Chapter 9 - Radioprotectors

Radioprotectors
Prepared by Du Le
MED PHYS 773 (2013)

Objectives
To introduce types of radioprotectors
(against indirect mode of ionizing
radiation) that are commonly used in
clinics as well as in radiation research.
To discuss pros and cons of each
radioprotector and its future direction (if
applicable)

Definitions
– Radioprotectors
– Dose reduction factor
Outline
Discovery and mechanism
– Cysteine (SH-compounds)
– Loman et al’s experiment on Thymine
– Development of more effective compounds
– Requirements of a radioprotector
Classification of Radioprotectors
– Prophylaxis (Pre-exposure protection )
Systemic Hypoxia
– Mitigation (shortly after exposure)
Radical Scavengers
– Amifostine
– Nitroxides
– Superoxide dismutase
– Selenium
– Treatment
Stem cell therapy
Intervention in the angiotensin pathway
Anti-inflammatory treatments
Growth factor treatments
Summary

Radioprotectors: Definition
Radioprotectors are agents or compounds
delivered prior or shortly after radiation
exposure with the intent of preventing or
reducing damage to normal tissues.
- Citrin et al., The oncologist (2010)

Dose reduction factor (DRF)
DRF is used to describe the effectiveness of a
radioprotector.
– Higher DRF, better protection to normal cells against ionizing radiation
DRF is expressed as the ratio of dose of radiation in the
presence of the drug to dose of radiation in the absence of
drug to produce a given level of lethality
Example: Considering the same mice population
– 100 Gy is 50% lethal dose in mice in presence of cysteine
– 80 Gy is 50% lethal dose in mice in absence of cysteine
– What is DRF?
100Gy
DRF
80Gy
1.
25

Discoveries of Radioprotectors
In 1948:
– Patt discovered that cysteine (a sulfhydryl
compound) could protect mice from effect of total
body exposure to x-rays if the drugs were
injected in large amount before radiation
Effect of cysteine on Thymine concentration was also
demonstration later
– Loman et al’s experiment (1970)
– Bacq discovered cysteamine could also protect
animals from total body irradiation
Cysteine
Cysteamine

Discoveries
(Cont.)
Patt’s results:
– Pre-treatment with
cysteine reduced
toxicity in total body
X irradiation.
– Increasing Cysteine
dose fo could
increase survival
rate.
– All injections were 5
mins before
irradiation.
H.M. Patt et al. Science (1949)

Patt’s results:
– Injection of cysteine immediately after exposure was ineffectual
– Similar high survival rate was obtained when cysteine was given 5
mins or 1 hr before exposure
– H.M. Patt et al. Science (1949)

Indirect action of Cysteine on Thymine
Loman et al.’s experiment (1970)
– Expose Thymine (5 X10 -4 M) in the excess of alcohol
radicals with dose rate of 46.5 Gy/ minute.
– Thymine concentrations were determined with a
spectrophotomer.
– The destruction of thymine was measured by following
the decrease in optical density as a function of radiation
dose rate per volume.
Optical density is proportional to concentration
– Small cystein concentration (10 -4 M) was added before
exposing for comparison with the control sample

Indirect action of Cysteine on Thymine (cont.)
Loman et al.’s results (1970)
No cystein added experiment
– Thymine concetration was strongly reduced
– Radicals produced from alcohol due to ionizing radiation were able
to destroy the thymine chromophore, with a rate constant of the
order of 105 M -1 sec -1
Cysteine added experiment
– Small or no change in Thynine concentration
– H-atom was transfered from the sulfhydryl compound to the
alcohol radicals, thus preventing the reaction of the organic radical
with thymine
– This type of indirect protection of a DNA constituent could be
importance for the radioprotection of DNA in living cell

Indirect action of Cysteine on Thymine (cont.)
If cysteine concentrations were increased, the bending
down of the dose-effect curve would be delayed
10 -4 M cystein
2*10 -4 M cystein
3*10 -4 M cystein
Irradiation of solutions of 5 X 10 -4 M Thymine + N20 +
0.5 M ethanol and (1) 10 -4 M cystein ; (2) 2x 10 -4 M
cystein; (3) 3x 10 -4 M cystein
- Loma et al. Radiation Reasearch (1970)

Development of more effective compounds
Cysteine is toxic and induces nausea and vomiting.
In 1959, U.S. Army initiated a program at Walter Reed Institute to
indentify and synthesize drugs capible of protecting individuals from
radiation enviroment without deliberating toxicity of cysteine.
– More than 4,000 compounds were synthesized and tested.
Toxicity of cysteine could be greatly reduced if the SH group was
covered by a phosphate group. Once in the cell, the phosphate group
will be stipped and SH group will scavenge for radicals
– 50% lethal dose of the compound can be doubled
– DRF can be greatly enhanced
Radiobiology for the Radiologist

