Purpose: RRx-001 is a novel anticancer drug that will be entering Phase III clinical trials as a pre-sensitizer or resensitizer to
chemotherapy and radiotherapy. To date, it has been very well tolerated, with the main adverse event being infusion-related
reactions, consisting primarily of discomfort, itching and tingling at the site of infusion. The purpose of this preclinical pilot
study was to study the antitumor efficacy and tolerability of oral administration of RRx-001 in a preclinical mouse model.
Methods: The maximum tolerated dose (MTD) of oral administration of RRx-001 was studied in normal C3H mice. For
antitumor efficacy study, mice bearing SCC VII tumors were administered by oral gavage with RRx-001 either alone or in
combination with external beam radiation therapy. Tumor growth delay time and body weight were used as the endpoints
for antitumor efficacy and systemic toxicity.
Results: The estimated MTD for oral RRx-001 in 10% dimethyl sulfoxide (DMSO) daily for 5 days was 10-20 mg/kg. In tumor-bearing mice, oral dosing of RRx-001 at a total equivalent dose of 60 mg/kg, given either daily, or every other day, or as a single dose, significantly inhibited tumor growth (p < 0.01 vs. vehicle control). There were no statistically significant differences in tumor growth delay time among three treatment regimens (P > 0.05). However, a single dose of 60 mg/kg
caused a 28% modality (2 death among 7 treated mice). When combined with local tumor radiation therapy, oral RRx-001 significantly increased the antitumor efficacy of radiation therapy (p = 0.02, combination vs. radiation alone). There were no obvious systemic or additional toxicities for combination of oral RRx-001 (10-20 mg/kg) and radiation therapy.
Conclusion: Oral administration of RRx-001 was safe and effective in the SCC VII tumor model in mice, and merits further study.
Keywords: RRx-001; anticancer drug; chemotherapy; efficacy; mouse tumor model
RRx-001 (1-bromoacetyl-3, 3-dinitroazetidine, also known
as ABDNAZ) is a first-in-class minimally toxic anticancer
agent with multiple mechanisms of action and synergistic
activity in combination with radiation and chemotherapy
[1-3]. It will be advanced to Phase III clinical trials shortly
for patients with advanced heavily pretreated refractory
cancers including small cell lung cancer (SCLC), highgrade
neuroendocrine carcinomas (HGNEC) and platinumresistant
ovarian cancer. In addition to anticancer activity,
RRx-001 has also shown promise as a chemoprotector and
radioprotector. When intravenously administered, RRx-001
rapidly binds to the reduced form of glutathione (GSH), the
principal scavenger of reactive oxygen/nitrogen species,
which results in the depletion of GSH, intracellular free
radical formation and tumor cell death [1,4].
In the previous preclinical studies and Phase I-II clinical trials,
intravenous administration of RRx-001 has demonstrated
tumor growth inhibition and prolonged survival in patients [1-
2,5]. Preclinically RRx-001 has been found to protect normal
tissues such as intestinal crypt stem cells, bone marrow and
skin tissues against chemotherapy- and ionizing irradiationinduced
damage and to promote survival [2,6]. RRx-001
activates nuclear factor erythroid 2-related factor 2 (Nrf2)
antioxidant signaling pathways , suggesting a mechanism
for its potential protective effects. In clinical trials, RRx-001
is minimally toxic with non-dangerous, transient infusion
related discomfort as the chief adverse event .
Oral dosing is a convenient and safe method for administration
of anticancer agents. Herein are reported the results of
preclinical pilot studies, which demonstrate that oral administration
of RRx-001 both as a single agent monotherapy and as
a combination therapy with radiotherapy is safe and effective.
Materials and Methods
C3H mice, male, 7-8 weeks old and 20-25 grams in body
weight were purchased from Taconic Biosciences. Mice were
housed in the Veterinary Service Center (VSC) of Stanford
University animal facilities. Mice were acclimated for 3-5
days before starting the experiment. All animal experiments
were approved by and complied with the regulations of the
Stanford University Animal Care Panel.
