Discovery of RRx-001, a Myc and CD47 Downregulating Small Molecule with Tumor Targeted Cytotoxicity and Healthy Tissue Cytoprotective Properties in Clinical Development

Article abstract of DOI:10.1021/acs.jmedchem.1c00599

After extensive screening of aerospace compounds in an effort to source a novel anticancer agent, RRx-001, a first-in-class dinitroazetidine small molecule, was selected for advancement into preclinical and clinical development. RRx-001 is a minimally tox

Full text of DOI:10.1021/acs.jmedchem.1c00599

pubs.acs.org/jmc
Drug Annotation
Discovery of RRx-001, a Myc and CD47 Downregulating Small
Molecule with Tumor Targeted Cytotoxicity and Healthy Tissue
Cytoprotective Properties in Clinical Development
Bryan Oronsky,* XiaoNing Guo, XiaoHui Wang, Pedro Cabrales, David Sher, Lou Cannizzo,
Bob Wardle, Nacer Abrouk, Michelle Lybeck, Scott Caroen, Arnold Oronsky, and Tony R. Reid
Cite This: J. Med. Chem. 2021, 64, 7261−7271
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: After extensive screening of aerospace compounds
in an effort to source a novel anticancer agent, RRx-001, a first-in-
class dinitroazetidine small molecule, was selected for advancement
into preclinical and clinical development. RRx-001 is a minimally
toxic small molecule with a distinct chemical structure and
mechanism of action. The paradox of RRx-001 is that it mediates
both antitumor cytotoxicity and normal tissue protection. The
question of exactly how RRx-001 does this, and by means of what
mechanism(s), depending on the route of delivery, intravenous or
intratumoral, are explored. RRx-001 is currently in phase 2 and 3
clinical trials for the treatment of multiple solid tumor
malignancies and as a supportive care drug.
the absence of any dose-limiting toxicities and no maximally
tolerated dose (MTD) has been reached. The most common
intravenous toxicity or side effect in phase 1 was a local,
temporary burning sensation and venous inflammation at the
site of administration.6
INTRODUCTION
■
RRx-001 is a first-in-class aerospace-based1 therapeutic,2 with a
chemical structure shown in Figure 1, containing an acyl
The discovery of RRx-001 was the product of a unique “hands
on” partnership between ATK, an American aerospace and
defense company, academia, venture capital, and biotech that
involved two parts serendipity and intuition, one part in vivo
observation, and one part novel combinatorial chemistry in
which a nitro group on the melt cast explosive, trinitroazetidine
(TNAZ), was replaced with an acyl bromide to render the
subsequent compound, RRx-001, less impact sensitive, and
more reactive with thiols. The decision to search the previously
unmined aerospace industry for potentially bioactive chemical
compounds was made in opposition to the so-called streetlight
effect which plagues the pharmaceutical industry and which
refers to an old joke about a drunk searching for his keys under a
streetlight, even though he lost them in the park, because “the
light is better here”.7 Accordingly, the tendency, which was not
followed with RRx-001, is to disproportionately focus on well-
Figure 1. Structure of RRx-001
bromide and a geminal dinitroazetidine group, in global phase 3
clinical trials3 that have demonstrated both selective antitumor
activity in several cancer types including small cell lung cancer
(SCLC),4 high-grade neuroendocrine carcinomas (HGNEC),
metastatic colorectal cancer (CRC), ovarian cancer, brain
metastases, and glioblastoma (GBM) as a single agent, as a
radiochemosensitizer, and as an immunosensitizer.5 Further, in
clinical trials, RRx-001 has demonstrated protection of the bone
marrow, kidneys, and gastrointestinal (GI) tract from chemo-
and radiation therapy and will additionally be entering phase 3
trials as a supportive care drug. To date, over 300 patients have
been treated in multiple phase 1, 2, and 3 trials with RRx-001 in
Received: April 1, 2021
Published: May 27, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271
7261

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Table 1. Compounds for Investigation of Structure−Activity Relationships (SARs)
Table 2. 4× Tumor Growth Delay of RRx-001 and Analogues
P value (t test)
ABDNAZ ADNAZ ABAZ TMAB
no. of mice 4× TGT (day) TGD (day)
CTL
Cys
CIADNAZ IADNAZ
control
ABDNAZ
ADNAZ
7
7
7
7
7
7
7
7
7
2.8 0.6
4.8 1.1
3.6 0.6
3.8 0.6
3.6 0.6
3.9 0.7
3.4 0.5
3.9 0.6
3.3 0.4
2.0 1.1
0.8 0.6
1.0 0.6
0.8 0.6
1.1 0.7
0.6 0.5
1.1 0.6
0.5 0.4
0.002
004
0.008
0.03
001
0.08
0.006
0.1
002
006
0.02
0.08
0.01
0.08
0.01
ABAZ
0.5
1.0
0.4
0.6
0.3
0.3
DNAZ-TMAB
ABDNAZ-cys
CIADNAZ
IADNAZ
A/F
0.4
0.8
0.2
0.8
0.1
0.4
0.6
0.3
0.3
0.2
1.0
0.1
0.1
0.7
0.06
known therapeutic targets and chemical structures, i.e., those
under the streetlight that are easiest to study and not on lesser
known (or unknown) therapeutic targets and chemical
structures, i.e., those beyond the streetlight that are hardest to
study but nevertheless potentially as or more important.
While target-based screening is the current predominant
paradigm in drug discovery, RRx-001 along with other energetic
compounds were assessed by in vitro and in vivo phenotypic
screens on the premise that their chemical structures,
unprecedented (and untested) in the annals of pharmaceutical
development, were not necessarily related to or predictive of
bioactivity.
In contrast to the target-based screening, phenotypic screens
involve a form of serendipity, because the mechanism of activity
of the drug candidate in question is unknown and, therefore,
difficult or impossible to predict in advance. The rationale to
evaluate energetic compounds in particular for therapeutic
activity is that the toxicological impacts of postdetonation
residues on flora and fauna (and even humans in some cases) are
already known and, therefore, potentially predictive of in vivo
safety, a cost-intensive preclinical hurdle that many drug
development candidates fail to clear, even with evidence of
significant anticancer activity.
Of all the energetic compounds that were tested, RRx-001
induced the most potent antiproliferative activity in vitro and
inhibited the growth of multiple tumor types in vivo both under
hypoxia and normoxia with minimal systemic toxicity.8 Finally,
the structure activity relationship (SAR) of RRx-001 was
studied, which demonstrated conclusively that replacement or
substitution of any part of RRx-001 decreased the anticancer
activity both in vivo and in vitro. These compounds are shown in
Table 1.
A syngeneic SCC VII tumor model in C3H mice was used to
test the efficacy of RRx-001 (ABDNAZ) and its analogues with
cisplatin (CDDP) as a positive control. All the compounds,
except CDDP, were injected intraperitoneally (ip) at an
equimolar dose to 12 mg/kg RRx-001 (ABDNAZ), every
other day for three times total. CDDP was dosed at 50%
7262
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
equimolar dose to 12 mg/kg RRx-001. Results showed that RRx-
001 was more toxic to tumors than its derivatives (P = 0.01−
0.08) but not more tumor-toxic than cisplatin. There was no
statistically significant difference in terms of tumor growth delay
time among the other compounds (Table 2) (Figures 2 and 3).
stimulation and upregulation of endogenous defense
mechanisms (Figure 4).
The objective of this review is to highlight the mechanisms by
which RRx-001 mediates its cytotoxic and chemoradioprotec-
tive activities, depending on the method of administration, i.e.,
intravenous, intratumoral or by hepatic artery infusion.
Figure 4. RRx-001 may act directly or indirectly on tumors, depending
on how it is administered.
Figure 2. SCCVII tumor growth curves
CHEMISTRY
■
The synthesis of RRx-001 is provided in Scheme 1. The
synthetic process of RRx-001 is highly hazardous and should not
be attempted by unauthorized parties. Multikilogram GMP
quantities of high purity RRx-001 API have been synthesized
from a two-step sequence of oxidative nitration of 1-tert-butyl-3-
hydroxymethyl-3-nitroazetidine
HMNAZ, 1, followed by acylative dealkylation and
functionalization with bromoacetyl bromide. Intermediate tert-
butyl-3,3-dinitroazetidine (TBDNAZ, 2) is achieved. The final
compound, RRx-001 (3), is isolated by extraction and purified
by recrystallization from ethyl acetate/hexane. Serious explosive
hazards are mitigated with the use of water extraction in place of
filtration.
Figure 3. Body weight of tumor-bearing mice on day 7.
RESULTS
■
While cisplatin at a 6.7 mg/kg dose given every other day × 3
(50% of equimolar dose of 12 mg/kg RRx-001) demonstrated
the most potent tumor inhibitory growth, it was very toxic and
resulted in 100% animal deaths (7/7) on day 8.
RRx-001 is a nongenotoxic alkylating agent, meaning that it
forms covalent adducts with thiol sulfurs but not amino
nitrogens in proteins and nucleic acids; hence, while RRx-001
depletes cellular stores of cysteine, thioredoxin, and glutathione
(GSH), which indirectly leads to nucleic acid oxidation,9 it does
not directly interact with DNA or RNA. The biological target of
RRx-001 is thiols, which leads to
Intravenously Administered RRx-001. As a nonpolar
electrophile, RRx-001 rapidly traverses the membrane of the red
blood cell (RBC) and reacts selectively with nucleophilic
sulfhydryl groups abundant in the RBC such as reduced
glutathione and the highly conserved β-globin chain cysteine 93
residue.10,11 These reactions, which proceed by nucleophilic
substitution of the α-bromine with a sulfur atom from a thiol
group, result in stable thioether bonds, as shown in Figure 5.
The rapid and irreversible binding of RRx-001 to hemoglobin
has several immediate and downstream consequences:
Immediate Consequence 1. Because RRx-001 binds to only
a small subpopulation of red blood cells (RBCs), there is no
anemia and no hematotoxicity.12
(1) Direct damage when RRx-001 is injected intratumorally
or given by hepatic artery infusion (HAI) from the
denaturation of proteins and the modification of kinases,
phosphatases, cell cycle regulators, fatty acids, cell
membranes, etc., as well as the potential for immune
cell activation due to antigen and danger signal release
from dying tumor cells/
(2) Indirect damage when RRx-001 is administered intra-
venously through reactive oxygen species/reactive nitro-
gen species (ROS/RNS) generation, formation of
inflammatory foam cell macrophages and vascular
normalization, which reprograms the tumor micro-
environment and potentiates synergistic interactions
with chemotherapy, targeted therapy, radiation, and
immunotherapy/
Immediate Consequence 2. Systemic toxicity is minimal,13
and no drug−drug interactions or induction/inhibition of
cytochrome P450 enzymes occur because RRx-001 is
immediately sequestered postinjection in red blood cells,
which selectively adhere to hypoxic tumor vasculature.
Immediate Consequence 3. Nitric oxide (NO)14,15 is
displaced from its binding site on β-cysteine 93, which leads
to localized infusional discomfort and venous inflammation with
direct IV administration, side effects that are ameliorated by a 10
min ex vivo incubation with an aliquot (12 mLs) of whole blood,
which is the current method of delivery.16
Immediate and Downstream Consequence 4. Modification
of Cys β93 with RRx-001 increases the oxygen affinity of the
RBC, which facilitates O2 delivery to more hypoxic areas of the
tumor.17
(3) Normal tissue protection from the cytotoxic effects of
radiation and chemotherapy through the compensatory
7263
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Scheme 1. RRx-001 Synthetic Scheme
(2) M2-to-M1 macrophage polarization:28 RRx-001-in-
duced tumor associated macrophage polarization from
protumor M2 to antitumor M1 increases vessel normal-
ization through decreased secretion of pro-angiogenic
factors, such as VEGF,29 adrenomedullin (ADM), platelet
derived growth factor (PDGF), and matrix metal-
loproteinases (MMPs).30
(3) Reverse Robin Hood syndrome (RRHS): RRHS, a term
that has been used to describe paradoxical changes in
cerebral hemodynamics after a stroke,31 refers in this
context to RRx-001 RBC-induced occlusion of hypoxic
tumor vessels which “steal” blood flow away from the
occluded, oxygen-poor vessels to nonoccluded, less
oxygen-poor ones.32 This redistribution of blood flow
leads to a regression or pruning of the dysfunctional
vasculature with a more homogeneous drug and oxygen
distribution as a result.33
Figure 5. RRx-001 reaction scheme for chemical covalent conjugation
of the bromoacetyl “warhead” to a sulfhydryl (in this case β-cysteine93)
on hemoglobin (Hb), which is enhanced by the inductive effect
(arrows) of the geminal dinitro groups on the azetidine ring. DNAZ,
dinitroazetidine.
Immediate Consequence 5. Hemoglobin undergoes dena-
turative precipitation, which results in the formation of
microvesicles (MVs) to remove the damaged hemoglobin
(Hb).18,19 These MVs, which are shed from the RBCs, deposit
iron, nitric oxide, heme, and lipid (because the RBC membrane
is comprised mainly of cholesterol and phospholipids) in healthy
tissues, inducing oxidative stress and the upregulation of
compensatory pathways to mitigate it. This adaptive upregula-
tion is basis for the chemoradioprotective effects of RRx-001, to
be discussed in more detail in the mechanism section below.
Immediate and Downstream Consequence 6. RRx-001-
bound red blood cells are more rigid and increase expression of
several tumor vascular adhesion molecules such as α4β1 integrin
(VLA-4), CD36 (thrombospondin receptor), Lu/BCAM
(CD239), ICAM-4, and phosphatidylserine (PS), facilitating
selective adhesion to the tumor endothelium and subsequent
phagocytosis by tumor associated macrophages (TAMs).20 This
phagocytosis is followed by subsequent degradation and release
of the contents of the RBC in the tumor parenchyma; the
released contents of the RBC include oxidized lipids, heme,
NO,21 iron, and RRx-001 metabolites, which account for the
benign safety profile of RRx-001, because all the cytotoxic
“action” takes place inside rather than outside of the tumor.
Cytotoxic Mechanisms. Vascular Normalization. Vascu-
lar normalization, a term coined by Jain,22 refers to “pruning”
rather than destruction of the morphologically aberrant, chaotic,
and leaky blood tumor vessels, to improve influx of anticancer
agents and oxygen. Clinical and preclinical evidence demon-
strates that RRx-001 administration at lower doses decreases
permeability, interstitial fluid pressure and vessel tortuosity,
enhances pericyte coverage, and improves perfusion, which lead
to improved entry of chemotherapy and its accumulation deep
within the tumor.
(a) Left shift of hemoglobin (Hb)-oxygen curve leads to
unloading of O2 in more hypoxic areas34 and a consequent
decrease of HIF-1α and VEGF (Figure 6)
Figure 6. RRx-001-induced vascular normalization mechanisms.
The molecular mechanisms by which lower dose RRx-001
drives vascular normalization effects23−25 and makes chemo-
therapy and radiation26 potentially more effective as a result are
3-fold:
(b) TAMs are skewed toward a pro-angiogenic phenotype,
which express high levels of VEGF;35 however, RRx-001-
mediated metabolite, iron, and heme delivery to the tumor
microenvironment elicits pro-inflammatory macrophages with
antiangiogenic properties36 (Figure 7).
(c) The sticky and more rigid RRx-001-bound red blood cells
that lodge in the hypoxic tumor vasculature provoke the reverse
Robin Hood-like steal of blood flow from the oxygen-poor to
more oxygen-rich regions of the tumor37 (Figure 8).
(1) Increased Hb oxygen affinity: RRx-001 increases the
oxygen affinity of hemoglobin, leading to unloading of O2
in more hypoxic areas, which lowers expressions of
hypoxia-inducible factor 1 alpha (HIF-1α) and vascular
endothelial growth factor (VEGF).27
7264
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
due to the promotion of immunosuppression, angiogenesis, and
chemoresistance; by contrast, the presence of M1 macrophages,
which secrete pro-inflammatory cytokines such as TNF-α, IL-
23, IL-1β, and IL-12, in the tumor microenvironment (TME), is
indicative of better outcomes.38 Therefore, one anticancer
strategy, given the inherent plasticity of macrophages and their
ability to switch between phenotypes depending on the
composition of the microenvironmental milieu is to target the
tumor-promoting M2-like TAMs and repolarize or reeducate
them to tumor-inhibiting M1-like macrophages.39
The covalent encapsulation of RRx-001 in red blood cells40
efficiently vectors it and its metabolites to the hypoxic tumor
vasculature, where multicellular adhesions between the RRx-
001-bound, oxidatively stressed RBCs, white blood cells
(WBCs), platelets, and endothelial cells culminate in vaso-
occlusion, hemolysis, and erythrophagocytosis by tumor
associated macrophages (TAMs) because oxidized and/or
hemolyzed RBCs are targets for TAMs, which are thought to
exit and reenter the tumor, shown in Figure 8B. Hence, RRx-001
might be properly called an “erythrophagoimmunotherapeu-
tic”.41
Figure 7. RRx-001-mediated metabolite, iron, and heme delivery to the
tumor microenvironment causes tumor-associated macrophage repola-
rization from an M2 (anti-inflammatory) phenotype to M1 phenotype,
eliciting pro-inflammatory macrophages with antiangiogenic proper-
ties.
Reactive oxygen species generated by hemoglobin denatura-
tion, RRx-001 metabolites and NO release oxidize lipoproteins
such as LDL and lipoprotein(a) within the RBC membrane,
facilitating erythrophagocytosis by tumor associated macro-
phages, with their subsequent conversion into lipid-laden foam
cells of the M1 phenotype, as seen in a serial patient biopsy in
Figure 9.
In the tumor, these M1-like foam cells initiate an
inflammatory response through the secretion of various pro-
inflammatory mediators such as IFN-γ, COX-2, iNOS, TNF-α,
IL-6, IL-1, and MCP-1 and reactive oxygen and nitrogen species,
and through their lysis, which leads to the formation of a necrotic
core42 that is seen in almost all RRx-001-treated patients,43 as
shown in Figure 10.
Recent data has demonstrated that these RRx-001-induced
foam cells overexpress PPAR-γ, which, in turn, inhibits the
transcription factor, MYC.44 Finally, the inhibition of MYC
reduces expression of CD47, the “don’t eat me” signal, which
functions as an immune checkpoint and drives phagocytic,
antitumor activity.45
A graphic summary of the intravenous mechanism of action is
shown in Figure 11.
In support of its immunotherapeutic activity, IV RRx-001 was
combined with 3 mg/kg nivolumab given every 2 weeks in a
single arm, open-label phase 1 pilot study (NCT02518958)
called PRIMETIME. Twelve patients with traditionally non-
checkpoint inhibitor-responsive tumor types received ≥1 dose
of RRx-001 and nivolumab. The combination was safe and well-
tolerated with preliminary evidence of anticancer activity based
on an objective response rate at 12 weeks of 25%
Protective Mechanisms. In addition to its cytotoxic
activity through vascular normalization, tumor associated
macrophage (TAM) polarization and CD47 downregulation,46
RRx-001 also mediates chemoradioprotective effects preclini-
cally and clinically. Preclinically, RRx-001 increases survival and
ameliorates renal, bone marrow, cardiac, and GI toxicity in
response to sublethal and lethal doses of radiation and
chemotherapy. Clinically RRx-001 has demonstrated potential
reduction of GI,47 bone marrow, and renal toxicities.48
A discussion of normal tissue protection in oncology
inevitably invites comparisons with the aminothiol, Amifostine,
approved as a chemo- and radioprotectant but not used in the
Figure 8. (A) Rigid RRx-001-bound red blood cells lodge in the
abnormal hypoxic tumor vasculature and provoke a reverse “Robin
Hood-like” steal of blood flow from the oxygen-poor to more oxygen-
rich regions of tumors. (B) Logjamming of the RRx-001 RBCs in the
tumor vasculature and their internalization by TAMs. Orange arrows
indicate red blood cells aggregating in the tumor.
M1 Foam Cell Transformation, Macrophage Polar-
ization, and Stimulation of Phagocytosis. Macrophages are
broadly categorized as pro-inflammatory M1 or anti-inflamma-
tory M2 types. M2-like tumor-associated macrophages (TAMs),
which are hallmarked by the release of IL-4 or IL-10, IL-13,
VEGF, and TGF-β and the expression of the immune
checkpoints, PD-1 and CD47, correlate with poor prognoses
7265
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Figure 9. Foam cells in a biopsy of an RRx-001-treated patient due to accelerated erythrophagocytic activity from RRx-001 metabolites. The three
windows are from left to right, pretherapy, after 6 weeks of RRx-001 and after 12 weeks of RRx-001. The orange arrows indicate the macrophages that
are overloaded with lipids.
burden of proof is on any subsequent chemoprotectant to verify
a lack of tumor protection.
In the case of RRx-001, multiple preclinical experiments50 and
clinical experience51−54 in several different tumor types
including head and neck cancer (H&N), small cell lung cancer
(SCLC), high-grade neuroendocrine carcinoma,55 glioblastoma
(GBM), melanoma brain metastases,56 colorectal cancer, and
ovarian cancer provide evidence in support of RRx-001’s
antineoplastic activity both as a single agent and in combination
with radiation and chemotherapy.
The differential RRx-001 red cell-based response between
normal and tumor tissue is likely a function of the expression,
“the dose makes the poison”. Whereas RRx-001 bound RBCs
adhere to the tumor vasculature prior to internalization by
TAMs, which deliver high-levels of iron, oxidized lipids
(oxLDLs) and RRx-001 metabolites directly to the tumor,
normal tissues only receive “microdoses” of the same iron,
oxidized lipids (oxLDLs), and RRx-001 metabolites from shed
RBC microvesicles (MVs). This MV deposition leads to an
upregulation of nuclear factor (erythroid-derived 2)-like 2
(Nrf2),57 p53, and peroxisome proliferator-activated receptor γ
(PPARγ) pathways, which protect against oxidative damage.
In this way, RRx-001 acts as an indirect cytoprotectant
through the induction of a state of compensatory gene
expression for DNA repair and anti-inflammation at low doses
that alleviates the burden of anticancer agent-mediated side
effects, while high doses delivered specifically to the tumor result
in cytotoxicity.58 This biphasic bell-shaped stress response with
RRx-001, in which beneficial effects are counterintuitively
induced at low doses while high doses result in toxicity,59 is
comparable to the dose response relationships, which exist with
other more familiar biologic stressors such as exercise (e.g.,
regular exercise vs overtraining), drinking (e.g., moderation vs
alcoholism), and eating (e.g., caloric restriction vs starvation).
The specific use of RRx-001 as a chemoradioprotectant will be
put to the test in a phase 3 trial to protect against oral mucositis
in first line H&N cancer.
Figure 10. Necrotic cores of RRx-001-treated tumors. (A) (top) PET-
CT scan of patient with pancreatic cancer showing extensive central
necrosis in liver and lung lesions. Necrosis was confirmed in the liver
lesion both in the rim and the center. (B) (bottom) PET-CT scan of
patient with metastatic sternal lesion (arrowed). Sternal metastasis is
largely necrotic and not very FDG avid, post RRx-001.
IV Pharmacokinetics. Because RRx-001 is extremely
rapidly metabolized, the glutathione metabolite (RRx-001-
GSH) was chosen as a surrogate for exposure to RRx-001. In the
phase I First-in-Man PK GCP study, RRx-001-GSH was
quantified in plasma predose, during infusion, at the end of
the infusion and time points after completion of the infusion.
Terminal half-life, which did not vary greatly across dose levels
or between sampling days was 27.6 min. The calculated average
AUCinf and, to a lesser extent, Cmax, for both day 1 and day 22/50
for each cohort, was dose dependent. However, because no
correlation between the PK parameters of RRx-001-GSH and
Figure 11. Red blood cell-based antitumor mechanism summary.
clinic in part due to the possibility, which has never been
definitively excluded, that Amifostine also protects tumors.49
Because of this potential nonspecificity/nonselectivity, the
7266
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
the activity and toxicity of RRx-001 is evident, RRx-001-GSH
levels only serve to reflect systemic exposure to RRx-001.
Intratumorally Administered RRx-001. As mentioned
previously, RRx-001 is an electrophile, which preferentially
binds to sulfur containing ligands60 rather than the hydroxy and
amino groups present in nucleic acids.61 Cellular thiols are
attractive anticancer targets because (1) the most abundant
thiol-containing peptides, reduced glutathione (γ-Glu-Cys-Gly;
GSH), and thioredoxin (Trx), are significantly upregulated in
cancers, which is thought to contribute to the development of
tumor cell chemo- and radioresistance and cell protection and
(2) so-called hyperreactive cysteines62 with enhanced nucleo-
philicity are found in diverse enzymatic classes such as
deubiquitinases, oxidoreductases, kinases, and in previously
“undruggable” oncoproteins.
Preclinically intratumoral (IT) administration and hepatic
artery infusion (HAI) of RRx-001 leads to highly selective tumor
cell killing via (a) caspase-3 activation, (b) PARP cleavage, (c)
cell cycle arrest, (d) superoxide/oxyradical ion generation, (e)
inhibition of proteasome-associated deubiquitinases, (f) protein
denaturation, (g) mitochondrial membrane depolarization, (h)
endoplasmic reticulum (ER) stress, and (i) activation of type-I
interferons (IFN-I).63 Moreover, in the event that RRx-001
“escapes” and enters the systemic circulation, as with IV
infusion, RRx-001 binds to hemoglobin on circulating red blood
cells and free thiols like reduced glutathione (GSH).
Consequently, toxicity from IT administration is anticipated
to be minimal.
RRx-001 also has the potential to induce immunogenic cell
death with the release of damage-associated molecular patterns
(DAMPs) from dying cancer cells, such as extracellular ATP,
calreticulin (CRT), and high mobility group box 1 protein
(HMGB1) through plasma membrane disruption due to
alkylation of exofacial thiols.64
The cytotoxic antiproliferative effects of RRx-001 (ABD-
NAZ) were studied on a panel of 12 cancer cell lines in vitro with
a CCK-8 cell counting kit. The IC50 values (concentration
required for 50% inhibition of cell growth) ranged from 1.8 to
6.0 μmol/L (Table 3). Comparison with cisplatin (CDDP)
showed that RRx-001 (ABDNAZ) had similar activity to CDDP
against four human cancer cell lines and SCC VII cells, with IC50
values of 2.6 1.6 μmol/L and 4.4 2.2 μmol/L for RRx-001
(ABDNAZ) and CDDP (P > 0.05), respectively.65
DISCUSSION AND CONCLUSIONS
■
The focus of this review is on the mechanistic effects of the
minimally toxic phase III immunotherapeutic anticancer agent
or “erythrophagoimmunotherapeutic”, RRx-001, which has
been used as an intravenous chemosensitizer, immunosensi-
tizer,66 and a radiosensitizer in clinical trials in multiple tumor
types including small cell lung cancer (SCLC), high-grade
neuroendocrine carcinomas (HGNEC), metastatic colorectal
cancer (CRC), brain metastases and glioblastoma (GBM), and
will be trialed as a single agent in leukemia and myelodysplastic
syndrome (MDS) as well as hepatocellular carcinoma (HCC)
via hepatic artery infusion. In addition to radiochemosensitiza-
tion and single agent activity, RRx-001 also acts as a
vasculoprotector and chemoprotector67 in nontransformed
cells.
RRx-001-related cytoprotection is a function of microvesicle
deposition from oxidatively stressed RBCs. This RBC-induced
microvesiculation, which delivers microdoses of heme,
hemoglobin, iron, nitric oxide, and RRx-001 metabolites to
normal tissues, activates multiple conserved protective enzy-
matic and signaling systems68 that increase adaptability and
stress tolerance.69 Akin to vaccine-induced immunity, RRx-001-
exposed patients are, therefore, theoretically “inoculated”
against larger future exposures of chemotherapy and/or
radiation.
The anticancer mechanisms of RRx-001 differ depending on
whether the route of administration is intravenous or intra-
tumoral. When administered intratumorally or by intrahepatic
artery infusion under balloon occlusion, RRx-001 covalently
alkylates and modifies the cysteine thiols of cancer-associated
proteins, which results in potent cytotoxicity and the potential
for immunogenic cell death (ICD). Intravenously, the main
mechanisms of action are vascular normalization, repolarization
of TAMs, and downregulation of the innate antiphagocytic
checkpoint, CD-47.70
Ironically, although RRx-001 is described as a novel, first-in-
class agent, its mechanism of induced red blood cell dysfunction
during intravenous administration is one of the most ancient by
analogy with the hemoglobin-based mechanism of defense used
by crocodiles, present since the Jurassic period from
approximately 200 million years ago.71,72 Despite the presence
of hazardous microbial concentrations in their swampy habitats,
crocodilian morbidity and mortality from bacterial infection
(and cancer) are uncommonly rare;73,74 antimicrobial and
anticancer protection is attributed in part to hemocidins,
proteolyzed ROS-producing residues of hemoglobin with
antimicrobial activity that are liberated from the red blood
cells of crocodiles (and other reptiles).75 These hemoglobin-
derived fragments from crocodiles potentially serve as
primordial archetypes for the cytotoxic and protective activity,
respectively, of the tumor-endothelial-bound RBCs and Hb-
laden microvesicles released from RRx-001-bound red blood
cells.
Table 3. IC50 of RRx-001 (ABDNAZ) against Cancer Cell
Lines in Vitro
IC50 of ABDNAZ against cancer cell lines in vitro
cell line
cell type
IC50 (pmol/L)
SD
a
SCC VII
22B
PANC-1
M21
U87
RKO
HT29
SNB75
MCF-7
A498
IMR32
A549
SCC
oral SCC
pancreatic carcinoma
melanoma
glioblastoma
colon carcinoma
colorectal adenocarcinoma
glioblastoma
breast adenocarcinoma
renal cell carcinoma
brain neuroblastoma
nonsmall cell lung carcinoma
1.8
2.3
2.3
2.6
2.7
3.0
3.4
3.8
4.0
4.9
5.1
6.0
0.3
0.5
0.7
0.5
0.6
0.4
0.3
1.4
0.5
1.3
The long evolutionary history of the crocodile suggests that an
important function of the RBC, and hemoglobin, may be to
produce cytotoxic effects in pathogenic states such as cancer and
counteractive effects in normal tissues that render them more
resistant to future stress.
1.8
a
NOTE: The SCC VII is a murine cancer cell line. All others are of
human origin.
7267
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
EXPERIMENTAL SECTION
ASSOCIATED CONTENT
* Supporting Information
Molecular strings and HPLC uploaded as supplementary files.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00599.
■
■
sı
Synthetic Chemistry. General Procedures. Step 1. Synthesis of
TBDNAZ. Deionized water, sodium hydroxide, and HMNAZ (1 equiv)
were added to a glass reaction vessel and the resulting solution was
cooled to 5 °C at which time a premade solution of potassium
hexanocyanoferrate (1 equiv)/sodium nitrite (4 equiv) in deionized
water was charged while maintaining the solution temperature at 5 °C.
The solution was agitated at 5 °C for NLT 0.5 h, at which time sodium
persulfate (2 equiv) was charged while maintaining the solution
temperature at NMT 20 °C. The solution was cooled to 5 °C and
agitated for NLT 2 h and was then allowed to warm to RT over NLT 1
h. After reaction completion was confirmed by IPC, dichloromethane
was charged, and the resulting biphasic mixture was agitated for NLT
0.25 h, allowed to settle, and then separated. The aqueous layer was
extracted a second time with dichloromethane, and the organic layers
were combined and sodium sulfate was charged to dry the solution. The
sodium sulfate was removed by filtration, and the dichloromethane was
removed via rotary evaporation under reduced pressure to a set volume
to give a solution of t-butyl-DNAZ of approximately 15 wt %, which was
used directly in the following step.
