Prodrugs targeting hypoxic cells William R. Wilson Auckland Cancer - - PowerPoint PPT Presentation

Prodrugs targeting hypoxic cells

William R. Wilson

Auckland Cancer Society Research Centre Th U i it f A kl d The University of Auckland

wr.wilson@auckland.ac.nz

My OCI mentors: 1978-79 Dick Hill y Gordon Whitmore Mike Rauth (Ian Tannock)

Hypoxia as a potential therapeutic target yp p p g

Nitro compounds Nitro compounds

NO2 NO2 NH2

1e reductase

NHOH NO 1 2e 2

1e reductase

O2 O2

R R R R R

Nitroso Hydroxylamine Amine Nitro 1e 2e 2e Nitroso Hydroxylamine Amine Nitro radical

2e reductase Protein thiols Potential cytotoxins Protein thiols Hypoxia probes Broadly similar redox chemistry for quinones, N-oxides and some transition metal complexes

The hippie phase The 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

• ther novel therapeutic agents licenced to Proacta.

The agents in question originate from my lab The agents in question originate from my lab Proacta funds research contracts in my lab

Bioreductive prodrugs Bioreductive prodrugs

S ll l l di t Small molecule direct oxygen sensors

DNA damage

PRODRUG DRUG

Molecular target

• r

Molecular target

O2

• Broad spectrum (multiple cell lineages)
• Stable enough to diffuse out of hypoxic

g yp regions (bystander effect)

The era of targeted agents The 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 gy selectivity of molecularly targeted agents (and dirty

• ld cytotoxics)

Bioreductive (hypoxia-activated) prodrugs prodrugs

No registered agents, but several in development: Tirapazamine Arom N-oxide Phase III SRI/Stanford Tirapazamine

• Arom. N oxide

Phase III SRI/Stanford AQ4N

• Aliph. N-oxide

Phase II Novacea PR-104 Nitro cmpd Phase II Proacta TH-302 Nitro cmpd Phase I Threshold p NLCQ-1 Nitro cmpd Preclinical Evanston Hosp SN 30000

• Arom. N-oxide

Preclinical Proacta SN 29730 Nitro cmpd Preclinical Proacta VPN 40541 Nitro cmpd Preclinical Vion

ASCO 2008: HeadSTART phase III trial P i l t t d d d HNSCC Previously untreated advanced HNSCC

Rischin et al J Clin Oncol 26: 2008 (May 20 Suppl) abstr LBA6008 Rischin et al., J Clin Oncol 26: 2008 (May 20 Suppl) abstr LBA6008 RT (70 Gy 7 wks) + Cisplatin (100 mg/m2 ) d1 wk 1,4,7 + Cisplatin (75 mg/m2 ) + TPZ (290 mg/m2) d1 wk 1,4,7 and 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 0.53-1.04 HR 0.74, 95% CI 0.53 1.04

• Patients not selected for the presence of hypoxia

Clinical proof of principle: Tirapazamine

P 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 Treatm ent PET hypoxia status P-value

RT + cisplatin RT + 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 i activity of tirapazamine

Gas Gas in in Gas Gas ou

• ut Gas

Gas i in

Multicellular layer (MCL)

Receiver Donor

(MCL)

230 x 500 x 500 µm region of R3230Ac tumour 11

Hicks et al., J. Natl Cancer Instit. 98: 1118-1128 (2006)

PK/PD guided lead optimisation of tirapazamine

N N+ O- N O

IMPROVED SOLUBILITY IMPROVED HYPOXIC SELECTIVITY

N+ O- N

IMPROVED HYPOXIC SELECTIVITY

15

IMPROVED PENETRATION OF HT29 MCLs

• n)

1.8

TPZ (133 μmol/kg)

IMPROVED HYPOXIC CELL KILL IN TUMOUR XENOGRAFTS SN 30000 ent Flux

10

nal to radiatio

1 0 1.2 1.4 1.6

( μ g) SN 30000 (600 μmol/kg)

Perce

5

TPZ

ll kill (addition

0.4 0.6 0.8 1.0

Percent Flux of urea internal standard (Corrected time axis) 5 10 15

HT29 SiHa H460 Log cel

0.0 0.2

PR-104 PR 104

Bystander effect Low K-value Improved extravascular transport

Patterson et al., Clin Cancer Res 2007 Hicks et al., IJROBP 2007

PR-104 combination chemotherapy: docetaxel

Androgen resistant prostate carcinoma xenograft (22RV1)

em

1000 1200 Control Docetaxel PR-104 Docetaxel + PR-104

me (mg) +/- se

600 800

Tumor volum

400 600 10 20 30 40 50 60 200

Time (days)

