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Pharminox SAB - 23 March 2009

Pharminox SAB - 23 March 2009. RHM Programme. RHM Programme – Agenda. Introduction Malcolm Stevens Biology/MOA Charlie Matthews Xenografts Data Charmaine Quarterman SAB Review Additional biology data Implications of conflicting xenograft data Development strategy

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Pharminox SAB - 23 March 2009

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  1. Pharminox SAB - 23 March 2009 RHM Programme

  2. RHM Programme – Agenda • Introduction Malcolm Stevens • Biology/MOA Charlie Matthews • Xenografts Data Charmaine Quarterman • SAB Review • Additional biology data • Implications of conflicting xenograft data • Development strategy • Approaches to back-ups

  3. Redox Homeostasis Modulator (RHM) programme Lead candidate PMX 2058 • Novel programme targeting redox homeostasis signalling pathways • Two chemical series: heterocyclic cyclohexadienones; (arylsulfonyl)indole-substituted quinols • Pharminox compounds inhibit thioredoxin (Trx), thioredoxin reductase (TrxR) and (downstream) HIF-1 and NFkB

  4. Lead candidate PMX 2058 - Profile PMX 2058 is a highly water-soluble pro-drug of the active moiety PMX 290 PMX 290 synthetic route already developed – short and straightforward PMX 290 has shown potent in vitro and in vivo activity in colorectal, renal and melanoma cell lines PMX 290 not developed due to poor solubility / oral bioavailability Rapid conversion of PMX 2058 to PMX 290 confirmed in vitro and in vivo PMX 2058 PMX 290

  5. RHM Programme Mechanism of action

  6. Overview of Key Data PMX 290 in vitro effects • Potent in vitro growth inhibition of cancer cell lines • G2/M cell cycle arrest and apoptosis induction Mechanism of action • ROS induction • Effects of Glutathione (GSH) depletion on PMX 290 activity • Evidence for TrxR inhibition through targeting of selenium residue • Conflicting evidence for TrxR inhibition • Apoptosis induction independent of Trx/ASK pathway • Other targets, e.g. HIF and ER stress Strictly confidential

  7. PMX 290 in vitro cell growth inhibition NCI 60 Cell Line Panel • Mean GI50 = 0.05 mM, Mean LC50 = 4.7 mM Activity in Resistant Cell Lines • PMX 290 active in colon cancer cell lines made resistant to methotrexate, cisplatin and doxorubicin Strictly confidential

  8.       p < 0.05  p < 0.001 Student’s T Test PMX 290 induces a G2/M cell cycle block Cell cycle analysis: HCT116 cells, 24h exposure Accumulation of cells in the G2/M phase of the cell cycle Population with sub G1 DNA content apparent DMSO 1 mM PMX 290 1 mM PMX 2058 Strictly confidential

  9. 13.7% 12.4% 38.2% 19.5% DMSO 1 mM PMX 290 1 mM PMX 2058 3.2% 1.8% PMX 290 induces apoptosis AnnexinV / propidium iodide analysis: HCT116: 24h, PMX 290 Caspase 3 and PARP cleavage ← PARP113 kDa ← cleaved PARP 89 kDa ← cleaved PARP24 kDa ← cleaved caspase 317/19 kDa ← D 0.1 0.5 1 5 0.1 0.5 1 5 0.5 mM PMX 290 PMX 2058 CPT CPT = camptothecin Strictly confidential

  10. 7h Induction of Reactive Oxygen Species (ROS) by PMX 290     Induction of ROS detected with H2DCF-DA by flow cytometry 16h  p < 0.05  p < 0.001 Strictly confidential

  11. Glutathione (GSH) depletion sensitises cells to PMX 290 HCT116 GSH depleted using buthionine L-sulfoximine Strictly confidential

  12. PMX 290 prevents binding of biotin-conjugated iodoacetamide (BIAM) that targets selenocysteine residue of TrxR • PMX 290 does not inhibit E. coli TrxR/Trx system or structurally similar yeast glutathione reductase • IC50s > 75 mM DMSO PMX 290 Time (min) 60 3 15 30 60 HRP- Streptavidin -ratTrxR PMX 290 is reported to be an inhibitor of mammalian thioredoxin reductase (TrxR) PMX 290 has been shown to be an irreversible inhibitor of NADPH-reduced mammalian thioredoxin reductase (DTNB assay). IC50 = 2.7 mM PMX 290 inhibits the NADPH/TrxR/Trx1 catalysed reduction of insulin IC50 = 7.9 mM Chew et al: FASEB Journal (2008): 22(6), 2072-83. Strictly confidential

