirculation, where plasma Hb leads to nitric oxide scavenging, oxidization, formation of reactive oxygen species, and inflammatory responses. A limited number of studies have investigated the implications of ECMO in the microcirculation, but more work is needed to minimize ECMO-induced reduction of microcirculatory perfusion and consequently oxygenation. The following review presents existing information on the implications of ECMO and CPB on microvascular function and proposes future studies to understand and leverage key mechanisms to improve patient outcomes.

The multi-kinase inhibitor Regorafenib, approved for the treatment of metastatic colorectal cancer, is poorly tolerated with a Grade 3/4 drug related adverse event rate of 54% resulting in frequent dose reductions and discontinuations. RRx-001 is a minimally toxic NLRP3 inhibitor small molecule with macrophage-repolarizing properties in Phase 3 clinical trials. Studies have demonstrated the inhibitory impact of M2 macrophages on the activity of tyrosine kinases, suggesting that the repolarization of macrophages by RRx-001 may enhance the activity of TKIs. The purpose of these experiments was to determine whether RRx-001 demonstrated in vitro and in vivo synergy with regorafenib in colorectal cancer and whether RRx-001 attenuated the toxicity of regorafenib. Tumor-bearing mice were randomized into four cohorts: RRx-001 alone, regorafenib alone, RRx-001 + regorafenib and control. RRx-001 demonstrated in vitro and in vivo synergy with regorafenib with attenuation of toxicity in colorectal cancer cell lines. These results provide a rationale to treat colorectal cancer with RRx-001 plus another tyrosine kinase inhibitor like regorafenib.

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.

Oxygen plays a central role in cardiac energy metabolism. At high altitude where the ambient oxygen level is low, we found EDNRB is associated with human hypoxia adaptation. Our subsequent study in global heterozygous knockout mice (Ednrb-/+) revealed that cardiac function was conserved in these mice when exposed to extreme hypoxia. The major goal of this study was (i) to determine the functional role of cardiomyocyte EdnrB in maintaining cardiac function under hypoxic stress and (ii) to validate the phenotypes we detected in Ednrb-/+ mice using EDNRB blockers. Unlike the global knockouts, cardiac-specific heterozygote (EdnrBflox/+) and homozygote (EdnrBflox/flox) EdnrB knockout mice were phenotypically normal. When treated with graded low levels of oxygen (10% and 5% O2), both EdnrBflox/+ and EdnrBflox/flox were hypoxia tolerant. The cardiac indexes at 10% and 5% O2 for EdnrBflox/+ were significantly higher and lactate levels were significantly lower when compared to the cre-negative controls (P

As the leading cause of cancer-related mortality, lung cancer is a worldwide health issue that is overwhelmingly caused by smoking. However, a substantial minority (~25%) of patients with non-small cell lung cancer (NSCLC) has never smoked. In these patients, activating mutations of the epidermal growth factor receptor (EGFR) are more likely, which render their tumors susceptible for a finite period to treatment with EGFR tyrosine kinase inhibitors (TKIs) and confer a better prognosis than EGFR wild-type NSCLC. On progression, due to the inevitable insurgence of resistance, TKIs are generally followed by second- or third-line salvage chemotherapy until treatment failure, after which no standard treatment options are available, resulting in a poor prognosis and a high risk of death. With the focus of clinical attention on treatment with TKIs, few studies on optimal salvage therapies, including cytotoxic chemotherapy, after failure of EGFR TKIs have been reported. Despite a paucity of available data, the aim of this review is to summarize the "no-man's land" of TKI-failed EGFR-mutated NSCLC and expand on alternative strategies as well as potential future directions.

In this study, because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology, genomics, and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, respectively, CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding, we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology.

The giant extracellular hemoglobin (erythrocruorin) from the earth worm (Lumbricus terrestris) has shown promise as a potential hemoglobin-based oxygen carrier (HBOC) in in vivo animal studies. An important beneficial characteristic of this hemoglobin (LtHb) is the large number of heme-based oxygen transport sites that helps overcome issues of osmotic stress when attempting to provide enough material for efficient oxygen delivery. A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation. The present study compares the NO-generating and NO bioactivity-preserving capability of LtHb with that of human adult hemoglobin (HbA) through several reactions including the nitrite reductase, reductive nitrosylation, and still controversial nitrite anhydrase reactions. An assignment of a heme-bound dinitrogen trioxide as the stable intermediate associated with the nitrite anhydrase reaction in both LtHb and HbA is supported based on functional and EPR spectroscopic studies. The role of the redox potential as a factor contributing to the NO-generating activity of these two proteins is evaluated. The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb. Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits.