1) Hypoxic cell sensitizers are agents that increase the lethal effects of radiation specifically on hypoxic tumor cells. An ideal sensitizer selectively sensitizes hypoxic cells at concentrations that do not greatly increase toxicity to normal tissues. 2) Several hypoxic cell sensitizers have been evaluated clinically including nitroimidazoles like misonidazole, metronidazole, and nimorazole. Nimorazole showed benefit for head and neck cancers with less toxicity. 3) Other approaches to overcoming tumor hypoxia include hyperbaric oxygen, carbogen breathing, blood transfusions, and bioreductive drugs that selectively kill hypoxic cells. A meta-analysis found that

1. HYPOXIC CELL SENSITIZERS
Dr Bharti Devnani
Moderator:-Dr Ritu Bhutani

2.  What is the Hypoxic cell sensitizer? What are the
characteristics of an ideal Radio sensitizer?
Enumerate all Radio sensitizers.

3. GOAL OF RADIATION THERAPY

4. CONCEPTS
 Two concepts fundamental to understanding the rationale for
modification of radiation response
1. Therapeutic ratio – defined as NTT/TLD
 Both of these parameters have sigmoid dose response curves
 As the separation between these curves increases, the likelihood
increases that treatment will be effective & not cause an
unacceptable level of morbidity
2. Efficacy & toxicity profile of the modifier – directly affect TR
 A radiosensitizing agent that exacerbates toxicity to same extent that
it improves efficacy – TR unchanged or worsened

6. RADIOSENSITISERS
Physical and Chemical (pharmacological) agents that increase the
lethal effects of radiation when administered in conjunction to
radiotherapy.

7. TYPES OF SENSITISERS
 Non hypoxic cell sensitisers(Halogenated
pyrimidines )
Differential effect is based on the premise that
tumor cells cycle faster and therefore incorporate
more of the drug than the surrounding normal
tissues.
 Hypoxic-cell sensitizers
Increase the radiosensitivity of cells deficient in
molecular oxygen(tumors) but have no effect on
normally aerated cells.

8. Oxygen fixes
the damage
by free radical to DNA.
(O2 fixation
hypothesis)
Why o2 is required?

9. CHARACTERISTICS OF AN IDEAL HYPOXIC CELL
SENSITIZER
1. Selectively sensitize hypoxic cells at concentration that would
result in acceptable normal tissue toxicity (differential effect)
2. Chemically stable & not subject to rapid metabolic break down
3. Highly soluble in water or lipids & must be capable of diffusing a
considerable distance through a nonvascularized cell mass to
reach the hypoxic cell
4. It should be effective at relatively low daily dose /# used in
conventional fractionated radiotherapy

10. METHODS TO SENSITIZE HYPOXIC
CELLS
Physical Chemical

11. METHODS TO SENSITIZE OR ELIMINATE HYPOXIC CELLS
1. Physical
 Overcoming hypoxia by eliminating it with treatment that increases
delivery of oxygen to tumor i.e. increases the oxygen carrying capacity
of blood and increasing the tumor blood flow
a) Hyperbaric oxygen
b) Carbogen with or without nicotinamide
c) ARCON
d) Hyperthermia

12. METHODS TO SENSITIZE OR ELIMINATE HYPOXIC CELLS
2. Chemical
a) Modifiers of Hb
b) Hypoxic cell sensitizers
c) Hypoxic cytotoxins or bioreductive dugs - Pharmacological
targeting of hypoxic cells – cytotoxic to hypoxic tumor cells
d) Biologic modifiers
e) Chemotherapeutic drugs

14. HYPERBARIC OXYGEN
 An increase in barometric pressure of the gas
breathed by the patient during radiotherapy is
termed ‘hyperbaric oxygen (HBO) therapy’.
 Pioneered by Churchill-Davidson in 1968 at St.
Thomas' Hospital in London.
 Patients were sealed in chambers filled with pure
oxygen raised to a pressure of 3 atm.

