Lecture by Prof. Kayne Williams in the context of the Course: "Tumour Hypoxia: From Biology to Therapy III". For the complete e-Course see http://www.myhaikuclass.com/MaastroClinic/metoxia

1. METOXIA Course
Bioreductive drugs and hypoxic cell
sensitisers
Kaye Williams
12th October 2012
This course is funded with the support of the METOXIA project
under the FP7 Programme.

2. Conventional chemotherapy
 Chemotherapy is the use of drugs that kill rapidly dividing
cells to treat cancer.
 Chemotherapy drugs are toxic to cancer cells, which take in
the drugs as they multiply.
 Once inside the cells, the drug kills the cell or prevents it from
dividing and forming new cells.
This course is funded with the support of the METOXIA project
under the FP7 Programme.

3. This course is funded with the support of the METOXIA project
under the FP7 Programme.
Chemotherapy is associated with a number of side effects,
mainly because chemotherapy drugs are toxic to all actively
dividing cells, not just cancer cells.
 Cells in the bone marrow, the lining of the stomach and
intestines, and the hair follicles are normally actively dividing.
Therefore, chemotherapy may result in a decrease in blood cell
counts, nausea, vomiting, diarrhoea, and loss of hair.
Conventional chemotherapy: problems

4. Targeted cancer therapies use drugs that block the growth and
spread of cancer by interfering with specific molecules involved
in carcinogenesis and tumour growth
Many of these therapies focus on proteins that are involved in
the signalling processes that regulate tumourigenesis.
Different classes of agent are in development or licensed,
including small molecule inhibitors and antibodies
This course is funded with the support of the METOXIA project
under the FP7 Programme.
Molecular targeted therapies

5. FDA approved molecular targeted therapies
 signal transduction inhibitors (Gleevec®, gastrointestinal
stromal tumour and chronic myeloid leukemia; Iressa® non-
small cell lung cancer)
 apoptosis (cell death)-inducing agents (Velcade®, multiple
myeloma)
 anti-angiogenics (Avastin®; colorectal carcinoma).
This course is funded with the support of
the METOXIA project under the FP7
Programme.

6. Targeting the tumour microenvironment-
hypoxia
This course is funded with the support of the METOXIA project
under the FP7 Programme.
 Normal tissues:
Hypoxia is rare
Median O2 concentration 3.1% to 8.7%
 Solid tumours:
Hypoxia is common
Greater than 80% of O2 readings below 0.3%
Hypoxia is a tumour-specific condition that can be
exploited in cancer therapy

7. This course is funded with the support of
the METOXIA project under the FP7
Programme.
How hypoxia develops in tumours

8. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Carbocyanin
Hoechst
Dynamic fluctuations in tumour perfusion

9. Why target tumour hypoxia?
This course is funded with the support of the METOXIA project
under the FP7 Programme.
 Regions of low oxygen present in majority of solid
tumours
 Hypoxic cells are viable and resistant to some forms
of chemotherapy and to radiotherapy
 Hypoxia drives genetic instability and is associated
with an aggressive disease phenotype

10. Significance of tumour hypoxia
This course is funded with the support of
the METOXIA project under the FP7
Programme.
Radiation Dose (Gy)
Etoposide (mM)
100 200 300 400 500
HT29
0.0001
0.001
0.01
0.1
1
Oxic Hypoxic
Promotion of genomic
instability
Aggressive phenotype
Radioresistance
Chemoresistance
Chronic
Acute

11. Targeting the tumour microenvironment
This course is funded with the support of
the METOXIA project under the FP7
Programme.
 A defining characteristic of all solid tumours analysed to date is the presence
of hypoxia (low oxygen)
Aggressive
disease
phenotype
Resistance to
therapy
Drug concentration
Proliferation
Nutrition
Oxygen
Capillary
Normal
oxygen
Low oxygen
(hypoxia)
Necrosis
(death)

12. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Gene expression
HYPOXIA
Oxygen effect
Hypoxia-selective prodrugs
Hypoxia induced transcription factors/
downstream effector molecules
Molecular targeted therapies
Low oxygen tension is present in all solid tumours and is
associated with treatment failure

13. Oxygen availability drives cellular response to radiotherapy
This course is funded with the support of the METOXIA project
under the FP7 Programme.
Indirect action
dominant for x-rays
Direct action
Image taken from Horsman and Overgaard in Basic Clinical Radiobiology (Ed. Steel)

