Prodrugs targeting hypoxic cells William R. Wilson Auckland Cancer Society Research Centre Th Ui it fA kl d The University of Auckland [email protected]
Oct 04, 2020
Prodrugs targeting hypoxic cells
William R. Wilson
Auckland Cancer Society Research CentreTh U i it f A kl dThe University of Auckland
My OCI mentors: 1978-79
Dick Hill
y
Gordon WhitmoreMike Rauth
(Ian Tannock)
Hypoxia as a potential therapeutic targetyp p p g
Nitro compoundsNitro compounds
NO2 NO2 NH21e reductase
NHOHNO1 2e 21e reductase
O2O2RR R R R
Nitroso Hydroxylamine AmineNitro
1e 2e 2e
Nitroso Hydroxylamine AmineNitroradical
2e reductaseProtein thiols
Potential cytotoxins
Protein thiols
Hypoxia probes
Broadly similar redox chemistry for quinones, N-oxides and some transition metal complexes
The hippie phaseThe hippie phase
Disclosure of conflict of interest
I am a founding scientist, stock holder, and consultant to Proacta Inc
I will discuss PR-104 (in clinical development by Proacta) and other novel therapeutic agents licenced to Proacta.
The agents in question originate from my labThe agents in question originate from my lab
Proacta funds research contracts in my lab
Bioreductive prodrugsBioreductive prodrugs
S ll l l di tSmall molecule direct oxygen sensors
DNA damage
PRODRUG DRUGMolecular target
or
Molecular targetO2
• Broad spectrum (multiple cell lineages)
• Stable enough to diffuse out of hypoxic g ypregions (bystander effect)
The era of targeted agentsThe era of targeted agents
• Molecularly targeted agents...with their >$100K QALYs
• Physiologically targeted agents– Hypoxia– Low pHe– Other microenvironmental features
Exploit pathophysiology to enhance tumour p p p y gyselectivity of molecularly targeted agents (and dirty
old cytotoxics)
Bioreductive (hypoxia-activated) prodrugsprodrugs
No registered agents, but several in development:
Tirapazamine Arom N-oxide Phase III SRI/StanfordTirapazamine Arom. N oxide Phase III SRI/StanfordAQ4N Aliph. N-oxide Phase II NovaceaPR-104 Nitro cmpd Phase II ProactaTH-302 Nitro cmpd Phase I Threshold p
NLCQ-1 Nitro cmpd Preclinical Evanston HospSN 30000 Arom. N-oxide Preclinical ProactaSN 29730 Nitro cmpd Preclinical ProactaVPN 40541 Nitro cmpd Preclinical Vion
ASCO 2008: HeadSTART phase III trialP i l t t d d d HNSCCPreviously untreated advanced HNSCC
Rischin et al J Clin Oncol 26: 2008 (May 20 Suppl) abstr LBA6008Rischin et al., J Clin Oncol 26: 2008 (May 20 Suppl) abstr LBA6008
RT (70 Gy7 wks)
+ Cisplatin (100 mg/m2 ) d1 wk 1,4,7+ Cisplatin (75 mg/m2 ) + TPZ (290 mg/m2) d1 wk 1,4,7and TPZ alone (160 mg/m2 ) d1,3,5 wk 2,3( g )
• 89 sites, 16 countries, 861 patients• Failed primary endpoint (OS)• RT deviations had adverse effect on treatment outcome• Trend in time to locoregional failure in patients without RT deviations
HR 0.74, 95% CI 0.53-1.04HR 0.74, 95% CI 0.53 1.04• Patients not selected for the presence of hypoxia
Clinical proof of principle: TirapazamineP i it f il i 92 d i d Primary site failure in 92 randomized
advanced H&N patients at Peter MacCallum Cancer Centre
Rischin et al., Int J Radiat Oncol Biol Phys 2007
PET Treatment
PET hypoxia status
P-valueRT +
cisplatinRT + cis
+TPZ
Non-Hypoxic 2/27 3/21 NS
Hypoxic 8/18 0/26 0.0002