15. HYPERBARIC OXYGEN

16.  Problems
 Feeling of claustrophobia
 Unconventional hypofractionated schemes
 Increase in late normal tissue damage(damage to
laryngeal cartilage in studies)
 Risk of fire
 Cumbersome
 Side effects - damage to the ears, sinuses and lungs
from the effects of pressure, temporary worsening of
myopia, acute central nervous system oxygen toxicity
(seizures)
Discarded due to introduction of better chemical radiosensitisers
that would achieve same end by simpler means

17. Clinical trial of HBO
 largest of the multicentre clinical trials of HBO is
performed by the British Medical Research Council.
Results:-
Benefit observed in (both LRC & OS)
 Advanced head and neck cancer
 Uterine cervix cancer
Benefit was not observed in
 bladder cancer;
Improvement in LRC
(6.6%) & survival

19. CARBOGEN
 Pure oxygen if breathed – vasoconstriction - closing down of
some blood vessels – defeats the object
 Carbogen – 95% O2 +5% CO2
 Rationale – addition of CO2 to gas breathing mixture - shift
the oxyHb association curve to right – facilitate unloading of
oxygen into most hypoxic tissues
 Simple attempt to overcome chronic hypoxia
 Can be given under normobaric condition.
Failed to show significant therapeutic gain (Horsman et al., 2007).

20. Diffusion limited chronic hypoxia
Hypoxia
Perfusion limited acute hypoxia

21. NICOTINAMIDE(B3)
Prevents the transient fluctuations in tumour blood
flow that lead to the development of acute hypoxia
(Horsman et al.,1990).
Benefit has been seen when combined with
 Hyperthermia,
 Perfluorochemical emulsions,
 Pentoxifylline and
 High oxygen-content gas breathing
(Horsman,1995).

22. ARCON
 Accelerated – to overcome proliferation
 Hyperfractionated – to spare late responding normal tissues
 Carbogen breathing – to overcome chronic hypoxia
 Nicotinamide – to overcome acute hypoxia

25. PHASE III TRIAL
 Laryngeal carcinoma(Netherland)
 Increase in regional control rate
 93% v/s 86%(p=.04)
 With equal toxicity

28. BLOOD TRANSFUSION
 Anemia – powerful adverse prognostic factor in pts of Ca Cervix, H&
N cancers & lung cancer
 Investigated in no. of studies
 1st clinical investigation – in advanced cervical cancer
 Transfusion to pts with low Hb levels - ↑ed oxygen tension within
tumor
 Transfusion to Hb level of 11g/dl or higher – improved survival
 Not been supported by data from controlled randomized trials
 H & N Cancer pts – 2 phase II trials from DAHANCA study group –
failed to demonstrate any benefit

29. ERYTHROPOETIN
 Low Hb concentration ↓es radiation response of tumors
 Two studies conducted in H & N cancers failed to show any benefit
 In one of the studies – pts who received erythropoetin showed
significantly poor outcome than those who did not
 ? Erythropoietin may stimulate tumor growth
 Schedules –
 Thrice weekly – 150 U/kg s.c
 Weekly - 40000 U s.c.

30. PERFLUOROCARBONS
 Artificial blood substances
 Small particles capable of carrying more oxygen or manipulating the
oxygen unloading capacity of blood
 Potential usefulness uncertain

31. HYPOXIC CELL SENSITISERS
 Nine different drugs have reached clinical
evaluation
 Misonidazole,
 Metronidazole,
 Benznidazole,
 Desmethyl-misonidazole
 Etanidazole,
 Pimonidazole
 Nimorazole,
 Ornidazole
 Rsu1069

32. Hypoxic Cell Radiosensitizers
The first candidate
to satisfy these criteria
was Misonidazole

33. Hypoxic Cell Radiosensitizers

34. METRONIDAZOLE
 1st generation 5-nitroimidazole
 Sensitizer Enhancement ratio - 1.2
 Formulations - 500 mg tablets or 500mg /100 ml solution
 Half life – 9.8 hrs
 Total cumulative dose not to exceed 54 gm/m2
 Multiple doses 6gm/m2 3 times/wk for 3- 4week
 Optimal time for administration - 4 hour before radiation
 Dose limiting toxicity –
 Gastrointestinal
 Sensory peripheral neuropathy