  13. Proposed model of TrxR inhibition Modified from Chew et al; FASEB Journal (2008): 22(6), 2072-83. Strictly confidential

  14. TrxR Actin 0 5 3 1 0.5 PMX 290 (µM) DMSO 0.5 1 3 5 PMX 290 (µM) PMX 290 inhibits TrxR in cells Thioredoxin reductase but not thioredoxin activity inhibited in cell lysates of HCT116 treated with PMX 290 Chew et al: FASEB Journal (2008): 22(6), 2072-83. Strictly confidential

  15. Difference in Trx/TrxR inhibition data NADPH/TrxR/Trx/insulin reduction assay Different concentrations/conditions to Chew paper but fundamentally similar assay Strictly confidential

  16. Whole Cell Lysates IP: Trx1 S—S Trx1 SH—SH Trx1 P ASK1  ASK1 ASK1 ASK1 ASK1 P SH—SH Trx1  Trx1 Trx1 S—S Inactive Active DMSO DMSO 5 M DMSO DMSO 5 M -Ab +Ab PMX -Ab +Ab PMX 290 290 H2O2, TNF-, cisplatin  p-p38 MEKK4/7 MEKK3/6  total p38 JNK p38  p-JNK   total-JNK  Apoptosis 0 1 2 3 4 5 6 7 24 h 0.5 mM PMX 290 Apoptosis Signal-Regulating Kinase 1 (ASK1) inhibition by Trx Strictly confidential

  17. ← dnASK Dominant negative ASK1 (dnASK) does not prevent apoptosis induced by PMX 290 ← actin K709R mutation leads to catalytically inactive, dominant negative ASK1 mock transfection dnASK1 Strictly confidential

  18. HIF-1 HRE binding prevented in MDA468 cells treated with PMX 290 • Inhibition of HIF-1 transactivation under normoxia and hypoxia * p = 0.01-0.05 ** p = 0.001-0.01 PMX 290 inhibits HIF dependent processes Reduction in hypoxia induced mRNAs of HIF target genes CA-IX, BNIP3, VEGF VEGF secretion inhibited at sub-toxic (sub-microM) concentrations of PMX 290. Reduction in CA-IX and BNIP3 protein expression under hypoxia. Jones et al; Clin. Cancer Research (2006): 12 (18), 5384 – 5394. Strictly confidential

  19. Increase in HIF-1 and HIF-2 protein levels in MDA468 breast and RCC4 renal cells grown under both normoxic and hypoxic conditions following PMX 290 treatment. • Cytoplasmic levels of HIF-1 increased in MDA468 cells • VHL independent • HIF hydroxylated for degredation • Stabilised by increase in HSP70 expression? PMX 290 MDA468 Jones et al; Clin. Cancer Research (2006) • HIF-1 protein effects not seen in HCT116 cells • Lower doses used HCT116 Ravizza R et al., Eur J Cancer (2009) PMX 290 increase HIF protein levels in MDA468 cells Strictly confidential

  20. PMX 290 inhibits HIF-1 transcriptional activity and potentiates 5FU induced apoptosis - 5FU + 5FU • HIF driven EGFP expression is inhibited by 0.25 mM PMX 290. • Potentiation of 5FU induced apoptosis under hypoxia Normoxic control Hypoxic control Hypoxic + PMX 290 Ravizza R et al., Eur J Cancer (2009) Strictly confidential

  21. PMX 290 reduces HIF-1 activity and enhances the response to 5FU in HCT116 spheroids spheroids monolayer Control 2.5 mM PMX 290 + vehicle + 2.5 mM PMX 290 • 3D HCT116 spheroid model • Sub-toxic concentrations of PMX 290 enhanced 5FU induced apoptosis Ravizza R et al., Eur J Cancer (2009) Strictly confidential

  22. 0.5 mM PMX 290, 24h, HCT116 cells • Some markers of oxidative stress markers (e.g. HO-1) • CHOP (DDIT3), Trib3, GRP78 (HSPA5/BiP), ATF3 all upregulated and involved in ER stress Induction of endoplasmic reticulum stress related mRNAs Strictly confidential

  23. Comparison of PMX 290 and oxidative stress inducer, Elesclomol Both induce ROS and apoptosis Elesclomol more potent at inducing ROS Elesclomol induced apoptosis is prevented by N-acetylcysteine PMX 290 activity unaffected by catalase overexpression Gene induction similarities/differences Strictly confidential