14. The dependence of radiosensitivity on
oxygen concentration
This course is funded with the support of the METOXIA project
under the FP7 Programme.
10 20 30 40 50 60 70 155 760
1
2
3
3mm Hg
or 0.5%
Air 100%
oxygen
Oxygen tension (mm Hg at 37°C)
Relative
radiosensitivity

15. Strategies to overcome tumour hypoxia:
hypoxic cell radiosensitisers
This course is funded with the support of
the METOXIA project under the FP7
Programme.
• Electron-affinic chemicals that mimic oxygen in fixing free-
radical damage
• Hypoxic cell sensitisers are not metabolised by the cells in the
tumour through which they diffuse
• Consequently, hypoxic cell sensitisers can penetrate further
than oxygen and reach all hypoxic cells in the tumour

16. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Radiation-response as a function of misonidazole administration
pre- or post- radiation treatment
C3H mouse mammary tumour measured 120 days after irradiation
Overgaard and Horsman, 1993

17. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Development of nitroimidazoles as hypoxic cell
radiosensitisers
Metronidazole
Misonidazole: more active, toxic (CNS toxicity dose limiting);
benefit in some cancer sub-groups
Etanidazole: equal activity to misonidazole, less toxic; no
clinical benefit
Nimorazole: less active, markedly less toxic; benefit in head-
and-neck cancer

18. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Actuarial estimated loco-regional tumour control in patients randomised
to receive nimorazole or placebo in conjunction with conventional
radiotherapy for carcinoma of the pharynx and supraglottic larynx.
Overgaard et al., 1998

19. Current Phase 3 nimorazole trial: IAEA-HypoX. Accelerated
Radiotherapy With or Without Nimorazole in Squamous Cell
Carcinoma of the Head and Neck
Danish Head and Neck Cancer Group
• Primary Outcome Measures: Locoregional control after curative
intended radiotherapy +/- Nimorazole
• Secondary Outcome Measures: Disease specific survival
• Overall survival
• Treatment related morbidity: Treatment related acute and late
morbidity related to radiotherapy and/or nimorazole treatment
This course is funded with the support of
the METOXIA project under the FP7
Programme.

20. Bioreductive drugs- hypoxic selective cytotoxins
This course is funded with the support of
the METOXIA project under the FP7
Programme.
“Exploit the reductive environment of tumours by
developing drugs that are reduced preferentially to
cytotoxic species in the hypoxic regions of tumours”
Rationale:
Activity depends not only on presence of hypoxia within a cell
but also upon the complement of reductase enzymes
Use in combination with agents that preferentially kill oxic cells

21. Classes of bioreductive agents
This course is funded with the support of
the METOXIA project under the FP7
Programme.
• Quinone antibiotics eg mitomycin C
• Nitroaromatics eg RSU1069 (developed from the
hypoxic-cell sensitiser misonidazole - normal tissue
cytoxicity prevented clinical usage); NLCQ-1
• di-N-oxides eg tirapazamine, AQ4N

22. Simplified schematic of bioreductive drug
activation
This course is funded with the support of
the METOXIA project under the FP7
Programme.
1 e - reductase
mediated
02
02
-
Toxic species
Prodrug
-
.

23. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Reference: C.J.Koch (1993)
Cancer Research 53:3992-3997
0.1 1 10 100
1
0.01
0.1
Oxygenpartial pressure (mmHg)
Surviving
fraction Intermediate O2 level
radiation
tirapazamine
AQ4N
NLCQ-1
RB6145/RSU1069

24. Prodrug Cytotoxic species Final product Enzymes thought
responsible
Benzotriazine
e.g. Tirapazamine
Free radical (SR4317) Non-toxic (SR4330) P450R; iNOS
Bi (and mono) functional
alkylating agents
e.g. MMC, porfiromycin,
RB 6145, E09
DNA-adduct Non-functional
reduced drug
P450R; DTD
Alkyl-amino-
Anthraquinones
e.g. Banoxantrone (AQ4N)
AQ4 Stable persistent
cytotoxin
Cytochrome P450s: 1A1,
2B6, 3A4; iNOS
NLCQ-1 Nitro (1ee), nitroso (2ee)
and hydroxylamine (4ee)
metabolites
Unknown, shows
weak
DNA-intercalating
activity
Cytochrome b5, other one
electron reductases
Phosphate esters of
dinitrobenzamide
mustards e.g. PR-104
(Proacta)
Stable cytotoxin Stable persistent
cytotoxin
Phosphatases to generate
alcohol then nitro-
reductases, including one-
electron reductases
This course is funded with the support of
the METOXIA project under the FP7
Programme.
McKeown SR, Cowen RL,
Williams KJ. Clinical Oncology,
19, 427-442 (2007).