P-value 0 008 NS
CONFIDENTIAL 10
P-value 0.008 NS
Extravascular transport limits therapeutic ti it f ti iactivity of tirapazamine
Gas in
Gas out Gas in
Multicellular layer(MCL)
Receiver
Donor (MCL)
230 x 500 x 500 µm region ofR3230Ac tumour
11Hicks et al., J. Natl Cancer Instit. 98: 1118-1128 (2006)
PK/PD guided lead optimisation of tirapazamine
NN+O-
NO IMPROVED SOLUBILITY
IMPROVED HYPOXIC SELECTIVITYN+
O-
N IMPROVED HYPOXIC SELECTIVITY
15
IMPROVED PENETRATION OF HT29 MCLs
on)
1.8 TPZ (133 μmol/kg)
IMPROVED HYPOXIC CELL KILL IN TUMOUR XENOGRAFTS
SN 30000
ent F
lux
10
nal t
o ra
diat
io1 0
1.2
1.4
1.6
( μ g)SN 30000 (600 μmol/kg)
Perc
e
5
TPZ
ll ki
ll (a
dditi
on
0.4
0.6
0.8
1.0
Percent Flux of urea internal standard(Corrected time axis)
5 10 150
HT29 SiHa H460
Log
cel
0.0
0.2
PR-104PR 104
Bystander effect
Low K-value
Improved extravascular transport
Patterson et al., Clin Cancer Res 2007Hicks et al., IJROBP 2007
PR-104 combination chemotherapy: docetaxelAndrogen resistant prostate carcinoma xenograft (22RV1)
em 1000
1200 Control Docetaxel PR-104 Docetaxel + PR-104
me
(mg)
+/-
se
600
800
Tum
or v
olum
400
600
0 10 20 30 40 50 600
200
Time (days)
Patterson et al. Clin Cancer Res, 2007
PR-104 (1100 mg/m2) + docetaxel (60 mg/m2 ) with G-CSF; q3wCSF; q3w
metastatic head and neck squamous cell ca
Confirmed partial response
Pretreatment 2nd cycle 3rd cyclePretreatment29 July 08
2nd cycle9 Sept 08
3rd cycle7 Oct 08
Single Agent Activity:PR 104 vs TirapazaminePR-104 vs Tirapazamine
SiHa human cervical ca xenografts (q4dx3)
val
80
100 PR-104 (1.8 g/kg)(P=0.012)
free
Surv
iv
60
Dis
ease
-f
40
%
0
20
Control (saline)
Tirapazamine (0.10 g/kg)(P=0.39)
Days post treatment0 20 40 60 80 100
0
Single Agent Activity:PR 104 vs conventional chemotherapyPR-104 vs conventional chemotherapy
H460 non small cell lung cancer xenografts, treated at the maximum tolerated dose of each agent (q4dx3)
100ControlPR 104
g (q )%
)
80
100 PR-104DocetaxelGemcitabineCisplatinCyclophosphamide
Sur
viva
l (%
40
60
Substantial oxic cell killing
20
Days post treatment initiation0 10 20 30 40 50 60 70 80 90 100
0
Delivery of a synthetic version of the E. coli nfsB nitroreductase (sNTR) using a Clostridial vectornitroreductase (sNTR) using a Clostridial vector
potentiates the activity of PR104 against SiHa tumors
ControlsNTR spores alone
PR104 alone
1
PR104 alone
sNTR+PR104
0 5 10 15 20 25 30
/
Days since spore injection
PR-104 given at 250 mg/kg days 2,9,16 following spores
Martin Brown, Stanford University
PR 104A is activated by a novel aerobicPR-104A is activated by a novel aerobic (2-electron) nitroreductase
Adam Patterson, PhD Chris Guise, PhD, ,
Large variations in aerobic metabolism of PR-104A t H&M b t diff t h t ll lito H&M between different human tumour cell lines
per 1
06 cel
ls
6 0 0
8 0 0
P R -1 0 4 MP R -1 0 4 H
per 1
06 cel
ls
6 0 0
8 0 0
P R -1 0 4 MP R -1 0 4 H
bolit
es fo
rmed
4 0 0
bolit
es fo
rmed
4 0 0
R-1
04A
met
ab
2 0 0
R-1
04A
met
ab
2 0 0
KOV
-3
A549
T-8
sa
Hep
G2
H46
0
SiH
a
HT2
9
anc-
01
22R
V1
FaD
u
DU
145
H52
2
H69
H12
99
A431
DA2
31
PC3
Hep
3B
aPac
a
CT1
16
H82
C33
A
A278
0
pmol
P
0
KOV
-3
A549
T-8
sa
Hep
G2
H46
0
SiH
a
HT2
9
anc-
01
22R
V1
FaD
u
DU
145
H52
2
H69
H12
99