35. MISONIDAZOLE
 2nd generation 2- nitroimidazole
 Higher electron affinity
 Sensitizer Enhancement ratio –
 1.4 with multiple dose of 2 gm/m2
 1.15 with 0.5mg/m2
 Formulations 500 and 100 mg tablets and capsules
 once or twice/wk for 5-6 wks
 Total cumulative dose not to exceed 12 gm/m2
 Optimal time for administration -- 4 hour before radiation
 Dose limiting toxicity-
 gastrointestinal
 Sensory peripheral neuropathy that progress to central
nervous system toxicity

36. DAHANCA 2
DAHANCA 2, showed a highly significant improvement in the
stratification subgroup of pharynx tumors, but not in
the prognostically better glottic carcinomas

37. ETANIDAZOLE (SR2508)
 3rd generation, analog of Misonidazole
 SER- 2.5-3 with dose of 12 g/m2
 Arthralgia seen more often with 48 hr continuous infusion
 1000mg/19.4 ml saline solution
 Total dose - 40.8 g/m2 at 1.7-2g/m2 3 times/wk for 6 wks
 30 min before radiation
Lesser neurotoxic due to
Shorter half life
Lower lipid solubility(less rapidly taken by the neural tissue)
No significant benefit was observed in two large head and neck
cancer trials, one in the USAand the other in Europe.

38. ETANIDAZOLE
 RTOG phase III study with Etanidazole in head and neck tumors
n- 521 patients
Conventionally fractionated RT RT
with Etanidazole 2mg/m2 with out Etanidazole
three times wk
 No grade III or IV central nervous system or peripheral neuropathy
was observed.
 The 2-year actuarial local tumor control was 40% in each arm, and
the survival was 41% and 43%, respectively, in the irradiation alone
and the irradiation plus etanidazole arms
No overall benefit when
Etanidazole added to
conventional radiotherapy

39. PIMONIDAZOLE
 4- nitroimidazole
 More potent than Misonidazole
 Uncharged at acid pH, thus promoting its accumulation in ischaemic
regions of tumours.
 Several – fold ↑ in tumor concentration
 Maximum tolerated dose – 750 mg/m2
 Dose limiting toxicity – CNS manifesting as disorientation & malaise
 A pimonidazole trial was started in uterine cervix, but was stopped when it
became evident that those patients who received pimonidazole showed a
poorer response.

40. NIMORAZOLE
 A 5-nitroimidazole of same structural class as metronidazole
 Administered in form of gelatin-coated capsules containing 500 mg
active drug
 Given orally 90 min prior to irradiation.
 Daily dose 1200 mg/m2 body surface
 Total dose should not exceed 40g/m2 or 75 g in total.
 Less effective radio sensitizer then Misonidazole or Etanidazole
 Less toxic, no cumulative neuropathy
 Large dose can be given
 dose-limiting toxicity is nausea and vomiting

41. NIMORAZOLE

42. NIMORAZOLE – DAHANCA 5
Significant improvement in
terms of LRC & OS
Nimorazole significantly improves the effect of
radiotherapeutic management of supraglottic and pharynx
tumors and can be given without major side-effects

43. As a consequence, nimorazole has now become part
of the standard treatment schedule for head and
neck tumours in Denmark.