Patterson et al. Clin Cancer Res, 2007

PR-104 (1100 mg/m2) + docetaxel (60 mg/m2 ) with G- CSF; q3w CSF; q3w

metastatic head and neck squamous cell ca Confirmed partial response Pretreatment

2nd cycle 3rd cycle

Pretreatment

29 July 08

2nd cycle

9 Sept 08

3rd cycle

7 Oct 08

Single Agent Activity: PR 104 vs Tirapazamine PR-104 vs Tirapazamine

SiHa human cervical ca xenografts (q4dx3)

val

80 100

PR-104 (1.8 g/kg)

(P=0.012)

free Surviv

60

Disease-f

40

%

20

Control (saline) Tirapazamine (0.10 g/kg)

(P=0.39)

Days post treatment

20 40 60 80 100

Single Agent Activity: PR 104 vs conventional chemotherapy PR-104 vs conventional chemotherapy

H460 non small cell lung cancer xenografts, treated at the maximum tolerated dose of each agent (q4dx3)

100

Control PR 104

g (q )

%)

80 100

PR-104 Docetaxel Gemcitabine Cisplatin Cyclophosphamide

Survival (%

40 60

Substantial oxic cell killing

20

Days post treatment initiation 10 20 30 40 50 60 70 80 90 100

Delivery of a synthetic version of the E. coli nfsB nitroreductase (sNTR) using a Clostridial vector nitroreductase (sNTR) using a Clostridial vector potentiates the activity of PR104 against SiHa tumors

Control sNTR spores alone PR104 alone

1

PR104 alone sNTR+PR104

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 aerobic PR-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 li to H&M between different human tumour cell lines

per 10

6 cells

6 0 0 8 0 0 P R -1 0 4 M P R -1 0 4 H

per 10

6 cells

6 0 0 8 0 0 P R -1 0 4 M P R -1 0 4 H

bolites formed

4 0 0

bolites formed

4 0 0

R-104A metab

2 0 0

R-104A metab

2 0 0 KOV-3 A549 T-8 sa HepG2 H460 SiHa HT29 anc-01 22RV1 FaDu DU145 H522 H69 H1299 A431 DA231 PC3 Hep3B aPaca CT116 H82 C33A A2780

pmol P

KOV-3 A549 T-8 sa HepG2 H460 SiHa HT29 anc-01 22RV1 FaDu DU145 H522 H69 H1299 A431 DA231 PC3 Hep3B aPaca CT116 H82 C33A A2780

pmol P

SK HCT H Pa 2 D H MD H Mia HC A SK HCT 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-104M PR-104H

H&M/106 cells

r

20 40 60 uction

• l

mol PR-104H

KR1 cluster

WT AKR1C1 AKR1C2 AKR1C3 AKR1B1 AKR1B10 NQO1 V5 TAG No V5 indu contro

pm

AK

proteins AKR1C3 AKR1B10 NQO1 Actin

AKR1C3 expression correlates with PR 104A bi t b li i it PR-104A aerobic metabolism in vitro

med per 10

6 cells

6 0 0 8 0 0 P R -1 0 4 M P R -1 0 4 H

med per 10

6 cells

6 0 0 8 0 0 P R -1 0 4 M P R -1 0 4 H

A metabolites form

4 0 0

A metabolites form

4 0 0 3 9 a 2 a 9 1 1 u 5 2 9 9 1 1 3 B a 6 2 A

pmol PR-104A

2 0 0 3 9 a 2 a 9 1 1 u 5 2 9 9 1 1 3 B a 6 2 A

pmol PR-104A

2 0 0 SKOV-3 A549 HCT-8 sa HepG2 H460 SiHa HT29 Panc-0 22RV FaDu DU145 H522 H69 H1299 A43 MDA23 PC3 Hep3B MiaPaca HCT116 H82 C33A A2780

AKR1C3 NQO1

SKOV-3 A549 HCT-8 sa HepG2 H460 SiHa HT29 Panc-0 22RV FaDu DU145 H522 H69 H1299 A43 MDA23 PC3 Hep3B MiaPaca HCT116 H82 C33A A2780

AKR1C3 NQO1 AKR1C3 NQO1 Q AKR1B10 β-actin Q AKR1B10 β-actin Q AKR1B10 β-actin

AKR1C3 is not a known it d t nitroreductase

St id h d t d Steroid hormone reductase and prostaglandin synthase:

• 3α-hydroxysteroid dehydrogenase (Type 2)
• 3α-hydroxysteroid dehydrogenase (Type 2)
• 17β-hydroxysteroid dehydrogenase (Type 5)

Androstendione → testosterone Estrone → estradiol Estrone → estradiol

• Prostaglandin F synthase

Diverts PGD2 from J series prostanoids to PGF2 Pure recombinant AKR1C3 catalyses PR-104A → PR-104H catalyses PR 104A → PR 104H Km ~ 30 µM

AKR1C3 uniquely reduces PR-104, t th bi d ti d not other bioreductive prodrugs

18

R1C3)

12 14 16 18

HCT116 AKR1C3 #1 HCT116 AKR1C3 #3

tio (WT/AKR

8 10 12

Nit d de

IC50 rat

2 4 6

Nitro cmpds Quinones N-oxid

Misonidazole Metronidazole RSU-1069 CB1954 Nitracrine PR-104A AQ4N Mitomycin C Porfiromycin EO9

Overexpression of AKR1C3 confers single agent sensitivity to PR-104 g y

HCT116 WT HCT116/AKR1C3

HCT116/AKR1C3 #1

800

HCT116 WT

800 600 800

± SEM; mm3) ± SEM; mm3)

600 800 200 400

• ur volume (Mean ±

Control

mour volume (Mean

200 400 Control 10 20 30 40

Tumo

CPA PR-104

Time from start of treatment (days) Time from start of treatment (days)

10 20 30 40

Tum

CPA PR-104

Endogenous expression of AKR1C3 in xenografts correlates with sensitivity to PR-104 monotherapy

4.0

PR-104 350 mg/kg IP

Clonogenic assay 18 hr later ll kill

2.5 3.0 3.5 H460 SiHa 6

AKR1C3 expression in xenografts

6

AKR1C3 expression in xenografts

AKR1C3 expression in xenografts

Log10 cel

1.0 1.5 2.0 A2780 A549 22Rv1 A549 C33A 22RVI HT29 SiHa A2780 H1299 H460 HCT116 A549 C33A 22RVI HT29 SiHa A2780 H1299 H460 HCT116

AKR1C3 β-ACTIN

0.0 0.5 HCT116 C33A H1299 HT29

Low AKR1C3 High AKR1C3 β-ACTIN IHC AKR1C3 AKR1C3

Tissue microarray, 2700 patients, 27 ti t 27 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 CYPOR CYPOR, iNOS, others

Determinants of sensitivity to bioreductive d prodrugs

Hypoxia markers Reductase

Hypoxia Reductases

Vascular disrupting agents profiling GDEPT

Intrinsic Sensitivity

R i fili

CONFIDENTIAL 29

Repair profiling, Repair inhibitors

Determinants of sensitivity to bioreductive d prodrugs

Hypoxia markers

Hypoxia

Vascular disrupting agents

Intrinsic Sensitivity

R i fili

CONFIDENTIAL 30

Repair profiling, Repair inhibitors

Radiation-activated prodrugs Radiation activated prodrugs

H2O e(aq)- + H + OH

RAD

Prodrug Prodrug Cytotoxin O2 O2

Radiation-activated prodrugs Radiation 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 challenge The 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 potent

cytotoxic effector with high efficiency on one- cytotoxic effector with high efficiency on one electron reduction

Transition metal complexes

Radiolytic activation in anoxic human plasma

Cl OMe OMe

SN 27892 oxic

N N O N H OMe

SN 27892 i

O N CoN N H H H SN 27892

Co(III) Cyclen2AzaCBI SN 27892 anoxic azaCBI

N H 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

20 Anoxic medium G = 48.8 nmol/J

gassing

MAC (µM) 10 15 Anoxic MCL G = 10.8 nmol/J

8 cm

ports

AM 5 Oxic MCL

MCL beam window

MCL Beam window

Radiation dose (Gy) 200 400 600

5 cm 8 mm

Determinants of sensitivity to bioreductive d prodrugs

Hypoxia markers IMRT

Hypoxia Radiation

Vascular disrupting agents IGRT SBRT

Intrinsic Sensitivity

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 sensitivity Radiolytic activation of prodrugs? – Radiolytic activation of prodrugs?

Acknowledgements Acknowledgements

• 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 Gu Yongchuan Gu – Kashyap Patel M ti B Ti S b

• Martin Brown

(Stanford)

• Tim Secomb

(U. Arizona)

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