A431
DA2
31
PC3
Hep
3B
aPac
a
CT1
16
H82
C33
A
A278
0
pmol
P
0
SK HC
T H Pa
2 D H
MD H
Mia
HC A
SK HC
T H Pa
2 D H
MD H
Mia
HC A
Affymetrix HG-U133 Plus2.0 array shows an aldo-keto reductase (AKR) cluster correlates with aerobic metabolism of PR-104A
160
Overexpression of AKR1C3 in HCT116
s
80
100
120
140 PR-104MPR-104H
H&
M/1
06 c
ells
r 0
20
40
60
uctio
nolm
ol P
R-1
04H
KR
1 cl
uste
r
WT
AK
R1C
1
AK
R1C
2
AK
R1C
3
AK
R1B
1
AKR
1B10
NQ
O1
V5 TAG
No
V5
indu
cont
ropm
AK
proteins
AKR1C3
AKR1B10
NQO1
Actin
AKR1C3 expression correlates with PR 104A bi t b li i itPR-104A aerobic metabolism in vitro
med
per
106 c
ells
6 0 0
8 0 0
P R -1 0 4 MP R -1 0 4 H
med
per
106 c
ells
6 0 0
8 0 0
P R -1 0 4 MP R -1 0 4 H
A m
etab
olite
s fo
rm
4 0 0
A m
etab
olite
s fo
rm
4 0 0
3 9 a 2 0 a 9 1 1 u 5 2 9 9 1 1 3 B a 6 2 A 0
pmol
PR
-104
A
0
2 0 0
3 9 a 2 0 a 9 1 1 u 5 2 9 9 1 1 3 B a 6 2 A 0
pmol
PR
-104
A
0
2 0 0
SKO
V-3
A549
HC
T-8
sa
Hep
G2
H46
0
SiH
a
HT2
9
Panc
-0
22R
V
FaD
u
DU
145
H52
2
H69
H12
99
A43
MD
A23
PC3
Hep
3B
Mia
Paca
HC
T116
H82
C33
A
A278
0
AKR1C3
NQO1
SKO
V-3
A549
HC
T-8
sa
Hep
G2
H46
0
SiH
a
HT2
9
Panc
-0
22R
V
FaD
u
DU
145
H52
2
H69
H12
99
A43
MD
A23
PC3
Hep
3B
Mia
Paca
HC
T116
H82
C33
A
A278
0
AKR1C3
NQO1
AKR1C3
NQO1Q
AKR1B10
β-actin
Q
AKR1B10
β-actin
Q
AKR1B10
β-actin
AKR1C3 is not a known it d tnitroreductase
St id h d t dSteroid hormone reductase and prostaglandin synthase:
• 3α-hydroxysteroid dehydrogenase (Type 2)• 3α-hydroxysteroid dehydrogenase (Type 2)
• 17β-hydroxysteroid dehydrogenase (Type 5)Androstendione → testosteroneEstrone → estradiolEstrone → estradiol
• Prostaglandin F synthaseDiverts PGD2 from J series prostanoids to PGF2
Pure recombinant AKR1C3 catalyses PR-104A → PR-104Hcatalyses PR 104A → PR 104H
Km ~ 30 µM
AKR1C3 uniquely reduces PR-104,t th bi d ti dnot other bioreductive prodrugs
18R
1C3)
12
14
16
18
HCT116 AKR1C3 #1HCT116 AKR1C3 #3
tio (W
T/A
KR
8
10
12
Nit d de
IC50
rat
2
4
6 Nitro cmpds Quinones
N-o
xid
Misonidazole
Metronidazole
RSU-1069
CB1954
Nitracrin
e
PR-104AAQ4N
Mitomycin C
Porfiromyci
nEO9
0
Overexpression of AKR1C3 confers single agent sensitivity to PR-104g y
HCT116 WT HCT116/AKR1C3
HCT116/AKR1C3 #1800
HCT116 WT800
600
800
± SE
M; m
m3 )
± SE
M; m
m3 )
600
800
200
400
our v
olum
e (M
ean
±
Control
mou
r vol
ume
(Mea
n
200
400
Control
0 10 20 30 400
Tum
o
CPA PR-104
Time from start of treatment (days)Time from start of treatment (days)
0 10 20 30 40
Tum
0
CPA PR-104
Endogenous expression of AKR1C3 in xenografts correlates with sensitivity to PR-104
monotherapy
4.0
PR-104 350 mg/kg IPClonogenic assay 18 hr later
ll ki
ll
2.5
3.0
3.5 H460
SiHa
6
AKR1C3 expression in xenografts
6
AKR1C3 expression in xenograftsAKR1C3 expression in xenografts
Log 1
0 ce
l
1.0
1.5
2.0
A2780
A549
22Rv1
A54
9
C33
A
22R
VI
HT2
9
SiH
a
A27
80
H12
99
H46
0
HC
T116
A54
9
C33
A
22R
VI
HT2
9
SiH
a
A27
80
H12
99
H46
0
HC
T116
AKR1C3β-ACTIN
0.0
0.5HCT116
C33AH1299
HT29
LowAKR1C3
HighAKR1C3
β-ACTIN
IHC
AKR1C3 AKR1C3
Tissue microarray, 2700 patients, 27 ti t27 tissue types
rmal
rmal
rmal
Hepatoma NSCLC Breast
Nor
Nor
Nor
Hepatoma NSCLC Breast
Phase II with sorafenib Phase II with docetaxel
PR-104A is not just a hypoxia-activated prodrug
AKR1C3