Oral administration of RRx-001
RRx-001 was obtained from ATK Aerospace Systems RRx-001
solution was prepared freshly and used on the same day. RRx-
001 was dissolved in DMSO and then diluted with double distilled
water to obtain a final solution of 2-6 mg/mL. The final
DMSO concentration was 5-10%. RRx-001 was administered
orally in a volume of 5-10 μL/gram body weight.
For oral administration of RRx-001, mice were firmly held in
an upright position and the neck and head are immobilized.
The head was slightly tilted back towards the spine with gentle
pressure from the gavage needle. The gavage needle was slid
into the animal mouth between the tongue and the front teeth
and slid down further into the esophagus. The RRx-001 solution
was slowly injected. After oral gavage, mice were monitored
for at least 10 minutes and observed for potential complications
or abnormal reactions.
Radiation therapy was delivered using a Polaris SC-500
250kVp X-ray machine (12.5 mA; half value layer, 0.5-mm
Cu). For local tumor irradiation, unanesthetized mice were
placed in individual lead jigs with tumors protruding through
a cut-out window at the rear of each jig and irradiated locally
to the tumors with 250 cGy per fraction per day for 5 consecutive
days at a dose rate of 2 Gy/min.
Mice were inoculated subcutaneously on the back with 5x105
squamous carcinoma SCC VII tumor cells in 0.05 ml Hank’s
solution. Ten (10) days after tumor implantation, mice with
tumors ranging from 150-250 mm3 in size were randomized
into treatment groups. There was no statistically significant
difference in tumor size and animal body weight among
groups on the first day of treatment (Day 0). The length and
width of the tumors were measured with calipers immediately
before treatment and three times a week thereafter until the
tumor volume reached at least four times (4X) the original
pre-treatment volume. Tumor volume (mm3) was calculated
according to the formula: tumor volume = π/6 x length x
width2. The data are expressed as percentage of pre-treatment
volume measured on Day 0 as a function of days from start of
the treatment. The tumor volume quadrupling time (4X TGT, in
days) was determined by a logarithmic regression analysis. The
tumor growth delay (TGD) time is the difference between the
4X TGT of treated tumors compared to that of untreated vehicle
control tumors. Both the 4X TGT and TGD time was calculated
for each individual animal, and then averaged for each group.
MTD of oral administration of RRx-001 in mice
Normal C3H mice were treated with RRx-001 administered
by oral gavage with RRx-001 at doses ranging from 10-
80 mg/kg, daily for 5 consecutive days. Following oral
administration, mice were monitored for mortality, physical
activity and body weight. Results showed that a daily dose
of 10 mg/kg was well tolerated and caused no mortality.
The highest daily dose of 80 mg/kg caused 100% mortality
(4 death of 4 mice total). The maximum tolerated dose
for daily oral administration for 5 days was estimated
at 10-20 mg/kg. The average body weight of mice after
oral dosing of 20-60 mg/kg RRx-001was decreased by
7-10% of pretreatment body weight between Days 2-9.
Antitumor efficacy of oral administration of RRx-001
The therapeutic efficacy of oral administration of RRx-001
in an immunocompetent syngeneic model of squamous cell
carcinoma SCC VII was studied. Tumor-bearing mice were
randomized into four groups and treated with oral RRx-001
at a total equivalent dose of 60 mg/kg. The treatment arms
are Group 1: 12 mg/kg, daily for 5 days on Day 0-4; Group
2: 20 mg/kg, every other day for 3 doses on Day 0, 2, and 4;
Group 3: 60 mg/kg, one single dose on Day 0; and Group 4:
10% DMSO daily for 5 days without oral RRx-001. Results
are shown in Table 1A and Figure 1A. Oral administration
of RRx-001 at these doses significantly inhibited tumor
growth (p < 0.01 vs. vehicle control) and produced a tumor
growth delay time of 1.3 -1.5 days. There was no statistically
significant difference in tumor growth delay time among three
RRx-001 treated groups. However, there were two mice that
died in Group 3 with a single dose treatment of 60 mg/kg.
Antitumor efficacy of RRx-001: oral vs. intravenous
Groups of mice with established SCC VII tumors were treated
with either oral gavage or intravenous injection of 10 mg/kg
RRx-001 every other day for 3 doses total from Days 0-4.
