Prodrugs targeting hypoxic cells William R. Wilson Auckland Cancer Society Research Centre Th The University of Auckland U i it f A kl d wr.wilson@auckland.ac.nz
My OCI mentors: 1978-79 y Dick Hill Gordon Whitmore Mike Rauth (Ian Tannock) Hypoxia as a potential therapeutic target yp p p g
Nitro compounds Nitro compounds NH 2 NO 2 NO 2 NO NHOH 1e reductase 1e reductase 1 1e 2e 2e 2e 2 R R R R R O 2 O 2 Nitro Nitro Nitroso Nitroso Hydroxylamine Hydroxylamine Amine Amine radical Potential cytotoxins 2e reductase Protein thiols 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 other 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 Small molecule direct oxygen sensors ll l l di t DNA damage or DRUG PRODRUG Molecular target Molecular target O 2 • 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 old cytotoxics)
Bioreductive (hypoxia-activated) prodrugs prodrugs No registered agents, but several in development: Tirapazamine Tirapazamine Arom N-oxide Arom. N oxide Phase III Phase III SRI/Stanford SRI/Stanford AQ4N Aliph. N-oxide Phase II Novacea PR-104 Nitro cmpd Phase II Proacta TH-302 Nitro cmpd p Phase I Threshold 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 Previously untreated advanced HNSCC P i l t t d d d HNSCC Rischin et al Rischin et al., J Clin Oncol 26: 2008 (May 20 Suppl) abstr LBA6008 J Clin Oncol 26: 2008 (May 20 Suppl) abstr LBA6008 + Cisplatin (100 mg/m 2 ) d1 wk 1,4,7 RT (70 Gy + Cisplatin (75 mg/m 2 ) + TPZ (290 mg/m 2 ) d1 wk 1,4,7 7 wks) and TPZ alone (160 mg/m 2 ) 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 Primary site failure in 92 randomized it f il i 92 d i d advanced H&N patients at Peter MacCallum Cancer Centre Rischin et al., Int J Radiat Oncol Biol Phys 2007 Treatm ent PET PET hypoxia P-value status RT + RT + cis cisplatin + TPZ Non- 2/ 27 3/ 21 NS Hypoxic Hypoxic 8/ 18 0/ 26 0.0002 P-value P-value 0 008 0.008 NS NS CONFIDENTIAL 10
Extravascular transport limits therapeutic activity of tirapazamine ti it f ti i Gas Gas in in out Gas Gas i in Gas Gas ou Donor Receiver Multicellular layer (MCL) (MCL) 230 x 500 x 500 µm region of R3230Ac tumour Hicks et al., J. Natl Cancer Instit. 98: 1118-1128 (2006) 11
PK/PD guided lead optimisation of tirapazamine O - N + IMPROVED SOLUBILITY N O IMPROVED HYPOXIC SELECTIVITY IMPROVED HYPOXIC SELECTIVITY N N N + O - IMPROVED HYPOXIC CELL KILL IN IMPROVED PENETRATION OF HT29 MCLs TUMOUR XENOGRAFTS 15 on) TPZ (133 μ mol/kg) μ 1.8 ( g) nal to radiatio SN 30000 (600 μ mol/kg) 1.6 SN 30000 ent Flux 1.4 10 1.2 Perce ll kill (addition 1.0 1 0 0.8 5 TPZ 0.6 0.4 Log cel 0 0.2 5 10 15 0.0 Percent Flux of urea internal standard (Corrected time axis) HT29 SiHa H460
PR-104 PR 104 Low K-value Bystander effect 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) Control 1200 Docetaxel PR-104 Docetaxel + PR-104 em 1000 me (mg) +/- se 800 Tumor volum 600 600 400 200 0 0 10 20 30 40 50 60 Time (days) Patterson et al . Clin Cancer Res , 2007
PR-104 (1100 mg/m 2 ) + docetaxel (60 mg/m 2 ) with G- CSF; q3w CSF; q3w metastatic head and neck squamous cell ca Confirmed partial response 2 nd cycle 2 nd cycle Pretreatment Pretreatment 3rd cycle 3rd cycle 29 July 08 9 Sept 08 7 Oct 08
Single Agent Activity: PR 104 vs Tirapazamine PR-104 vs Tirapazamine SiHa human cervical ca xenografts (q4dx3) 100 PR-104 (1.8 g/kg) (P=0.012) val 80 free Surviv 60 Disease-f 40 % Tirapazamine (0.10 g/kg) 20 (P=0.39) Control (saline) 0 0 0 20 40 60 80 100 Days post treatment