  24. PMX 290 in vitro effects • Potent in vitro growth inhibition of cancer cell lines • G2/M cell cycle arrest and apoptosis induction Mechanism of action • PMX 290 induces ROS in sensitive cells and GSH depletion sensitises cells to PMX 290 • Evidence for TrxR inhibition through targeting of selenium residue • Conflicting evidence for TrxR inhibition • Apoptosis induction independent of Trx/ASK pathway Summary Strictly confidential

  25. PMX 290 HIF effects • Conflicting cell type-specific alterations of HIF protein levels but… • Consistent inhibition of HIF transcriptional activity at sub toxic doses in monolayer and 3D cell models • Inhibition of HIF transactivation activity • Inhibition of VEGF secretion by hypoxic cells • Potentiation of 5-FU induced apoptosis in hypoxic monolayers and 3D spheroids PMX 290 compared to molecules with similar MOA • MOA distinct from that of oxidative stress inducer Elesclomol Summary Strictly confidential

  26. RHM programme Xenografts & development strategy

  27. PMX 2058: Rationale for Recent Preclinical Evaluation Review of existing xenograft data with PMX 290 and PMX 2058 Borderline to modest efficacy in the colon (HCT116), breast (MDA-MB-435) and renal (CAKI-1) xenografts studied. Other models more sensitive to PMX 2058? Mechanism of action Some uncertainty associated with primary target – data not entirely supportive of PMX 290 being a Trx/TrxR inhibitor. Consistent evidence supporting inhibition of HIF-1 signalling. Data available for other agents PX-12 (Trx inhibitor) – Phase I data provides evidence of stable disease in patients with pancreatic carcinoma. PX-478 (selective HIF-1 inhibitor) – preclinical efficacy in broad spectrum of tumour types including small cell lung, prostate, pancreatic and ovarian carcinomas. Strictly confidential

  28. Xenograft studies in HCT116, MDA-MB-435 and CAKI-1 show modest activity. NCI mean LC50 data • ACHN more sensitive than CAKI-1 • Other agents that target Trx-1 (PX-12) and HIF-1 signalling (PX-478) show efficacy in pancreatic models. • Further in vitro studies to select more sensitive cell lines: • Panc-1 IC50: 104nM • BxPC3 IC50: 272nM • MIA PaCa-2 GI50: 313nM • ACHN IC50: 107nM Colon Renal Breast HCT 116 CAKI-1 MDA-MB-435 LC50 values relative to overall mean (vertical line) Wells et al J Med Chem 46 532-541, 2003 PMX 290: In Vitro Antitumour Activity Strictly confidential

  29. PMX 2058: Target Clinical Indications? Colorectal carcinoma Existing xenograft data demonstrates modest activity. Pancreatic carcinoma Clear unmet clinical need. Existing therapies offer small increase in survival - Low regulatory hurdle but particularly challenging area Approval of erlotinib in combination with gemcitabine based on increase in median survival from 5.9 to 6.4 months when compared to gemcitabine alone. Renal carcinoma A number of drugs approved Sunitinib (Sutent) – median TTP 11 months compared to 5 months with IFN. Sorafenib (Nexavar) – 39% OS improvement compared to placebo at initial review. Temsirolimus (Torisel) – median survival 10.9 months compared to 7.3 months with IFN. Bevacizumab (Avastin) – with IFN increases survival from 5.4 months to 10.2 months. Clinical need remains but crowded landscape Melanoma / Others? – PMX 290 potent in methotrexate, cisplatin and doxorubicin resistant cell lines but not in histotypes that are clinically relevant. Await further data. Strictly confidential

  30. PMX 2058: Pancreatic Cancer as Target Indication? (1) Pancreatic tumours – particularly hypoxic Pancreatic ductal adenocarcinomas – 4 fold increase in Trx Good potency of PMX 290 in Panc-1 and BxPC3 cells PX-12 (Trx Inhibitor) - development deprioritized Sept 08 Ongoing Phase II study in advanced pancreatic cancer 33% patients with elevated plasma Trx-1 (>ULN: 18ng/ml) achieved stable disease. No patients with normal level of plasma Trx-1 achieved stable disease Plasma Trx-1 may be a suitable predictor of response - patient selection Plasma VEGF decreases – may be a biomarker of response Strictly confidential

  31. PMX 2058: Pancreatic Cancer as Target Indication? (2) PX-478 PMX 290 HIF signalling Decrease Decrease Hypoxia-mediated VEGF Decrease Decrease Normoxic VEGF No change ? HIF-1 protein levels Decrease Increase HIF-2 protein levels Unchanged Increase Cell growth inhibition (differing experimental conditions): MCF-7 25µM3µM HT-29 30µM 5µM PX-478 (selective HIF-1 inhibitor) Ongoing Phase I trial Preclinical studies in Panc-1 xenograft model 20mg/kg x 5 (p.o) affords ~ 60% TGI. Enhanced efficacy in combination with radiation. Pancreatic cancer likely target indication. Strictly confidential