Step 2. Synthesis of RRx-001. To the 15 wt % solution of t-butyl-
DNAZ in a glass vessel at 20 °C, was charged boron trifluoride etherate
(0.1 equiv) followed by the slow addition of bromoacetyl bromide (1
equiv) over NLT 1 h while maintaining the reaction mixture
temperature at NMT 25 °C. The reaction mixture was then agitated
at 25 °C for NLT 12 h until reaction completion was confirmed by IPC
testing. To the reaction mixture was charged dichloromethane and
deionized water, and the resulting biphasic mixture was allowed to stir
at 20−25 °C for NLT 2 h and the layers were than separated. To the
organic layer was charged deionized water, and after agitation for NLT
0.5 h the layers were separated and the dichloromethane extracts were
combined and sodium sulfate was added to dry the solution. After
filtration of the sodium sulfate, ethanol was charged and the solvent was
removed using rotary evaporation under reduced pressure to a set
volume of solvent remained. The resulting slurry was filtered and
washed with ethanol to give a wet cake of crude RRx-001, which was
then charged to a clean glass reaction vessel to which ethyl acetate was
charged. The mixture was stirred at room temperature for NLT 1 h until
complete dissolution was achieved, at which time heptane was charged
over NLT 0.5 h. The resulting slurry was filtered and the cake was
washed with heptane and the material was dried in vacuo to a constant
weight giving white crystalline RRx-001 in 32% overall yield (from
HMNAZ) in >99% purity by HPLC.
RRx-001 and Cisplatin Experiment Animal Data. For the study
illustrated in Table 2 and Figures 2 and 3, all animal experiments were
conducted accordingly to the Guide for the Care and Use of Laboratory
Animals (U.S. National Research Council, 2010). Mice (C3H, male,
aged 7−8 weeks and weighing 22−25 g) sourced from Charles River
were inoculated subcutaneously in the back with 5 × 105 SCC VII
tumor cells suspended in 0.05 mL of Hank’s solution. Ten days post
injection, mice were randomized into nine groups (tumor size range
50−300 mm3). On treatment day 0, no statistically different size in
tumors was observed among groups. Tumors were measured (length
and width) before treatment and after (3× per week) until tumor
volumes reached 4 times the day 0 volume. The formula used to
calculate tumor volume in mm3: (tumor volume = π/6 × length ×
width2). Final data are expressed as % of pretreatment volume (day 0
measurement) as a function of days from start of treatment. Exponential
regression analysis was used to calculate the quadrupling tumor growth
time (4× TGT, in days). Tumor growth delay (TGD) time was defined
as the difference between the treated tumors’ 4× TGT compared to the
same for control tumors (not treated). The 4× TGT and TGD times
were determined for each animal, after which an average was calculated
for each group. Body weight measurements were conducted 3× per
week.
(PDF)
(CSV)
AUTHOR INFORMATION
Corresponding Author
■
Bryan Oronsky − EpicentRx Inc., La Jolla, California 92037,
United States; orcid.org/0000-0002-3940-6329;
Phone: (858) 947-6635; Email: boronsky@epicentrx.com;
Fax: (858) 724-3080
Authors
XiaoNing Guo − SciClone Pharmaceuticals Co., Ltd., Huangpu,
Shanghai 200020, China
XiaoHui Wang − SciClone Pharmaceuticals Co., Ltd.,
Huangpu, Shanghai 200020, China
Pedro Cabrales − Department of Bioengineering, University of
California San Diego, La Jolla, California 92093, United
States; orcid.org/0000-0002-8794-2839
David Sher − Department of Radiation Oncology, UT
Southwestern Medical Center, Dallas, Texas 75390, United
States
Lou Cannizzo − Department of Space Systems, Northrop
Grumman Corporation, Falls Church, Virginia 22042, United
States
Bob Wardle − Department of Space Systems, Northrop
Grumman Corporation, Falls Church, Virginia 22042, United
States
Nacer Abrouk − EpicentRx Inc., La Jolla, California 92037,
United States
Michelle Lybeck − EpicentRx Inc., La Jolla, California 92037,
United States
Scott Caroen − EpicentRx Inc., La Jolla, California 92037,
United States
Arnold Oronsky − InterWest Partners, Los Altos, California
94022, United States
Tony R. Reid − EpicentRx Inc., La Jolla, California 92037,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00599
Author Contributions
All authors contributed to the conceptualization, data collection
and analysis, drafting, writing and editing of this manuscript.
Notes
The authors declare the following competing financial
interest(s): EpicentRx Inc. funds research of RRx-001. Authors
B.O., S.C., and T.R. are employed by EpicentRx, Inc.
ACKNOWLEDGMENTS
EpicentRx, Inc. funds research of RRx-001.
■
ABBREVIATIONS USED
■
ABDNAZ, RRx-001; ADM, adrenomedullin; CDDP, cisplatin;
CRC, colorectal cancer; GBM, glioblastoma; GI, gastro-
intestinal; GSH, glutathione; H&N, head and neck cancer;
HAI, hepatic artery infusion; Hb, hemoglobin; HCC,
7268
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
(13) Oronsky, B.; Carter, C.; Scicinska, A.; Oronsky, A.; Oronsky, N.;
Lybeck, M.; Scicinski, J. Medical Machiavellianism: the tradeoff
between benefit and harm with targeted chemotherapy. Oncotarget.
2016, 7 (8), 9041−5.
(14) Huang, Z.; Fu, J.; Zhang, Y. Nitric oxide donor-based cancer
therapy: advances and prospects. J. Med. Chem. 2017, 60, 7617.
(15) Fens, M. H.; Larkin, S. K.; Oronsky, B.; Scicinski, J.; Morris, C.
R.; Kuypers, F. A. The capacity of red blood cells to reduce nitrite
determines nitric oxide generation under hypoxic conditions. PLoS One
2014, 9 (7), No. e101626.
(16) Caroen, S.; Oronsky, B.; Carter, C.; Lybeck, M.; Oronsky, A.;
Reid, T. Rationale and necessity for delivery of RRx-001, a Myc and
CD47 antagonist, by intravenous blood mix. Expert Opin. Drug Delivery
2020, 17 (6), 741−742.
(17) Fens, M. H.; Cabrales, P.; Scicinski, J.; Larkin, S. K.; Suh, J. H.;
Kuypers, F. A.; Oronsky, N.; Lybeck, M.; Oronsky, A.; Oronsky, B.
Targeting tumor hypoxia with the epigenetic anticancer agent, RRx-
001: a superagonist of nitric oxide generation. Med. Oncol. 2016, 33 (8),
85.
(18) Cirrik, S.; Ugurel, E.; Aksu, A. C.; Oronsky, B.; Cabrales, P.;
Yalcin, O. Nitrite may serve as a combination partner and a biomarker
for the anti-cancer activity of RRx-001. Biorheology 2019, 56 (4), 221−
235.
(19) Cabrales, P.; Reid, T.; Oronsky, B.; Scicinski, J.; Chauchan, D.;
Parker, C.; Caroen, S.; Fanger, G. RRx-001 an EXO-based anti-cancer
agent in phase 2 clinical trials. ISEV2014 Educational Event:
Extracellular Vesicles and Biofluid Biopsies/Companion Diagnostics
Conference; ISEV, 2014.
(20) Cabrales, P.; Carter, C.; Oronsky, B.; Reid, T. RRx-001 is a phase
3-ready small molecule which immunonormalizes the tumor micro-
environment. Society for Immunotherapy of Cancer (SITC) Annual
Meeting Nov 7−11, 2018, Washington, DC; Society for Immunotherapy
of Cancer (SITC), 2018.