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) g (q ) Control 100 100 PR 104 PR-104 Docetaxel Gemcitabine Cisplatin Cyclophosphamide 80 %) Survival (% 60 Substantial oxic cell killing 40 20 0 0 10 20 30 40 50 60 70 80 90 100 Days post treatment initiation
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 PR104 alone sNTR+PR104 1 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 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 to H&M between different human tumour cell lines t 6 cells 6 cells pmol P pmol P R-104A metab R-104A metab bolites formed bolites formed per 10 per 10 H&M b t 2 0 0 2 0 0 4 0 0 4 0 0 6 0 0 6 0 0 8 0 0 8 0 0 0 0 SK SK KOV-3 KOV-3 A549 A549 HCT HCT T-8 sa T-8 sa H H HepG2 HepG2 H460 H460 SiHa SiHa diff HT29 HT29 Pa Pa anc-01 anc-01 2 22RV1 2 22RV1 FaDu FaDu D D DU145 DU145 t h H522 H522 H69 H69 H1299 H H H1299 A431 A431 MD MD DA231 DA231 t PC3 PC3 Hep3B H Hep3B H Mia Mia aPaca aPaca HC HC CT116 CT116 P R -1 0 4 H P R -1 0 4 H P R -1 0 4 M P R -1 0 4 M H82 H82 C33A C33A ll li A A2780 A2780 A
Affymetrix HG-U133 Plus2.0 array shows an aldo-keto reductase (AKR) cluster correlates with aerobic metabolism of PR-104A Overexpression of AKR1C3 in HCT116 160 s H&M/10 6 cells PR-104M 140 PR-104H 120 100 80 mol PR-104H 60 40 20 uction pm 0 r KR1 cluster ol No V5 indu contro WT AKR1C1 AKR1C2 AKR1C3 AKR1B1 AKR1B10 NQO1 V5 TAG AK proteins AKR1C3 AKR1B10 NQO1 Actin
PR-104A aerobic metabolism in vitro PR 104A AKR1C3 expression correlates with 6 cells 6 cells pmol PR-104A pmol PR-104A A metabolites form A metabolites form med per 10 med per 10 AKR1B10 AKR1B10 AKR1B10 AKR1C3 AKR1C3 AKR1C3 β -actin β -actin β -actin NQO1 NQO1 NQO1 2 0 0 2 0 0 4 0 0 4 0 0 6 0 0 6 0 0 8 0 0 8 0 0 Q Q Q 0 0 SKOV-3 SKOV-3 3 3 A549 A549 9 9 HCT-8 sa HCT-8 sa a a HepG2 HepG2 2 2 H460 H460 0 0 SiHa SiHa a a HT29 HT29 9 9 bi Panc-0 Panc-0 1 1 22RV 22RV 1 1 FaDu FaDu u u DU145 DU145 5 5 H522 H522 2 2 H69 H69 9 9 H1299 H1299 9 9 t b li A43 A43 1 1 MDA23 MDA23 1 1 PC3 PC3 3 3 Hep3B Hep3B B B MiaPaca MiaPaca a a HCT116 HCT116 6 6 P R -1 0 4 H P R -1 0 4 H P R -1 0 4 M P R -1 0 4 M H82 H82 2 2 C33A C33A A A i A2780 A2780 0 0 it
AKR1C3 is not a known nitroreductase it d t St Steroid hormone reductase and id h d t d prostaglandin synthase : • 3 α -hydroxysteroid dehydrogenase (Type 2) • 3 α -hydroxysteroid dehydrogenase (Type 2) • 17 β -hydroxysteroid dehydrogenase (Type 5) Androstendione → testosterone Estrone → estradiol Estrone → estradiol • Prostaglandin F synthase Diverts PGD 2 from J series prostanoids to PGF 2 Pure recombinant AKR1C3 catalyses PR-104A → PR-104H catalyses PR 104A → PR 104H Km ~ 30 µM
AKR1C3 uniquely reduces PR-104, not other bioreductive prodrugs t th bi d ti d 18 18 HCT116 AKR1C3 #1 16 HCT116 AKR1C3 #3 R1C3) 14 tio (WT/AKR 12 12 10 8 de Nitro cmpds Nit d N-oxid IC 50 rat Quinones 6 4 2 0 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 WT HCT116/AKR1C3 #1 800 800 800 800 ± SEM; mm3) ± SEM; mm3) 600 600 mour volume (Mean our volume (Mean ± 400 400 200 200 Control Control Tum Tumo CPA CPA PR-104 PR-104 0 0 0 10 20 30 40 0 10 20 30 40 Time from start of treatment (days) Time from start of treatment (days)
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