25. This course is funded with the support of
the METOXIA project under the FP7
Programme.
RSU1069 Tirapazamine
Influence of hypoxia on the response of KHT murine
tumour cells to RSU1069 or tirapazamine
Air Air
Anoxia
Anoxia

26. Effect of TPZ treatment on radiation response of
SCCVII mouse carcinoma: growth delay
This course is funded with the support of
the METOXIA project under the FP7
Programme.
Brown et al, Int.J.Radiat. Oncol. Biol.
Phys, 20, 457-461, 1991
Tumour
volume
(mm
3
)
103
102
101
0 25 50
Days after first treatment
TPZ+ x-ray
X-ray +TPZ
Saline
TPZ
X-ray alone
8x2.5Gy

27. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Calu-6
Glut-1 AQ4
50mm
16mm
Pre-clinical validation of methods for detecting AQ4 in tumour samples
that have subsequently been used in clinical trials
Standards
RT112
AQ4N
AQ4
Control
RT112
AQ4N
AQ4
Untreated control
tumour
Standards
Calu-6 24h post
60mg/kg
AQ4N
In vivo activation of the hypoxia-targeted cytotoxin AQ4N
(banoxantrone) in human tumour xenografts
Williams KJ*, Albertella MR*, et al. Mol Cancer
Therapeutics, 8, 3266-3275 (2009).

28. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Tumour
volume
(mm
3
)
Time (days)
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
1200
800
400
0
80
40
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
AQ4N +Cis
80
40
0
1200
800
400
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
AQ4N
80
40
0
1200
800
400
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
Cisplatin
80
40
0
1200
800
400
0
Tumour
volume
(mm
3
)
Time (days)
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
1200
800
400
0
80
40
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
1200
800
400
0
1200
800
400
0
80
40
0 80
40
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
AQ4N +Cis
80
40
0
1200
800
400
00
400
800
1200
0 20 40 60 80
10 x 2 Gy
AQ4N +Cis
80
40
0 80
40
0
1200
800
400
0
1200
800
400
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
AQ4N
80
40
0
1200
800
400
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
AQ4N
80
40
0
1200
800
400
0
10 x 2 Gy
AQ4N
80
40
0 80
40
0
1200
800
400
0
1200
800
400
0
0
400
800
1200
0 20 40 60 80
10 x 2 Gy
Cisplatin
80
40
0
1200
800
400
00
400
800
1200
0 20 40 60 80
10 x 2 Gy
Cisplatin
80
40
0 80
40
0
1200
800
400
0
1200
800
400
0
AQ4N enhances chemo-radiotherapy in human tumour
xenografts
Williams KJ*, Albertella MR*, et al. Mol Cancer
Therapeutics, 8, 3266-3275 (2009).

29. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Phase III Trial data: Tirapazamine plus cisplatin versus
cisplatin alone in non-small cell lung cancer
Cisplatin Tirapazamine+cisplatin
Number patients 218 219
Total responders 30 (13.7%) 60 (27.5%)

30. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Phase I/II Trial data: concurrent tirapazamine, cisplatin and
radiotherapy
• complete pelvic control of disease in 13 out of 15 patients
treated (87%)
• suggested better efficacy than standard regimes that
achieve 70-75% control rates at 6 months
Phase I/II: Locally advanced cervical cancer, 6 month follow-up:
Rischin et al,J Clin Oncol, 19, 535-542, 2001
Phase I: Advanced head and neck cancer:
• 3-year local progression free rate 88% reported although
trial was not designed to evaluate efficacy
Craighead et al, Int J Rad Onc Biol Phys, 48, 791-795, 2000

31. This course is funded with the support of
the METOXIA project under the FP7
Programme.
PATIENTS AND METHODS:
Patients with previously untreated stage III or IV head and neck cancers
Treatment:
1. Radiotherapy (70 Gy in 7 weeks) concurrently with CIS (100 mg/m(2))
on day 1 of weeks 1, 4, and 7 (CIS)
2. Radiotherapy (70 Gy in 7 weeks) concurrently with CIS (75 mg/m(2))
plus TPZ (290 mg/m(2)/d) on day 1 of weeks 1, 4, and 7 and TPZ alone
(160 mg/m(2)/d) on days 1, 3, and 5 of weeks 2 and 3 (TPZ/CIS).
The primary end point was overall survival (OS).
Tirapazamine, cisplatin, and radiation versus cisplatin and
radiation for advanced squamous cell carcinoma of the
head and neck (TROG 02.02, HeadSTART): a phase III trial
of the Trans-Tasman Radiation Oncology Group.
Rischin D, et al J Clin Oncol.
2010;28 :2989-95.