(21) Oronsky, B. T.; Knox, S. J.; Scicinski, J. J. Is Nitric Oxide (NO)
the Last Word in Radiosensitization? A Review. Transl Oncol. 2012, 5
(2), 66−71.
hepatocellular carcinoma; HGNEC, high-grade neuroendocrine
carcinomas; HIF-1α, hypoxia-inducible factor 1 alpha;
HMNAZ, 1-tert-butyl-3-hydroxymethyl-3-nitroazetidine;
MMPs, matrix metalloproteinases; MTD, maximally tolerated
dose; MVs, microvesicles; NO, nitric oxide; oxLDLs, oxidized
lipids; PDGF, platelet derived growth factor; PPARγ, perox-
isome proliferator-activated receptor-gamma; PS, phosphatidyl-
serine; RBC, red blood cell; RRHS, reverse Robin Hood
syndrome; SCLC, small cell lung cancer; TAMs, tumor
associated macrophages; TBDNAZ, tert-butyl-3,3-dinitroazeti-
dine; TME, tumor microenvironment; TNAZ, trinitroazetidine;
VEGF, vascular endothelial growth factor; WBCs, white blood
cells
REFERENCES
■
(1) Oronsky, B.; Scicinski, J.; Ning, S.; Peehl, D.; Oronsky, A.;
Cabrales, P.; Bednarski, M.; Knox, S. Rockets, radiosensitizers, and
RRx-001: an origin story part I. Discov. Med. 2016, 21 (115), 173−80.
(2) Ning, S; Bednarski, M; Oronsky, B; Scicinski, J.; Saul, G; Knox, S.
Dinitroazetidines are a novel class of anticancer agents and hypoxia-
activated radiation sensitizers developed from highly energetic
materials. Proceedings: 102nd Annual Meeting 2011, Apr 2−6, 2011,
Orlando, FL; AACR. 2011; Vol. 71, 676.
(3) Oronsky, B.; Reid, T. R.; Larson, C.; Caroen, S.; Quinn, M.;
Burbano, E.; Varner, G.; Thilagar, B.; Brown, B.; Coyle, A.; Ferry, L.;
Abrouk, N.; Oronsky, A.; Scribner, C. L.; Carter, C. A. REPLATINUM
Phase III randomized study: RRx-001 + platinum doublet versus
platinum doublet in third-line small cell lung cancer. Future Oncol.
2019, 15 (30), 3427−3433.
(4) Morgensztern, D.; Rose, M.; Waqar, S. N.; Morris, J.; Ma, P. C.;
Reid, T.; Brzezniak, C. E.; Zeman, K.; Padmanabhan, A.; Hirth, J.; Spira,
A. I.; Trepel, J. B.; Padda, S. K. RRx-001 followed by platinum plus
etoposide in patients with previously treated small-cell lung cancer. Br.
J. Cancer 2019, 121 (3), 211−217.
(22) Jain, R. K. Normalization of tumor vasculature: an emerging
concept in antiangiogenic therapy. Science 2005, 307 (5706), 58−62.
(23) Oronsky, B.; Scicinski, J.; Cabrales, P.; Minchinton, A. RRx-001,
an epigenetic-based radio- and chemosensitizer, has vascular normal-
izing effects on SCCVII and U87 tumors. Clin. Epigenet. 2016, 8, 53.
(24) Kim, M. M.; Parmar, H. A.; Schipper, M.; Devasia, T.; Aryal, M.
P.; Kesari, S.; O’Day, S.; Morikawa, A.; Spratt, D. E.; Junck, L.;
Mammoser, A.; Hayman, J. A.; Lawrence, T. S.; Tsien, C. I.; Aiken, R.;
Goyal, S.; Abrouk, N.; Trimble, M.; Cao, Y.; Lao, C. D. BRAIN-
STORM: A Multi-Institutional Phase 1/2 Study of RRx-001 in
Combination With Whole Brain Radiation Therapy for Patients With
Brain Metastases. Int. J. Radiat. Oncol., Biol., Phys. 2020, 107 (3), 478−
486.
(25) Scicinski, J.; Oronsky, B.; Ning, S.; Minchinton, A.; Knox, S. RRx-
001 modulates intratumor blood flow in SCCVII and U87 tumors.
Cancer Res. 2012, 72, 4371.
(26) Oronsky, B. T.; Knox, S. J.; Scicinski, J. Six degrees of separation:
the oxygen effect in the development of radiosensitizers. Transl Oncol.
2011, 4 (4), 189−98.
(27) Oronsky, B.; Scicinski, J.; Reid, T.; Knox, S. Beyond
antiangiogenesis: Vascular modulation as an anticancer therapy-a
review. Trans. Onc. 2012, 5, 133.
(5) Knox, S.; Ning, S.; Peehl, D.; Oronsky, B.; Scicinski, J. RRx-001
combined with anti-PD-L1 antibody increases the complete response
rate in a preclinical myeloma model. AACR-NCI-EORTC International
Conference 2015: Molecular Targets and Cancer Therapeutics, November
5−9, 2011, Boston, MA; AACR-NCI-EORTC, 2015.
(6) Reid, T.; Oronsky, B.; Scicinski, J.; Scribner, C. L.; Knox, S. J.;
Ning, S.; Peehl, D. M.; Korn, R.; Stirn, M.; Carter, C. A.; Oronsky, A.;
Taylor, M. J.; Fitch, W. L.; Cabrales, P.; Kim, M. M.; Burris, HA Rd;
Lao, C. D.; Abrouk, N. E. D.; Fanger, G. R.; Infante, J. R. Safety and
activity of RRx-001 in patients with advanced cancer: a first-in-human,
open-label, dose-escalation phase 1 study. Lancet Oncol. 2015, 16 (9),
1133−1142.
(7) Streetlight Effect. Wikipedia, 2017; https://en.wikipedia.org/
wiki/Streetlight_effect (accessed 2017-02-11).
(8) Ning, S.; Bednarski, M.; Oronsky, B.; Scicinski, J.; Saul, G.; Knox,
S. J. Dinitroazetidines are a novel class of anticancer agents and hypoxia-
activated radiation sensitizers developed from highly energetic
materials. Cancer Res. 2012, 72 (10), 2600−8.
(9) Raghunand, N.; Scicinski, J.; Guntle, G. P.; Jagadish, B.; Mash, E.
A.; Bruckheimer, E.; Oronsky, B.; Korn, R. L. Magnetic resonance
imaging of RRx-001 pharmacodynamics in preclinical tumors.
Oncotarget. 2017, 8 (60), 102511−102520.
(28) Oronsky, B.; Paulmurugan, R.; Foygel, K.; Scicinski, J.; Knox, S.
J.; Peehl, D.; Zhao, H.; Ning, S.; Cabrales, P.; Summers, T. A., Jr; Reid,
T. R.; Fitch, W. L.; Kim, M. M.; Trepel, J. B.; Lee, M. J.; Kesari, S.;
Abrouk, N. D.; Day, R. M.; Oronsky, A.; Ray, C. M.; Carterg, C. A. RRx-
001: a systemically non-toxic M2-to-M1 macrophage stimulating and
prosensitizing agent in Phase II clinical trials. Expert Opin. Invest. Drugs
2017, 26 (1), 109−119 Jan.
(29) Scicinski, J.; Oronsky, B.; Ning, S.; Minchinton, A.; Knox, S.
Antivascular activity of RRx-001 in SCCVII and U87 tumors. 15th
International Symposium on Anti-Angiogenic Therapy, Jan 31- Feb 2,
2013, La Jolla, CA, 2013.
(10) Scicinski, J.; Oronsky, B.; Cooper, V.; Taylor, M.; Alexander, M.;
Hadar, R.; Cosford, R.; Fleischmann, T.; Fitch, W. L. Development of
methods for the bioanalysis of RRx-001 and metabolites. Bioanalysis
2014, 6 (7), 947−56.
(11) Fitch, B.; McLaughlin, T.; Fens, M.; Scicinski, J.. High resolution
MS proves that the developmental cancer drug, RRx-001, alkylates the
hemoglobin beta chain. Western Regional Meeting 2013; American
Chemical Society, 2013; abstract WRM 267.
