Novel pharmacological strategies to
reduce acute radiation injury
© Maaike Berbée, Maastricht 2011
Layout: Tiny Wouters
Production: Datawyse | Universitaire Pers Maastricht
ISBN: 978 94 6159 080 0
The work presented in this thesis was supported by the United States' NIH/NIAID, NIH/NCI,
Department of Defense/Defense Threat Reduction Agency, and Veterans Administration, and
by the Maastro Cancer Foundation.
Novel pharmacological strategies to
reduce acute radiation injury
Ter verkrijging van de graad van doctor aan de Universiteit Maastricht,
op gezag van de Rector Magnificus, Prof. mr. G.P.M.F. Mols,
volgens het besluit van het College van Decanen,
in het openbaar te verdedigen
op vrijdag 21 oktober 2011 om 14.00 uur
Prof. dr. Ph. Lambin
Prof. dr. M. Hauer‐Jensen, University of Arkansas for Medical Sciences, USA
Dr. L.C.H.W. Lutgens
Prof. dr. F.C.S. Ramaekers (voorzitter)
Dr. H.J.N. Andreyev, The Royal Marsden Hospital, UK
Prof. dr. A.A.M. Masclee
Prof. dr. D. De Ruysscher
γ‐tocotrienol ameliorates intestinal radiation injury and reduces
vascular oxidative stress after total body irradiation by an
HMG‐CoA reductase‐dependent mechanism
Pentoxifylline enhances the radioprotective properties of
γ‐tocotrienol: differential effects on the hematopoietic,
gastrointestinal, and vascular system
Mechanisms underlying the radioprotective properties of
γ‐tocotrienol: comparative gene expression profiling in
tocol‐treated endothelial cells
Reduction of radiation‐induced vascular nitrosative stress by
the vitamin E analog γ‐tocotrienol: evidence of a role
Novel strategies to ameliorate radiation injury:
a possible role for tetrahydrobiopterin
The somatostatin analog SOM230 (Pasireotide) ameliorates injury
of the intestinal mucosa and increases survival after total
body irradiation by inhibiting exocrine pancreatic secretion
Preclinical evaluation of SOM230 as a radiation mitigator in
a mouse model: post‐exposure time window and mechanisms
General discussion 145
Dankwoord / Acknowledgements 173
List of publications 177
Curriculum Vitae 181
Exposure to ionizing radiation, whether in non‐clinical emergency situations or during
radiotherapy for cancer treatment, may cause substantial injury to human tissues.
Therefore, there is an urgent need for the development of pharmacological strategies
that can prevent or reduce radiation‐induced injury. To date, due to the threat of
malevolent radiation events such as terrorist attacks, significant effort is put into the
development of agents to be used as radiation countermeasures in non‐clinical
emergency situations. Some of these agents, however, may qualify as “dual utility”
drugs that can both be used as radiation countermeasure and during radiotherapy,
and thereby also benefit cancer treatment.
The main aim of the research described in this thesis was to identify novel
radioprophylactic and/or mitigating agents and to unravel their mechanism of action.
Both the vitamin E analog γ‐tocotrienol and the novel somatostatin analog SOM230
(Pasireotide) were proven to reduce acute radiation injury and to have significant
potential for clinical use as dual utility drugs. Moreover, we were able to identify
important features of the mechanisms through which these agents exert their effect.
Exposure to ionizing radiation
Human exposure to high doses of ionizing radiation, i.e., radiation with enough energy
to detach electrons from atoms or molecules, may occur both in non‐clinical
emergency situations and in clinical settings (Figure 1.1).
Non clinical exposure to high doses of ionizing radiation may have various causes. The
threat of terroristic acts using radioactive material or nuclear warheads has become
more prominent in the recent past. An act of radiological terrorism may
simultaneously expose large numbers of people to high doses of ionizing radiation and
thereby cause a mass casualty situation. Other potential causes of public radiation
exposure include the occurrence of major accidents in nuclear power plants.
Accidents like the recent accident in Fukushima or the Chernobyl accident from 1986
have shown that people present in the area near the facility may be exposed to
significant radiation doses. The management of situations of non clinical radiation
exposure can be highly complex due to its unexpectedness, uncertainties with regard
to dose levels, the high numbers of people exposed at the same time, and the
occurrence of combined injury such as radiation injury plus blast injury.
In the medical field, ionizing radiation is used in both diagnostic and treatment
procedures. Low to moderate doses of ionizing radiation are used in various
radiological imaging modalities such as X‐ray radiography and computed tomography
(CT). Clinical exposure to high radiation doses predominantly occurs in the field of
radiotherapy. Radiotherapy is, next to surgery, one of the two most effective
treatment modalities for cancer. Annually, about 7.5 million radiotherapy treatments
are executed worldwide and about half of all cancer patients is estimated to receive
radiotherapy at some point in their treatment process1.