CYPORCYPOR, iNOS, others
Determinants of sensitivity to bioreductive dprodrugs
Hypoxia markers Reductase Hypoxia Reductases
Vascular disrupting agents
profiling
GDEPT
IntrinsicSensitivity
R i fili
CONFIDENTIAL 29
Repair profiling,Repair inhibitors
Determinants of sensitivity to bioreductive dprodrugs
Hypoxia markers
HypoxiaVascular disrupting agents
IntrinsicSensitivity
R i fili
CONFIDENTIAL 30
Repair profiling,Repair inhibitors
Radiation-activated prodrugsRadiation activated prodrugs
H2O e(aq)- + H + OHRAD
Prodrug Prodrug
CytotoxinO2O2
Radiation-activated prodrugsRadiation activated prodrugs
Unique advantages:Unique advantages:
• Dual specificity (hypoxia+radiation targeting)Dual specificity (hypoxia radiation targeting)
• Independent of enzyme expression– Universal for all tumours– Exploits hypoxia in necrotic regions
• RT clearest evidence for resistance due to hypoxia (especially important in SBRT?)
The challengeThe challenge
• Low yield of radiation induced reducing radicals during radiotherapy
T i l f ti t d RT (2 G /f ) 0 6 l/k– Typical fractionated RT (2 Gy/fr): 0.6 µmol/kg– (SBRT provides greater opportunity)
• Requires prodrugs capable of releasing a potentcytotoxic effector with high efficiency on one-cytotoxic effector with high efficiency on oneelectron reduction
Transition metal complexesRadiolytic activation in anoxic human plasma
ClOMe
OMe
SN 27892 oxic
N
NO
NH OMe
SN 27892 iO
N CoNN
H
H
HSN 27892
Co(III) Cyclen2AzaCBI
SN 27892 anoxic
azaCBINH
H Yield of effector= 0.075 µmol/J(0 075 M/G )
Ahn et al., Biochem Pharmacol 71: 1683-1694 (2006)
(0.075 µM/Gy)
But yields are 5x lower again in tissue
20Anoxic mediumG = 48.8 nmol/J
gassing
MA
C (µ
M)
10
15
Anoxic MCLG = 10.8 nmol/J8 cm
ports
AM
5
Oxic MCLMCLbeam window MCLBeam window
Radiation dose (Gy)0 200 400 600
0
5 cm
8 mm
Determinants of sensitivity to bioreductive dprodrugs
Hypoxia markers IMRTHypoxia Radiation
Vascular disrupting agents
IGRTSBRT
IntrinsicSensitivity
R i fili
CONFIDENTIAL 36
Repair profiling,Repair inhibitors
Targeting hypoxia with prodrugs:A th t?Are we there yet?
• Powerful theoretical justification for hypoxia as a target across multiple tumour types
• Only now are the tools becoming available to exploit this target properly. g y– Extravascular transport (including active metabolites)– Hypoxia imaging– Reductase (and other molecular) profiling– Reductase (and other molecular) profiling– Complementary strategies to manipulate these determinants of
sensitivityRadiolytic activation of prodrugs?– Radiolytic activation of prodrugs?
AcknowledgementsAcknowledgements
• My lab • Bill Denny (U. Auckland)– Adam Patterson– Chris Guise
– Moana Tercel– Mike Hay
– Maria Abbattista– Frederik Pruijn
K i Hi k
– Jeff Smaill– Graham Atwell
– Kevin Hicks– Rachelle Douglas– Yongchuan GuYongchuan Gu– Kashyap Patel
M ti B Ti S b• Martin Brown(Stanford)
• Tim Secomb(U. Arizona)
Harper staffer quits over plagiarized 2003 speech on Iraq
CBC News, 30 Sept 2008
A staff member [Owen Lippert] resigned and apologized Tuesday for writing a [ pp ] g p g y gspeech read by Stephen Harper in 2003 as leader of the Opposition that plagiarized from an address days earlier by then Australian prime minister John Howard.