Development of more effective
compounds (cont.)
Two typical compounds of more than 4,000 synthesized:
– Cystaphos (WR-638): used during Cold War
– Amifostine (WR-2721):
Most effective radioprotector of those synthesized
Good protetcion to blood organ, DRF for 30-day dealth in mice nearly
maximum value of 3
Carried and used by US astronauts if a solar event occur
Radiobiology for the Radiologist

Requirements for Radioprotectors
Should be selective in protecting normal tissues
from radiotherapy without protecting tumor
tissue.
Should be delivered with minimal toxicity.
Should protect normal tissues that are
considered sensitive such that acute or late
toxicities in these tissues are either dose-limiting
or responsible for a significant reduction in
quality of life
- Citrin et al., The oncologist (2010)

Recall: Events following radiation exposure
- Citrin et al. The Oncologist (2010)

Classification of Radioprotector (indirect
mode protection)
Prophylaxis
(Pre-exposure protection)
Mitigation
(during or shortly after
exposure)
Treatment
Systematic Hypoxia
Amifostine
Nitroxides
Superoxide dismutase
Selenium
Stem cell therapy
Intervention in the
angiotensin pathway
Anti-inflammatory treatments
Growth factor treatments

Prophylaxis

Recall: Oxygen Effect on Survival Curve
Cells are much more sensitive to x-rays in the presence of
molecular oxygen than in its absence (i.e., under hypoxia).
Introduction hypoxia could be expected to reduce radiosensitivity in normal
tissues
Radiobiology for the radiologists, Chap. 6

Systemic Hypoxia (cont.)
Systemic reduction of oxygen partial pressure can be achieved by
breathing air with a reduced oxygen concentration
The protection factor : dose required for a specific effect with
reduced oxygen compared with the dose giving the same effect
with normal oxygen breathing
In single-dose: protection factors in the range of 1.2 –1.4 have
been observed.
In radiotherapy: systemic hypoxia is associated with an increase
in the fraction of hypoxic cells within the tumour
– → an increase in tumour radioresistance
– → Precluded for the amelioration of radiotherapy complications

Example: effect of BW12C on tumour
hypoxia
An increase in the binding of oxygen to haemoglobin by BW12C (5-[2formyl-3-hydroxyphenoxy]
pentanoic acid) - an agent for the treatment
of sickle cell anaemia → reducing availability of oxygen in normal
tissues, with protection factors between 1.0 and 1.3.
Normal blood cells
Sickle cells
http://www.nhlbi.nih.gov

Honess’ experiments
Example (cont.)
Either RIF-1 or KHT tumor was injected to mice legs
Tumour-carrying-mice were injected with BW12C (70mg/kg) 30 mins before
being exposed to dose rate at 67.4 cGy/min.
Results:
– BW12C has no effect on survival of unradiated cells.
– BW12C-injected tumours are more sensitive to radiation.
Dose (Gy)
- Honess et al Br. J. Cancer (1991), 64, 715-722

Classification of Radioprotector (indirect
mode protection)
Prophylaxis
(Pre-exposure protection)
Mitigation
(during or shortly after
exposure)
Treatment
Systematic Hypoxia
Amifostine
Nitroxides
Superoxide dismutase
Selenium
Stem cell therapy
Intervention in the
angiotensin pathway
Anti-inflammatory treatments
Growth factor treatments

Mitigators
(Radical Scavengers)
Free radicals are responsible for large amount of damage
caused by ionizing radiations
– Mitotic cell death
– Tissue hypoxia
– Late effects
Fibrosis
Vascular damage, organ damage
Radiation mitigators aim to scavenge free radicals to
prevent expression of toxicity
For a mitigator to protect cells from radical damage,
migator need to be present at time of radiation and in a
sufficient concentration to compete with radicals
- Citrin et al. The Oncologist (2010)

Amifostine
Organic thiophosphate compound that has been suggested for
amelioration of radiation effects in a variety of normal tissues.
The only radioprotective drug approved by U.S. Food and Drug
Administration for use in radiation therapy.
Sold under trade name Ethyol for use in prevention of xerostomia in
patients treated for head and neck cancer.
The drug is given 30 mins before treatment to exploit the slower rate
at which the drug penetrates tumors ralative to normal tissues
Also used to protect kidneys from side effects of chemotherapy for
treatment of ovarian cancer
More info http://www.nlm.nih.gov/

Amifostine (cont.)
Is a phosphorothioate that does not
permeate cells due to its terminal
phosphorothioic acid group and is used
as a “prodrug”.
– Administered in inactive form
– Converted into drug in metabolic process
When dephosphorylated by enzyme
alkaline phosphatase, aminofostine is
converted to active metabolite designate
WR-1065
WR-1065 enters normal cells by diffusion
to scavenge free radicals generated by
ionzing radiations.
Optimized high dose for cytoprotection is
400 mg/kg → significant side effects
– Hypocalcemia: low serium calcium in
blood
– Nausea/vomiting
More info http://www.nlm.nih.gov/

Nitroxides
Are stable free radicals, have an unpaired electron and
consist of a six- member ring piperidine derivative
Radiation pros:
– Is among most promising agent for future use as radioprotector.
In labaroratory, nitroxides was used to protect cells when
exposed to oxidative stress such as H 2O 2
– Preclinical studies have shown
that the oxidized form of a
nitroxide is a radioprotector in
both in vitro and in vivo models.
Radiation cons:
– Although the hydroxylamine exhibits antioxidant activity, it might
protect tumor as well as normal tissue against ionizing radiation
- Citrin et al. The Oncologist (2010)

Superoxide dismutase (SOD)
Superoxide radicals
– Reactive form of oxygen with an extra electron produced after
exposing cells to ionizing radiation
– Wreak havoc on the cell
– Cause mutations in DNA or attack enzymes that make amino acids
and other essential molecules
- http://www.rcsb.org
Superoxide dismutase:
– Produced by most cells to detoxify superoxide radicals
– Pros:
Catalyzes the conversion of superoxide to oxygen and hydrogen
peroxide and functions as an antioxidant during normal conditions
and after radiation
– Cons:
SOD is a large molecule, does not freely enter into cells
– Animal studies have used gene therapy to increase the levels of
SOD in tissues to be irradiated to prevent radiation fibrosis
(scarring and hardening of tissue inside the body or on skin)
→ Additional works needed to determine effectiveness for clinical trial

Selenium
Atomic number 34, exist in brick-red powder but forms black solid bead
when melt.
Stimulates glutathione peroxidase, which can reduce the level of toxic
oxygen compounds (free radicals) in cells irradiated with ionizing radiation.
Is essential to good health but
required only in small
amounts : daily value is 70
micrograms (mcg) (FDA)
- http://ods.od.nih.gov/factsheets/Selenium-HealthProfessional/
Radiation pros: In laboratory, sodium selenite were shown to
– Clearly increase animal survival after total-body irradiation of rats
– Served as protection of salivary glands by sodium selenite has also
been found in preclinical studies in rats.
Currently, clinical data do not provide a basis for any recommendation
either in favour of or against selenium supplementation in cancer patients

Classification of Radioprotector (indirect
mode protection)
Prophylaxis
(Pre-exposure protection)
Mitigation
(during or shortly after
exposure)
Treatment
Systematic Hypoxia
Amifostine
Nitroxides
Superoxide dismutase
Selenium
Growth factor treatments
Stem cell therapy
Intervention in the
angiotensin pathway
Anti-inflammatory treatments

Growth factor treatments
Growth factor: (Goustin et al. Cancer Research 1986)
– a complex family of polypeptide hormones
– produced by the body to control growth, division and maturation of blood
cells by the bone marrow.
– Regulate the division and proliferation of cells and influence the growth
rate of some cancers
Exogenous growth factors
– Activate or stimulate tissues specific endogenous signalling cascades.
– Haematopoietic growth factor
– Keratinocyte growth factor
Growth factor signalling
– has been shown to change after radiation exposure
– Inhibition of upregulated signalling cascades may be applied either by
antibodies against the growth factor or the respective receptors
– Tumour necrosis factor-α signalling
– Transforming growth factors β- signaling

Haematopoietic growth factors
Produced by bone marrow stromal cells, which reside in close proximity
to hemopoietic precursors
Consist: granulocyte colony-stimulating factor (G-CSF) and granulocyte
macrophage colony-stimulating factor (GM-CSF)
Radiation Pros:
– Stimulation of progenitor cells by G-CSF or
GM-CSF has been demonstrated in
numerous preclinical and clinical studies i.e.
management of leukopenia in cancer
patients
– Might be used to improve radiation effects in
the bone marrow and oral mucosa.
Radiation Cons:
– Recent guidelines recommend not to apply
GM-CSF mouthwashes for the prevention of
oral mucositis in the transplant setting
– Tumour-protective effects of haematopoietic
growth factors have also been demonstrated
experimentally for various tumour types
Influence on multi organ systems
http://www.ncbi.nlm.nih.gov