Exposure to high doses of ionizing
radiation may occur both in controlled and
less controlled settings.
intentionally exposed to high doses of
ionizing radiation in a controlled manner.
In radiotherapy, ionizing radiation is used
in order to induce tumor cell kill.
In case of radiological/nuclear incidents or
attacks, exposure occurs in a less
radiation exposure is often unexpected. As
a consequence, there may be little to no
information about the actual radiation
dose received or about the dose
In such cases,
incidents or attacks
Normal tissue radiation injury
Ionizing radiation may cause injury to biological tissues. It may induce both
morphological and functional changes in exposed organs.
After non‐clinical exposure to high radiation doses, normal tissue radiation injury may
result in substantial morbidity, often resulting in radiation‐induced death. During
radiotherapy, side effects are caused by the exposure of non‐cancerous, i.e., normal,
tissues to ionizing radiation. Although novel technical advances in treatment delivery
have enabled us to more selectively irradiate the region of interest/tumor, normal
tissue radiation toxicity remains the most important dose limiting factor of
Normal tissue radiation injury is traditionally divided in acute and chronic radiation
injury. Acute or early radiation toxicity can be observed shortly after radiation
exposure. Chronic or late radiation injury is generally defined as injury present or
occurring at least 90 days after being irradiated. The latency period of chronic
radiation toxicity may be months up to years.
Although the time of manifestation might be the most obvious difference between
early and delayed radiation injury, there are distinct biological differences as well.
Acute radiation injury
Acute toxicity is mainly seen in high turnover tissues, i.e., tissues with a high
proliferation rate combined with a high cell loss rate. In such tissues, acute radiation
injury is caused by a radiation‐induced decrease in cell proliferation resulting in an
inadequate supply of cells to compensate for continuous cell loss. Besides inadequate
cells supplies, early radiation‐induced inflammatory changes are of importance in the
pathogenesis of acute radiation toxicity as well.
Due to high intrinsic cell turnover, organ systems like the hematopoietic system and
gastro‐intestinal (GI) tract are highly sensitive to acute radiation injury. Radiation‐
induced bone marrow failure may result in pancytopenia with lethal hemorrhage or
infections as a result. In the intestinal compartment, the epithelial lining of the
mucosa is comprised of a single layer of enterocytes that constitutes the most
extensive and important barrier between the body’s interior and the external
environment. Post‐irradiation enterocyte depletion causes breakdown of the mucosal
barrier, mucositis and secretory diarrhea (Figure 1.2). Consequentially, radiation‐
induced hematopoietic and GI‐injury are the two main determinants of survival after
accidental total body radiation exposure.
Figure 1.2 a. Radiation exposure induces a dose‐dependent decrease in proliferating intestinal crypt cells
(measured 3.5 days after radiation exposure). The crypt cells are the tissue stem cells of the
b. Microscopic image of rodent small intestine before radiation exposure.
c. Microscopic image of rodent small intestine 3.5 days after 8 Gy radiation exposure.
Radiation exposure induces a decrease in proliferating crypt cells resulting in enterocyte
depletion. The intestinal mucosal surface area is decreased after radiation exposure.
In clinical situations, acute GI mucosal injury is a common side effect of radiotherapy
for head and neck, thoracic, and abdominal/pelvic tumors. In order to provide
adequate care and to compensate for massive fluid and electrolyte loss, hospital
admission if often necessary in patients suffering from radiotherapy‐induced GI‐
toxicity. Clinically relevant bone marrow toxicity with consequential pancytopenia is a
less frequent side effect of radiotherapy. It is limited to cases in which a major part of
the hematopoietic systems is in the radiation field or to patients with impaired bone
marrow function to start with.
Late radiation injury
Chronic radiation injury is an important issue among cancer survivors2‐5. It may occur
in almost any organ system and may severely affect quality of life. Even though
current radiotherapy approaches can generally prevent the development of severe
morphological and structural changes, like chronic ulcerations and fistulae, delayed
changes in organ function may still have disabling effects. In general, chronic radiation
injury is a progressive condition with few therapeutic options.
The pathogenesis of delayed radiation injury is complex and involves functional
changes in parenchymal and vascular cells as well as chronic inflammatory changes. In
contrast to the earlier belief that acute and chronic normal tissue injuries are
unrelated, several studies have shown that part of the chronic effects are
consequential to acute injury. Since the development of delayed injury involves a
02468 1012 1416
Crypt cell survival (%)
Radiation dose (Gy)
A B C
cascade of events, the evolution of chronic toxicity may be modulated by intervening
in the cascade using pharmacological strategies.
