Prodrugs targeting hypoxic cells

William R. Wilson

Auckland Cancer Society Research CentreTh U i it f A kl dThe University of Auckland

wr.wilson@auckland.ac.nz

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

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