A control group of mice with tumors received 10% DMSO
orally. Seven animals were used in each group. Results showed
that both regimens of oral and intravenous dosing of RRx-001
inhibited tumor growth and produced the tumor growth delay
times of 1.2 ± 0.4 days and 3.0 ± 1.4 days for oral and intravenous
dosing, respectively (p < 0.01 compared with Control group).
However, at the same dose level, intravenous dosing was more
effective in inhibition of tumor growth than oral dosing in
terms of the tumor growth delay time (p < 0.05). Both oral and
intravenous administration of RRx-001 were well tolerated.
Combination therapy of oral RRx-001 and radiotherapy
Mice bearing SCC VII tumors were treated with an oral dose
of 10 mg/kg RRx-001 or 250 cGy local tumor radiation,
either alone or in combination daily for 5 consecutive days.
As shown in Table 1B and Figures 1B, oral dosing with 10
mg/kg RRx-001 produced a moderate inhibition of tumor
growth (P = 0.01 vs. Control). Radiation therapy alone
inhibited tumor growth and produced a tumor growth delay
time of 0.9 ± 0.7 days (P = 0.01 vs. Control). When the use
of oral RRx-001 and radiation were combined, the tumor
growth delay time was significantly increased from 0.5–0.9
days for either modality alone to 3.1 ± 1.8 days for RRx-
001 plus radiation (P < 0.01 vs. Control). More importantly,
oral RRx-001 did not increase the systemic toxicities of
radiotherapy as measured by the body weight change of mice
Toxicity of oral RRx-001
A daily oral dose of 10 mg/kg for 5 days was well tolerated.
Cumulative toxicity was not observed. Following oral
administration, there were no unexpected deaths, and no
obvious change in the general appearance, skin reaction, or
daily activity of mice. There were no signs of severe systemic
toxicities following oral dose of 10 mg/kg daily for 5 days.
These experiments demonstrate that orally administered
RRx-001 is safe and inhibits tumor growth in tumor-bearing
mice. Evidence of anticancer activity with RRx-001 has
already been demonstrated clinically and preclinically via the
intravenous route [1-2,5]. The reason to study the effects of
oral administration is two-fold: 1) to circumvent the infusion
related reactions (chiefly discomfort) associated with RRx-
001 and 2) for the purpose of radiation protection. In the
chaotic aftermath of a radiation accident or radiological
terrorism, protection of emergency responders, military
personnel and the at-risk civilian population with an agent
like RRx-001 requires oral or subcutaneous delivery rather
than intravenous administration, which is highly impractical
and inconvenient. In addition, since acute radiation
syndrome (ARS) occurs after whole-body or significant
partial-body irradiation and involves the gastrointestinal
tract, oral administration may preferentially protect the
intestinal epithelium. In general, the disadvantage of oral
administration is poor absorption and/or bioavailability.
In the case of RRx-001, these experiments demonstrate
that orally administered RRx-001 in mice is bioavailable,
well tolerated and active. Future studies will further assess pharmacokinetics, bioavailability, food interaction and toxicity.
The purpose of this pilot study was to evaluate the tolerability
and antitumor efficacy of oral administration of RRx-001 both
alone and in combination with radiation therapy in mice. All
available clinical data with RRx-001 are based on intravenous
therapy; however, oral administration is a more practical
alternative. Oral administration of RRx-001 at its MTD dose
daily was well tolerated and significantly inhibited tumor
growth. When combined with radiation therapy, oral RRx-001
increased the antitumor efficacy of radiation therapy without
increasing the radiation-induced systemic toxicities. Hence,
RRx-001 can be safely administered orally in mice.
In the quest to find minimally toxic, orally bioavailable
‘dual function’ agents that selectively sensitize tumors
to radiotherapy and also preferentially protect normal
tissues, RRx-001 is potentially an ideal candidate.
However, further work is required to determine the
suitability of RRx-001 for oral formulation development.
Compliance with ethical standards
Conflict of interest
B. Oronsky, J. Scicinski and C.A. Carter are employees
of Epicent Rx, and S.J. Knox is a founder of Epicent Rx.
The authors have no other relevant affiliations or financial
involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or
materials discussed in the manuscript apart from those
All animal experiments were approved by and complied
with the regulations of the Stanford University Animal
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