Keratinocyte growth factor (KGF, palifermin)
Synthesized by mesenchymal cells (fibroblasts or connective tissues).
Forms epithelium in epithelialization-phase of wound healing. The
target cells are the epithelial cells in a variety of tissues.
Radiation pros:
– The factor has been tested in preclinical models for its potential to
ameliorate radiation effects in oral mucosa, skin, intestine, lung and
urinary bladder. Positive effects have been found in all studies.
– Treatment with KGF could result in a highly significant reduction in
the incidence and duration of oral mucositis in patients receiving
total body irradiation
Radiation cons:
– The mechanisms through which KGF acts remain currently unclear.
– More studies are needed

Epidermal growth factor (EGF) signalling
EGF
– protein that is thought to be involved in
mechanisms: normal cell growth, oncogenesis,
and wound healing
– a small 53 amino acid residue long protein that
contains three disulfide bridges (Cys6-Cys20,
Cys14-Cys31, and Cys33-Cys42 )
Radiation pros
– EGF receptor is overexpressed in a variety of tumours and represent
one specific target for improving the tumour effects of radiotherapy.
Radiation cons
-http://www-nmr.cabm.rutgers.edu/photogallery/proteins
– Animal models showed that targeting of EGFR may also modify
normal-tissue effects of radiotherapy.
More research works are necessary to prove effectiveness of EGF

Tumour necrosis factor-α signalling
Tumour necrosis factor-α(TNFα) is a vital cytokine involved in inflammation,
immunity, and cellular organisation.
– First isolated from the serum of mice infected with Bacillus Calmette-Guérin treated with
endotoxin
– Replicate the ability of endotoxin to induce tumour necrosis
– TNF is capable of initiating a tumour apoptotic response (with or without an immune
mediated mechanism)
– Stimulated interest in the use of TNF for the prevention and treatment of cancer
Radiation Pros
- P.W. Szlosarek et al. The Lancet Oncology (2003)
– Upregulation in normal tissues by irradiation has been demonstrated and is usually
considered to promote the radiation response of these normal tissues.→ inhibition of
TNF-α signalling might be beneficial
– Drugs directed against TNF- α signalling (e.g. infliximab or Remicade) are already used
clinically for treating Crohn’s disease - chronic inflammatory condition of the
gastrointestinal tract from the end of the small bowel to the beginning of the colon
(http://www.ccfa.org)
Radiation Cons
– Experiments on mouse kidney showed that treatment with infliximab significantly
aggravate kidney injury (radiation nephropathy) →more tests in relevant preclinical
models before clinical testing is undertaken

Transforming growth factors β- signaling
(TGF)
TGF-beta: is a potent regulatory cytokine with diverse effects on hemopoietic
cells.
– Maintains tolerance in immune system via the regulation of lymphocyte proliferation,
differentiation, and survival.
– Controls the initiation and resolution of inflammatory responses through the regulation
of chemotaxis, activation, and survival of lymphocytes, natural killer cells, dendritic
cells, macrophages, mast cells, and granulocytes
Signaling from TGF-beta through its transmembrane receptor serine threonine
kinases plays a complex role in carcinogenesis, having both tumor suppressor
and oncogenic activities
– Tumor cells have ability to alter TGF receptors functions to compromise tumor
suppressor activity of TGF-beta, increase its oncogenic functions → inhibit TGF-beta
signaling
Inhibit the activation of TGF-β from its latent form is regulated by the integrin
alpha(v)beta6.
However, treatment of irradiated mice with a monoclonal antibody against this
integrin has prevented fibrosis
- De Caestecker et al. J.National Canc. Inst. (2000)

Transplantation of bone marrow
Stem cell therapy
– Potential to improve oral mucositis in the mouse
– Transplantation during daily fractionated irradiation resulted in a reduction in mucosal
reactions
- Dorr et al (unpublished data)
Transplantation of mesenchymal stem cells (MSC)
– MSCs are found in bone marrow and other tissues such as liver and lung. MSCs
differentiate to form cartilage, bone, tendons, muscle, and skin.
– In mouse oral mucosa, daily fractionated irradiation has significantly reduced the
incidence of confluent oral mucositis - Haagen and Dörr (unpublished data)
– Applied successfully as part of the therapy of skin lesions in patients after radiation
accidents -Lataillade et al., 2007
Mobilization of bone marrow stem cells
– Release of stem cells from the bone marrow can be stimulated by granulocyte colonystimulating
factor (G-CSF) which affects both haematopoietic and mesenchymal stem
cells.
– Administration of G-CSF resulted in a clear reduction of radiation-induced mucositis
after single-dose irradiation in mouse mucosa.
- Dorr et al. (unpublished data)