(12) Oronsky, B.; Scicinski, J.; Ning, S.; Peehl, D.; Oronsky, A.;
Cabrales, P.; Bednarski, M.; Knox, S. RRx-001, A novel dinitroazetidine
radiosensitizer. Invest. New Drugs 2016, 34 (3), 371−7 Jun.
7269
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
́
(30) Jarosz-Biej, M.; Kaminska, N.; Matuszczak, S.; Cichon, T.;
(46) Cabrales, P. RRx-001 Acts as a Dual Small Molecule Checkpoint
Inhibitor by Downregulating CD47 on Cancer Cells and SIRP-α on
Monocytes/Macrophages. Transl. Oncol. 2019, 12 (4), 626−632.
(47) Oronsky, B.; Goyal, S.; Kim, M. M.; Cabrales, P.; Lybeck, M.;
Caroen, S.; Oronsky, N.; Burbano, E.; Carter, C.; Oronsky, A. A Review
of Clinical Radioprotection and Chemoprotection for Oral Mucositis.
Transl. Oncol. 2018, 11 (3), 771−778.
Pamula-Pilat, J.; Czapla, J.; Smolarczyk, R.; Skwarzynska, D.; Kulik, K.;
Szala, S. M1-like macrophages change tumor blood vessels and
microenvironment in murine melanoma. PLoS One 2018, 13 (1),
No. e0191012.
(31) Alexandrov, A. V.; Sharma, V. K.; Lao, A. Y.; Tsivgoulis, G.;
Malkoff, M. D.; Alexandrov, A. W. Reversed Robin Hood syndrome in
acute ischemic stroke patients. Stroke 2007, 38 (11), 3045−3048.
(32) Oronsky, B. T.; Knox, S. J.; Scicinski, J. Six degrees of separation:
the oxygen effect in the development of radiosensitizers. Transl Oncol.
2011, 4 (4), 189−98.
(33) Cabrales, P.; Oronsky, B.; Scicinski, J. Enhanced uptake and
accumulation of Temozolomide and irinotecan in orthotopically-
implanted gliomas by vascular priming with RRx-001. AACR 107th
Annual Meeting 2016, April 16−20, 2016, New Orleans, LA; AACR,
2016.
(34) Das, D; Ray, A; Song, Y; Richardson, P; Oronsky, B; Scicinski, J;
Chauhan, D; Anderson, K The hypoxia-selective epigenetic agent, RRx-
001, triggers apoptosis and overcomes drug-resistance in multiple
myeloma cells. ASH 2015, December 5−8, 2015, Orlando, FL, 2015.
(35) Szala, S.; Mitrus, I.; Sochanik, A. Can inhibition of angiogenesis
and stimulation of immune response be combined into a more effective
antitumor therapy? Cancer Immunol. Immunother. 2010, 59, 1449−
1455.
(36) Das, D. S.; Ray, A.; Das, A.; Song, Y.; Tian, Z.; Oronsky, B.;
Richardson, P.; Scicinski, J.; Chauhan, D.; Anderson, K. C. A novel
hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and
overcomes drug resistance in multiple myeloma cells. Leukemia 2016,
30 (11), 2187−2197.
(37) Oronsky, B.; Ning, S.; Knox, S.; Cabrales, P.; Minchinton, A.;
Kuypers, F.; Fens, M.; Santini, J.; Reid, T.; Scicinski, J. RRx-001: A
novel hypoxia activated nitric oxide generating vascular disrupting
agent (VDA). 15th International Symposium on Anti-Angiogenic Therapy,
Jan 31−Feb 2, 2013, La Jolla, CA USA, 2013.
(38) Jayasingam, S. D.; Citartan, M.; Thang, T. H.; Mat Zin, A. A.;
Ang, K. C.; Ch’ng, E. S. Evaluating the Polarization of Tumor-
Associated Macrophages Into M1 and M2 Phenotypes in Human
Cancer Tissue: Technicalities and Challenges in Routine Clinical
Practice. Front. Oncol. 2020, 9, 1512.
(39) Cabrales, P.; Caroen, S.; Oronsky, A.; Carter, C.; Trepel, J.;
Summers, T.; Reid, T.; Oronsky, N.; Lybeck, M.; Oronsky, B. The
macrophage stimulating anti-cancer agent, RRx-001, protects against
ischemia-reperfusion injury. Expert Rev. Hematol. 2017, 10 (6), 575−
582.
(40) Cabrales, P.; Scicinski, J.; Reid, T.; Kuypers, F.; Larkin, S.; Fens,
M.; Oronsky, A.; Oronsky, B. A look inside the mechanistic black box:
Are red blood cells the critical effectors of RRx-001 cytotoxicity? Med.
Oncol. 2016, 33 (7), 63.
(41) Oronsky, B.; Carter, C.; Caroen, S.; Scribner, C.; Oronsky, A.;
Reid, T. R. (2020). RRx-001, a first-in-class small molecule inhibitor of
MYC and a downregulator of CD47, is an ″erythrophagoimmunother-
apeutic″. OncoImmunology 2020, 9 (1), 1746172.
(42) Carter, C. A.; Schmitz, B.; Peterson, P. G.; Quinn, M.; Degesys,
A.; Jenkins, J.; Oronsky, B.; Scicinski, J.; Caroen, S.; Reid, T. R.;
Cabrales, P.; Brzezniak, C. Immune Reactivity and Pseudoprogression
or Tumor Flare in a Serially Biopsied Neuroendocrine Patient Treated
with the Epigenetic Agent RRx-001. Case Rep. Oncol. 2016, 9 (1), 164−
70.
(43) Brzezniak, C.; Schmitz, B. A.; Peterson, P. G.; Degesys, A.;
Oronsky, B. T.; Scicinski, J. J.; Caroen, S. Z.; Carter, C. A. RRx-001-
Induced Tumor Necrosis and Immune Cell Infiltration in an EGFR
Mutation-Positive NSCLC with Resistance to EGFR Tyrosine Kinase
Inhibitors: A Case Report. Case Rep. Oncol. 2016, 9 (1), 45−50.
(44) Oronsky, B.; Reid, T. R.; Oronsky, A.; Caroen, S.; Carter, C. A.;
Cabrales, P. Brief report: RRx-001 is a c-Myc inhibitor that targets
cancer stem cells. Oncotarget. 2018, 9 (34), 23439−23442.
(45) Oronsky, B.; Carter, C.; Reid, T.; Brinkhaus, F.; Knox, S. J. Just
eat it: A review of CD47 and SIRP-α antagonism. Semin. Oncol. 2020,
47 (2−3), 117−124.
(48) Oronsky, B.; Oronsky, N.; Cabrales, P. Platelet inhibitory effects
of the Phase 3 anticancer and normal tissue cytoprotective agent, RRx-
001. J. Cell. Mol. Med. 2018, 22 (10), 5076−5082.
(49) Hensley, M. L.; Schuchter, L. M.; Lindley, C.; Meropol, N. J.;
Cohen, G. I.; Broder, G.; Gradishar, W. J.; Green, D. M.; Langdon, R. J.;
Mitchell, R. B.; Negrin, R.; Szatrowski, T. P.; Thigpen, J. T.; VonHoff,
D.; Wasserman, T. H.; Winer, E. P.; Pfister, D. G. American Society of
Clinical Oncology clinical practice guidelines for the use of chemo-
therapy and radiotherapy protectants. J. Clin. Oncol. 1999, 17, 3333−
3355.
(50) Scicinski, J.; Fisher, G.; Carter, C.; Cho-Phan, C.; Kunz, P.; Ning,
S.; Knox, S.; Oronsky, B.; Caroen, S.; Parker, C.; Fanger, G.; Reid, T.
The Development of RRx-001, A Novel Nitric-Oxide-Mediated
Epigenetically Active Anticancer Agent. Redox Biol. 2015, 5, 422.
(51) Brzezniak, C.; Oronsky, B.; Trepel, J.; Summers, T. A., Jr.;
Cabrales, P.; Lee, M. J.; Day, R.; Jha, S.; Caroen, S.; Zeman, K.; Ferry,
L.; Harmer, C.; Oronsky, N.; Lybeck, M.; Lybeck, H. E.; Brown, J. F.;
Reid, T. R.; Carter, C. A. RRx-001 Priming of PD-1 Inhibition in the
Treatment of Small Cell Carcinoma of the Vagina: A Rare
Gynecological Tumor. Case Rep. Oncol. 2017, 10 (1), 276−280.