44. DEVELOPMENT OF NITROIMIDAZOLES
Metronidazole
Misonidazole
more active,
toxic, benefit in
subgroup
Etanidazole
less toxic, not
active
Nimorazole
less active,
much less
toxic, benefit
in H& N
cancer

45. Summary of nitroimidazoles trials

46. NEWER NITROIMIDAZOLES
Doranidazole
 promising preliminary results were obtained in a
phase III study with intraoperative radiotherapy in
advanced pancreatic cancer
Sanazol
 which in an International Atomic Energy Agency
multicentre randomized trial (Dobrowsky et al.,
2007) in cervical cancer was found to significantly
increase local control and survival following radical
radiotherapy

47. OVERGAARD META-ANALYSIS
 10,779 patients
 83 RCT
 Hyperbaric oxygen (HBO) (28 trials),
 Hypoxic radiosensitizers (52 trials),
 Oxygen or carbogen breathing (3 trials),
 Blood transfusion (1 trial).
The tumor sites were
 Bladder (16 trials),
 Uterine cervix (15 trials),
 Central nervous system (13 trials),
 Head and neck (24 trials),
 Lung" (11 trials),
 Esophagus (2 trials),
 Mixed (2trials).

48. RESULTS OF META-ANALYSIS
Statistically significant benefit in LRC(4.7%) & OS (2.7%)

49. Locoregional tumor control was stastically significant in H&N
(p=0.0002)

51. HYPOXIC CYTOTOXINS BIOREDUCTIVE DRUGS
 Elimination of radioresistant hypoxic cells by selectively killing them.
 These compounds undergo intracellular reduction to form active
cytotoxic species, primarily under low oxygen tensions.
 Maximum cytotoxicity to cells at maximum distance from tumor blood
vessels
 Overcome major cause of resistance of solid tumors – inadequate
oxygenation & drug delivery to tumor cells distant to blood vessel

52. Quinone
antibiotics
• MMC
• EO9
• porfiromycin
Nitroaromatic
compounds
• Misonidazole
• (Rb-6145)
• Nlcq-1, Cb1954,
Sn23862
• Pr-104
Benzotriazine
di-N-oxides
• Tirapazamine
• chlorambucil N-
oxide
• AQ4N
(banoxantrone),

53. QUINONE ANTIBIOTIC - MITOMYCIN C
 Prototype bioreductive drug
 Used as chemotherapy agent for many years
 Cytotoxic to relative radio resistant hypoxic cells
 But the differential cytotoxicity between hypoxic and oxygenated cells
, however is small
 Acts as an alkylating agent after intracellular activation & inhibits
DNA – DNA cross linking, DNA depolymerization
 Dose limiting toxicity – cumulative myelosuppression
 Mitomycin C plays an important role in conjunction with radiotherapy
and 5FU, the definitive, chemoradiation squamous cell carcinoma
of the anus

54. PORFIROMYCIN
 A mitomycin C derivative
 Provides greater differential cytotoxicity between hypoxic and
oxygenated cells in vitro
Phase III study
 Compared patients treated with conventionally fractionated
radiation plus mitomycin C versus radiation plus porfiromycin
 The median follow-up - >6 years. Hematologic and non-
hematologic toxicity was equivalent in the two treatment arms
 Mitomycin C was superior to porfiromycin with respect to 5-year
local relapse-free survival (91.6% vs. 72.7%; p = 0.01)

55.  Local-regional relapse-free survival (82% vs. 65.3%; p = 0.05)
 Disease-free survival (72.8% vs. 52.9%; p = 0.03)
 There were no significant differences between the two arms with
respect to overall survival (49% vs. 54%) or distant metastasis-free
rate (80% vs. 76%)
 Their data supported the continuing use of mitomycin C as an
adjunct to radiation therapy in advanced head and neck cancer and
will become the control arm for future studies
PORFIROMYCIN…

56. TIRAPAZEMINE (SR 4233)
 Highly selective toxicity against hypoxic cells both in vivo and vitro
 MOA- Drug is reduced by intracellular reductases to form highly
reactive radical - produces both double & single strand breaks in DNA
 Analysis of DNA and chromosomal breaks after hypoxic exposure to
Tirapazemine suggests that DNA double-strand breaks are the
primary lesion causing cell death
 Efficacy depends on no. of effective doses that can be administered
during course of RT & presence of hypoxic tumor cells
 S/E – nausea & muscle cramping