Angiotensin converting enzyme (ACE)
inhibitors
Angiotensin:
– is a peptide hormone derived from angiotensinogen (serum globulin
produced in liver). Its main role is to constrict blood vessels causing high
blood pressure
– In the kidney, angiotensin converting enzyme (ACE)-induced hypertension
contributes to the development of the radiation response.
– Therefore, ACE inhibitors, such as captopril, and antagonists of the
angiotensin II type 1 (AT1) and type 2 (AT2) receptor, have been tested for
their potential to mitigate or treat late radiation effects, particularly in kidney
and lung.
Radiation pros of ACE inhibitors
– Were shown to prevent kidney sequelae of irradiation in rat models (Moulder et al.,
2007)
– Effective in the prevention of radiation fibrosis - scarring and hardening of tissue
inside the body or on the skin (Molteni et al., 2000).
Cons
– Used in combination with chemotherapy which may alter radiation effect.
– Validation of the results with ACE inhibitor is desirable

Glucocorticoids
Anti-inflammatory treatments
– Steroid hormones present in almost every vertebrate
animal cell
– Bind to the glucocorticoid receptor.
– Effect on metabolism:
Synthesis of glucose from amino acid
Stimulation of fat breakdown in adipose tissue
– The vast majority of glucocorticoid activity in most mammals is from cortisol
– Cortisol binds to the glucocorticoid receptor in the cytoplasm and the
hormone-receptor complex is then translocated into the nucleus
– Applied as symptomatic, supportive treatment in order to manage oedema
and pain associated with the inflammatory component of radiation-induced
side-effects.
– No conclusive results are available for this class of drugs for specific targeting
of inflammatory processes in order to prevent radiotherapy side effects

Anti-inflammatory treatments (cont.)
Non-steroidal anti-inflammatory drugs (NSAIDs)
– Pros:
– Cons:
Block the cyclooxygenase enzymes (COX) and reduce prostaglandins
throughout the body
Prostaglandins are produced within the body's cells by the enzyme
cyclooxygenase. They promote inflammation, pain, fever
- http://www.medicinenet.com
Used for the symptomatic management of inflammatory signs of (early)
radiation side-effects : acetylic salicylic acid
Cause prolongation of renal failure in experiment on mouse kidney
Since Prostaglandins can protect stomach from acid damage and
support blood clotting, reducing Prostaglandins with NSAIDs may
cause bleeding and stomach diseases

Summary
Radioprotectors are chemicals that reduce the biologic effect of
radiation.
Mechanism of action of radioprotection is the scavenging of free
radicals to prevent normal tissue damage
Based on the biology of the response of the tissues to irradiation,
radiation protection approaches can be categorized as prophylaxis,
mitigation or treatment.
Amifostine remains the only agent currently in clinical use as a
radioprotector. Most other agents are predominantly experimental.
A number of other candidate compounds will be tested in future
years as a way to reduce radiation induced normal tissue toxicity
and complications.
Most promising, with first clinical studies, are the interaction with
growth factor signaling, and treatment with stem cell therapy

References
http://www.ccfa.org/what-are-crohns-and-colitis/what-is-crohns-disease/
http://www-nmr.cabm.rutgers.edu/photogallery/proteins/htm/page3.htm
http://www.medicinenet.com/nonsteroidal_antiinflammatory_drugs/article.htm
http://www.rcsb.org/pdb/101/motm.do?momID=94
Patt, H. M., Tyree, E. B., Straube, R. L., & Smith, D. E. (1949). Cysteine protection against
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Wilkins. Chap 9
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Citrin, D., Cotrim, A. P., Hyodo, F., Baum, B. J., Krishna, M. C., & Mitchell, J. B. (2010).
Radioprotectors and mitigators of radiation-induced normal tissue injury. The
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NAIR, C. K., PARIDA, D. K., & NOMURA, T. (2001). Radioprotectors in
radiotherapy. Journal of radiation research, 42(1), 21-37.
Honess, D. J., Hu, D. E., & Bleehen, N. M. (1991). BW12C: effects on tumour hypoxia,
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De Caestecker, M. P., Piek, E., & Roberts, A. B. (2000). Role of transforming growth
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Thank you !