Pharmacological interventions: prophylaxis, mitigation, and
Pharmacological interventions to reduce acute radiation injury are urgently needed.
These agents can be divided in 3 groups depending on the time of administration
relative to the time of radiation exposure and the occurrence of symptoms.
Prophylactic agents or protectors are administered before radiation exposure;
mitigators are administered shortly after exposure, but before symptoms arise; and
treatments are given after the appearance of symptoms6 (Figure 1.3).
Figure 1.3 Schematic figure adapted from Stone et al.6 depicting the timing of prophylactic, mitigating,
and symptomatic strategies in relation to the moment of radiation exposure and the
occurrence of symptoms.
Prophylaxis and mitigation
To date, clinically applicable pharmacological strategies to prevent or mitigate acute
radiation injury are scarce, if existent at all. Most research on agents to reduce
radiation injury has focused on radioprophylaxis. Many different compounds such as
various free radical scavengers, anti‐oxidants, cytokines, thiols, and steroids have
been tested as radioprophylactic agents7,8. Only the thiol‐containing compound
amifostine has been proven to be an effective and applicable radioprotectant in
humans. Unfortunately, the use of amifostine is hampered by a narrow therapeutic
window and severe side effects, such as nausea, vomiting and hypotension9. As a
consequence, in the US, amifostine has only been approved for clinical use in head
and neck cancer patients undergoing radiotherapy. Because of its toxicity profile,
amifostine should not be used as a radioprophylactic agent in non‐clinical situations.
The Food and Drug Administration (FDA) has not approved the use amifostine as a
radioprotectant for first responders in radiological/nuclear accidents or attacks.
Hence, currently there are no effective pharmacological radioprophylactic or
migrating strategies to reduce radiation toxicity in non‐clinical emergency situations.
During radiotherapy, some radioprophylaxis may be possible in certain patient groups
pre‐exposure radiation symptoms
prophylaxis mitigation treatment
using amifostine. However, no effective mitigating therapies are available for this
Currently, post‐exposure symptom management is the sole therapy that can be
offered to personnel at risk for radiation exposure or victims of such exposure, and
almost the only available intervention for patients receiving radiotherapy10. For the
radiation‐induced hematopoietic syndrome, characterized by pancytopenia, relatively
adequate therapies are available and promising new treatment modalities are under
development11,12. Treatment of pancytopenia, either symptomatic by transfusing red
blood cells and platelets or by stimulating bone marrow recovery using hematopoietic
cytokines or stem‐cell transplantation, reduces lethality of radiation‐induced
hematopoietic syndrome. In contrast, no effective strategies are available to treat
acute intestinal radiation toxicity.
Novel approaches are needed to develop: (a) safer and more efficient
radioprophylactic agents, and (b) agents that can mitigate radiation injury after
radiation exposure has occurred. Even though it is not unthinkable that a
radioprotective strategy may act both as a radioprophylactic and a mitigating strategy,
it is to be expected that, once developed, there will be substantial differences in the
mechanisms by which radioprophylactic agents and mitigating agents exert their
effect. An ideal radioprophylactic agent is expected to be a strong cytoprotector at
the time of radiation exposure. It is likely to interfere directly with the initial
radiochemical event and to prevent radiation‐induced DNA strand breaks. In contrast,
a mitigating agent is supposed to regulate the downstream pathophysiological
manifestation of radiation injury. It should act on the radiation injury cascade and
thereby prevent the development of further injury.
Since relatively effective strategies to treat hematopoietic radiation injury are already
available, gastro‐intestinal radiation injury is gaining importance as a determinant of
radiation induced death. Therefore, there is particular interest in developing novel
agents that can prevent or reduce intestinal radiation toxicity.
At this moment in time, the socio‐political climate tends to be supportive towards
research programs to develop novel radiation countermeasures for emergency
situations. This trend does not only provide novel opportunities and funds to develop
interventions for non‐clinical radiation exposure, but may also benefit cancer
treatment. It may lead to the development of “dual utility” drugs that can both be
used as radiation countermeasure and in patients undergoing radiotherapy. The field
of radiotherapy may therefore benefit greatly from novel advances made in the
development radiation countermeasures.
However, it needs to be noted that special care should be taken when developing
radioprophylactic or mitigating agents to be used during or after radiotherapy. In
order to improve the therapeutic index, i.e., the likelihood of tumor cure compared to
normal tissue damage, the agents are to protect non cancerous tissues without
hampering the effect of radiotherapy on tumor cell injury.