57. TIRAPAZEMINE
 Hypoxic/cytotoxicity ratio – ratio of
drug concentration under aerated and
hypoxic condition required to produce
same cell survival
 Unlike the oxygen-mimetic sensitizers,
tirapazamine-mediated therapeutic
enhancement occurs both when the
drug is given before or after irradiation.
 Tirapazamine can also enhance the
cytotoxicity of cisplatin

58. N= 121 STAGE III/IV SCC OF THE HEAD AND NECK
RANDOMIZED TO RECEIVE DEFINITIVE RADIOTHERAPY (70 GY
IN 7 WEEKS)
Tirapazamine On day 2 of weeks 1, 4, and 7,
290 mg/m2 was administered for 2 hours,
followed 1 hour later by cisplatin 75 mg/m2 for 1 hr
followed immediately by radiotherapy
In addition, tirapazamine 160 mg/m2 was given
before radiation three times/week in weeks 2 and 3
Cisplatin 50 mg/m2 was given
before radiotherapy on day 1 of
weeks 6 and 7 of radiotherapy
and
Fluorouracil 360 mg/m2/d was
given by continuous infusion from
day 1 - 5 (120-hour infusion) of
weeks 6 and 7 of radiotherapy
Arm 2. n-58Arm 1,n-62

60.  Three-year failure-free survival rates were 55% with TPZ/CIS and
44% with chemo RT( p .16)
 Three-year locoregional failure-free rates were 84% in the
TPZ/CIS arm and 66% in the chemo RT arm (p .069)
 Toxicity
 More febrile neutropenia and grade 3 or 4 late mucous membrane
toxicity were observed with TPZ/CIS
 Compliance with protocol treatment was satisfactory on both arms

61. Markers of hypoxic cells

62. MARKERS OF HYPOXIC CELLS
Radioactive labeled nitroimidazoles
Bioreduction
Deposition of
radionuclide
Quickly excreted
Without breaking
down
Hypoxia Aerobic tissue

63. RADIOACTIVE LABELED NITROIMIDAZOLES
 Nitroimidazole can be labeled with I 123
 Hypoxic region of tumour can be visualized with single photon
emission computed tomography
 Now, tumor Hypoxia can be detected by [18F]-Misonidazole
Positron Emission( FMISO-PET) in Patients With Advanced Head
and Neck Cancer imaging
 Noninvasive procedure that can be used as predictive assay in
individual patients
 The availability of methods to detect significant areas of hypoxia can
allow selection of patients who may benefit from method of
overcoming hypoxia

64. CONCLUSION
 1896 -First radiotherapy treatment.
 1909 -First clinical observation by Gollwald Schwarz
showing Reduced blood flow caused radioresistance.
 1953- First experimental observation of potential
importance of hypoxia in radiotherapy.
 1955 -First observation of hypoxia in human tumors.
 1955 -First hyperbaric treatment.
 1968- Results from first randomized trial.
 1976 -First randomized study with hypoxie
radiosensitizer.
 19,95- More than 10000 patients in 83 randomized
trials. Metaanalysis shows highly significant survival and
local control benefit.
 2013- Still no impact on general dinical practice.

65. NOVEL DRUGS
 5 promising redox modulators are in development.
 Tirapazamine
 AQ4N
 RSR13 facilitates delivery of oxygen to tumor cells,
thereby rendering them more sensitive to radiation.
 Motexafin gadolinium, with a porphyrin-like
structure, selectively accumulates in tumor cells
and thereby enhances radiation-induced DNA
damage.
 HIF-1 inhibitors target a transcription factor that
regulates hypoxia related events and cell survival.

66. HIF-1 TARGETING AGENTS
 Soluble guanylyl cycle activators (e.g., YC-1) (84,
85),
 HSP90 inhibitors (e.g., geldanamycin radicicola)
(80, 81, 86),
 PI3K inhibitors (e.g., wortmannin, LY294002)
 mTOR inhibitors(e.g., Rapamycin, CCI-779)
 Microtubule modifiers (e.g., 2-methyloxyestradiol,
vincristine,Taxol)
 topoisomerase I inhibitors