32. RESULTS:
• Eight hundred sixty-one patients were accrued from 89 sites in 16
countries.
• In an intent-to-treat analysis, the 2-year OS rates were 65.7% for CIS and
66.2% for TPZ/CIS (TPZ/CIS--CIS: 95% CI, -5.9% to 6.9%).
• There were no significant differences in failure-free survival, time to
locoregional failure, or quality of life.
CONCLUSIONS:
We found no evidence that the addition of TPZ to chemoradiotherapy, in
patients with advanced head and neck cancer not selected for the
presence of hypoxia, improves OS.
This course is funded with the support of
the METOXIA project under the FP7
Programme.
TROG 02.02, HeadSTART

33. This course is funded with the support of
the METOXIA project under the FP7
Programme.
PURPOSE: To report the impact of radiotherapy quality on outcome in a
large international phase III trial evaluating radiotherapy with
concurrent cisplatin plus tirapazamine for advanced head and neck
cancer.
PATIENTS AND METHODS: The protocol required interventional review
of radiotherapy plans by the Quality Assurance Review Center (QARC).
All plans and radiotherapy documentation underwent post-treatment
review by the Trial Management Committee (TMC) for protocol
compliance.
Secondary review of noncompliant plans for predicted impact on tumor
control was performed.
Critical impact of radiotherapy protocol compliance and quality
in the treatment of advanced head and neck cancer: results
from TROG 02.02.
Peters LJ, et al J Clin Oncol.
2010; 28:2996-3001.

34. RESULTS:
 25.4% of the patients had noncompliant plans
 47% of noncompliant plans (12% overall) had deficiencies with a
predicted major adverse impact on tumor control.
 Major deficiencies were highly correlated with number of patients
enrolled at the treatment center (less patients, worse RT)
 In patients who received at least 60 Gy, those with major
deficiencies (n = 87) had a markedly inferior outcome compared
with those whose treatment was initially protocol compliant.
This course is funded with the support of
the METOXIA project under the FP7
Programme.
Critical impact of radiotherapy protocol compliance …..
These results demonstrate the critical importance of radiotherapy
quality on outcome of chemoradiotherapy in head and neck cancer.
Centers treating only a few patients are the major source of quality
problems.
Peters LJ, et al J Clin Oncol.
2010; 28:2996-3001.

35. Tirapazamine failure
• Poorly given radiotherapy
• Poorly managed drug toxicity
• No patient selection based on tumour hypoxia
This course is funded with the support of
the METOXIA project under the FP7
Programme.
Cohorts of patients with hypoxic tumours that could have
been benefiting from the treatment would not be easily
identified

36. Normoxia
necrosis
RB6145/NLCQ-1
TPZ
O2
Chronic hypoxia
Intermediate hypoxia
Tirapazamine is not an ideal drug- it has
poor biodistribution

37. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Hicks et al Clin Cancer Res. 2010
New Tirapazamine analogues for clinical
development- SN30000
TPZ
SN30000
SN29751
Multicellular layer model
Drug passing through-
TPZ is worst

38. This course is funded with the support of
the METOXIA project under the FP7
Programme.
Summary
• Hypoxic cells are found in all solid tumours
• Hypoxic cells cause resistance to radio and mainly types of
chemotherapy
• Hypoxic cells can be targeted
• Classic radiosesnitisers mimic the affect of oxygen- nimorazole shows
clinical benefit
• Bioreductive drugs are selectively toxic towards hypoxic cells
• Phase II data has shown promise, but Phase III trials disappointing-
need to select patients with hypoxic tumours and manage RT and
drug appropriately
• New bioreductives are entering the clinic:
Need to ensure we get the right drugs in to the patients that are most
likely to benefit to ensure failure of TPZ is not repeated