(52) Kim, M. M.; Parmar, H.; Cao, Y.; Knox, S. J.; Oronsky, B.;
Scicinski, J.; Lawrence, T. S.; Lao, C. D. Concurrent whole brain
radiotherapy and RRx-001 for melanoma brain metastases. Neuro
Oncol. 2016, 18 (3), 455−6.
(53) Carter, C. A.; Degesys, A.; Oronsky, B.; Scicinski, J.; Caroen, S.
Z.; Oronsky, A. L.; Reid, T.; Cabrales, P.; Roswarski, J. Flushing Out
Carcinoid Syndrome: Beneficial Effect of the Anticancer Epigenetic
Agent RRx-001 in a Patient with a Treatment-Refractory Neuro-
endocrine Tumor. Case Rep. Oncol. 2015, Z8 (3), 461−5.
(54) Reid, T.; Oronsky, B.; Scicinski, J.; Scribner, C. L.; Knox, S. J.;
Ning, S.; Peehl, D. M.; Korn, R.; Stirn, M.; Carter, C. A.; Oronsky, A.;
Taylor, M. J.; Fitch, W. L.; Cabrales, P.; Kim, M. M.; Burris, HA Rd;
Lao, C. D.; Abrouk, N. E. D.; Fanger, G. R.; Infante, J. R. Safety and
activity of RRx-001 in patients with advanced cancer: a first-in-human,
open-label, dose-escalation phase 1 study. Lancet Oncol. 2015, 16 (9),
1133−1142.
(55) Oronsky, B.; Ma, P. C.; Morgensztern, D.; Carter, C. A. Nothing
But NET: A Review of Neuroendocrine Tumors and Carcinomas.
Neoplasia. 2017, 19 (12), 991−1002.
(56) Kim, M. M.; Parmar, H.; Cao, Y.; Pramanik, P.; Schipper, M.;
Hayman, J.; Junck, L.; Mammoser, A.; Heth, J.; Carter, C. A.; Oronsky,
A.; Knox, S. J.; Caroen, S.; Oronsky, B.; Scicinski, J.; Lawrence, T. S.;
Lao, C. D. Whole Brain Radiotherapy and RRx-001: Two Partial
Responses in Radioresistant Melanoma Brain Metastases from a Phase
I/II Clinical Trial: A TITE-CRM Phase I/II Clinical Trial. Transl Oncol.
2016, 9 (2), 108−113.
(57) Ning, S.; Sekar, T. V.; Scicinski, J.; Oronsky, B.; Peehl, D. M.;
Knox, S. J.; Paulmurugan, R. Nrf2 activity as a potential biomarker for
the pan-epigenetic anticancer agent, RRx-001. Oncotarget. 2015, 6 (25),
21547−56.
(58) Mikhed, Y.; Görlach, A.; Knaus, U. G.; Daiber, A. Redox
regulation of genome stability by effects on gene expression, epigenetic
pathways and DNA damage/repair. Redox Biol. 2015, 5, 275−289.
(59) Hoffmann, G. R. A Perspective on the Scientific, Philosophical,
and Policy Dimensions of Hormesis. Dose-Response 2009, 7 (1), 1−51.
(60) Raghunand, N.; Scicinski, J.; Oronsky, B.; Guntle, G.;
Bruckheimer, E.; Korn, R. RRx-001 oxidation of redox sensitive protein
thiols in tumors measured by Gd-LC7-SH enhanced MRI in preclinical
tumor models. Cancer Res. 2014, 74, 2068.
(61) Scicinski, J.; Oronsky, B.; Fitch, W.; Taylor, M.; Luo, G.; Musick,
T.; Marini, J.; Adams, C.; Schicker, M.; Gohdes, M.; McKenzie, D.;
Ridgewell, R. Disposition of 14C-RRx-001 in rats after a single
7270
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
intravenous administration and in blood from rats, dogs, monkeys, and
humans. Drug. Metabol. Rev. 2011, abstract P81.
(62) Feng, W.; Liu, G.; Xia, R.; Abramson, J. J.; Pessah, I. N. Site-
selective modification of hyperreactive cysteines of ryanodine receptor
complex by quinones. Mol. Pharmacol. 1999, 55, 821−831.
(63) Zhao, H.; Ning, S.; Nolley, R.; Scicinski, J.; Oronsky, B.; Knox, S.
J.; Peehl, D. M. The immunomodulatory anticancer agent, RRx-001,
induces an interferon response through epigenetic induction of viral
mimicry. Clin. Epigenet. 2017, 9, 4.
(64) Raghunand, N.; Scicinski, J.; Oronsky, B.; Guntle, G.;
Bruckheimer, E.; Korn, R. Abstract 2068: RRx-001 oxidation of redox
sensitive protein thiols in tumors measured by Gd-LC7-SH enhanced
MRI in preclinical tumor models. Cancer Res. 2014, 74, 2068.
(65) Ning, S.; Bednarski, M.; Oronsky, B.; Scicinski, J.; Saul, G.; Knox,
S. J. Dinitroazetidines are a novel class of anticancer agents and hypoxia-
activated radiation sensitizers developed from highly energetic
materials. Cancer Res. 2012, 72 (10), 2600−8.
(66) Carter, C.; Caroen, S.; Oronsky, B.; Quinn, M. F.; Williams, J.;
Brzezniak, C. Phase I pilot study of RRx-001 + nivolumab in patients
with traditionally non-checkpoint inhibitor-responsive cancers (PRI-
METIME). J. Clin. Oncol. 2020, 38, No. e15119.
(67) Oronsky, B.; Scicinski, J.; Ning, S.; Peehl, D.; Oronsky, A.;
Cabrales, P.; Bednarski, M.; Knox, S. RRx-001, A novel dinitroazetidine
radiosensitizer. Invest. New Drugs 2016, 34 (3), 371−7.
(68) Sonneborn, J. S. Mimetics of Hormetic Agents: Stress-Resistance
Triggers. Dose-Response 2010, 8, 97−121.
(69) Calabrese, E. J. Evidence that hormesis represents an
″overcompensation″ response to a disruption in homeostasis.
Ecotoxicol. Environ. Saf. 1999, 42 (2), 135.
(70) Cabrales, P. RRx-001 Acts As a Dual Small Molecule Checkpoint
Inhibitor By Downregulating CD47 on Cancer Cells and SIRPα on
Monocytes/Macrophages. Transl. Oncol 2019, 12 (4), 626−632.
(71) Schwab, I. R.; O’Connor, G. R. The lonely eye. Br. J. Ophthalmol.
2005, 89 (3), 256.
(72) van Hoek, M. L. Antimicrobial peptides in reptiles.
Pharmaceuticals 2014, 7 (6), 723−53.
(73) Preecharram, S.; Jearranaiprepame, P.; Daduang, S.;
Temsiripong, Y.; Somdee, T.; Fukamizo, T.; Svasti, J.; Araki, T.;
Thammasirirak, S. Isolation and characterisation of crocosin, an
antibacterial compound from crocodile (Crocodylus siamensis)
plasma. Anim. Sci. J. 2010, 81 (3), 393−401.
(74) Ariel, E.; Ladds, P. W.; Roberts, B. L. Mycobacteriosis in young
freshwater crocodiles (Crocodylus johnstoni). Aust. Vet. J. 1997, 75
(11), 831−833.
(75) Jandaruang, J.; Siritapetawee, J.; Thumanu, K.; Songsiriritthigul,
C.; Krittanai, C.; Daduang, S.; Dhiravisit, A.; Thammasirirak, S. The
effects of temperature and pH on secondary structure and antioxidant
activity of Crocodylus siamensis hemoglobin. Protein J. 2012, 31, 43−
50.
7271
https://doi.org/10.1021/acs.jmedchem.1c00599
J. Med. Chem. 2021, 64, 7261−7271