During the last few years, several promising novel radioprophylactic and
radiomitigating agents have been discovered. Considerable effort has been made to
identify the mechanisms by which these agents confer radioprotection. In order
improve the efficacy of these agents and to develop novel, even more effective
strategies, it is essential to identify and understand the mechanisms by which these
agents exert their effect. A profound understanding of the mechanisms that confer
radioprotection is necessary to make further progress in the development of effective
countermeasures against radiation injury and subsequent lethality.
Purpose and outline of the thesis
The main aim of this thesis was to identify novel radioprophylactic and/or mitigating
agents and to unravel their mechanism of action.
Considering the increasing importance of intestinal radiation toxicity as a determinant
of radiation‐induced death, we were particularly interested in identifying agents that
could prevent or reduce intestinal radiation injury.
When starting the research described in this thesis, we had 2 main hypotheses:
1. The vitamin E analog γ‐tocotrienol protects against total body irradiation (TBI)‐
induced injury through mechanisms depending on inhibition of 3‐hydroxy‐3‐
methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase.
2. The novel somatostatin analog SOM230 (Pasireotide) reduces TBI‐induced
mortality by inhibiting the secretion of pancreatic enzymes and thereby reducing
post‐irradiation intestinal injury.
The results of the research related to the first hypothesis will be described in chapter
2‐6. Chapter 7 and 8 will show the outcome of studies that were driven by the second
In general, the studies presented in this thesis were focused on the development of
radiation countermeasures to be used before or after radiological emergencies.
However, the described agents are strong candidates for development as “dual utility”
drugs, that is, they could be used both as radiation countermeasure and clinically to
make radiation therapy safer and more effective.
The first part of the thesis will focus on the vitamin E analog γ‐tocotrienol (GT3). There
is significant interest in developing vitamin E analogs as radioprophylactic agents,
Introduction 15 Download full-text
because of their potent anti‐oxidant properties and lack of performance degrading
side effect. GT3 has recently been shown to be more effective in decreasing total
body irradiation‐induced death than α‐tocopherol, the most abundant and commonly
used vitamin E analog13.
In chapter 2, we confirm that a single prophylactic dose of GT3 greatly reduces
lethality after TBI14. Moreover, we show that in addition to stimulating hematopoietic
recovery, GT3 also reduces radiation‐induced intestinal injury and vascular oxidative
stress. Interestingly, GT3 appears to exert its effect on vascular free radical production
through inhibition of 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase.
Chapter 3 shows that the beneficial effect of GT3 on acute radiation toxicity can be
enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX).
Combined treatment with GT3 and PTX increases post‐TBI survival over GT3 alone by
a mechanism that may depend on induction of hematopoietic stimuli. GT3+PTX does
not reduce gastro‐intestinal toxicity or vascular oxidative stress compared to GT3
alone. Furthermore, we showed that the beneficial effects on post‐irradiation
mortality of either drug alone or in combination on post‐irradiation mortality do not
require the presence of the endothelial nitric oxide synthase (eNOS).
In order to improve our understanding of the mechanisms responsible for the
differences in radioprotective potential of the various vitamin E analogs or so called
tocols, and to elucidate why GT3 is a more potent radioprotectant than most other
tocols, we performed a series of gene expression experiments of which the results are
reported in chapter 4. GT3 was far more potent in inducing gene expression changes
than α‐tocopherol and γ‐tocopherol, the 2 other tocols that were used. GT3 induced
multiple changes in functional gene clusters known to be of importance in de cellular
response to radiation exposure.
In chapter 5 evidence is provided for a possible role for tetrahydrobiopterin (BH4) in
the mechanism by which GT3 reduces post‐irradiation vascular oxidative stress15. BH4
is an essential cofactor for all nitric oxide synthase (NOS) enzymes and a critical
determinant of NOS function. Under conditions of oxidative stress, such as after
radiation exposure, the availability of BH4 might be reduced due to rapid oxidation of
the molecule. Insufficient availability of BH4 leads to uncoupling of endothelial NOS
(eNOS). In an uncoupled state, eNOS will produce the highly oxidative radical
superoxide and peroxynitrite at the cost of nitric oxide. We have shown that BH4
levels are indeed reduced in the early post‐irradiation phase and that exogenous
administration of BH4, like GT3, reduces post‐irradiation vascular oxidative stress.
Moreover, GT3 was proven to reduce the expression of guanosine triphosphate
cyclohydrolase 1 (GTPCH) feedback regulatory protein (GFRP), one of the key
regulatory proteins in BH4 metabolism. GT3 may thus exert some of its beneficial
effects on radiation‐induced vascular oxidative stress by counteracting the decrease in
Since inadequate supplies of BH4 in the early post‐irradiation phase may play an
important role in the pathogenesis of radiation‐induced endothelial dysfunction and