  32. PMX 2058 Efficacy: Panc-1 Xenografts (1) Efficacy data supported by limited PK and PD data. Strictly confidential

  33. PMX 2058 Efficacy: Panc-1 Xenografts (2) Bold type – statistically significant (p<0.05) Strictly confidential

  34. PMX 2058 Efficacy: Panc-1 Xenografts (2) Insert available PK data – awaited Insert available PD data (HIF-1, VEGF, GLUT-1 and CD31) - awaited. Strictly confidential

  35. PMX 2058 Efficacy: BxPC3 Xenografts Insert updated efficacy data – awaited. Efficacy data supported by limited PK and PD data – data to be generated. Strictly confidential

  36. PMX 2058: Rationale for Recent Preclinical Evaluation Back-up slides Strictly confidential

  37. PMX 290: Efficacy • Significant activity i.p. in 3/3 xenograft models: Colon (HCT 116), melanoma (MDA-MB-435) and renal (CAKI-1) HCT 116, MDA-MB-435 CAKI-1 • 150 / 200 mg/kg d 7 100 / 150 mg/kg d 7, 14 • 25 / 50 mg/kg d 7-11 25 / 50 mg/kg d 7-11 Strictly confidential

  38. PMX 290 Efficacy: CAKI-1 Xenografts (s.c) Route of administration: I.P Species: Ncr nu/nu mice 100 / 150 mg/kg d 7, 14 25 / 50 mg/kg d 7-11 • Borderline and transient inhibition of tumour growth except at 50mg/kg/d x 5 • 50mg/kg/d x 5 results in maximal T/C of 52% (day 35). • All doses, except 25mg/kg/d x 5, result in diarrhoea • 50mg/kg/d x 5 causes a body weight loss of 13%. Strictly confidential

  39. PMX 290 Efficacy: HCT116 Xenografts (s.c) Route of administration: I.P Species: NMRI nu/nu mice 150 / 200 mg/kg d 7 25 / 50 mg/kg d 7-11 • Toxic deaths at 150mg/kg and 200mg/kg – after treatment. Prevented planned second dose. • Toxic deaths associated with diarrhoea. Inflamed gut at autopsy. • Maximal T/C of 39% (day 14) at 50mg/kg/d x 5 • 50mg/kg/d x 5 causes a body weight loss of 12%. Strictly confidential

  40. PMX 290 Efficacy: HCT116 Xenografts (s.c) * Efficacy following administration of 40mg/kg/day i.p attained significance: p  0.05 T/C: 36% (Day 21) Strictly confidential

  41. PMX 290 Toxicity: HCT116 Tumour-Bearing Mice Mice: NMRI (nu/nu); HCT116 (colon) xenografts Treatment period: Daily on Days 11 – 15. Inoculation: 107 cells / mouse – Day 0 (Subcutaneous). Vehicle: Saline containing 10%v/v DMSO and 0.05%v/v Tween 80. Blood parameters determined on Day 15 (n = 5). a Day on which maximum response observed in parentheses. T = Size of tumours in treated mice; C = Size of tumours in non-treated, control mice. Responses in bold type attain statistical significance with respect to control group (p < 0.05). b Day(s) on which toxic deaths observed in parentheses. Strictly confidential

  42. PMX 290 Efficacy: MDA-MB-435 Xenografts (s.c) Route of administration: I.P Species: NMRI nu/nu mice 150 / 200 mg/kg d 7 25 / 50 mg/kg d 7-11 • Toxic deaths at 150mg/kg and 200mg/kg – after treatment. Prevented planned second dose. • Toxic deaths associated with diarrhoea. Inflamed gut at autopsy. • Maximal T/C of 35% and 32% (day 14) after 150mg/kg and 200mg/kg respectively • 150mg/kg and 200mg/kg causes body weight loss of 12% and 17% respectively. Strictly confidential

  43. PMX 290 Efficacy: MDA-MB-435 Xenografts (s.c) Efficacy following administration of 40mg/kg/day i.p attained significance: p  0.05 Maximal T/C: 47% (Day 16) Strictly confidential

  44. PMX 290 Toxicity: MDA-MB-435 Tumour-Bearing Mice Mice: NMRI (nu/nu); MDA-MB-435 (breast) xenografts Treatment period: Daily on Days 6 – 10. Inoculation: 107 cells / mouse – Day 0 (Subcutaneous). Vehicle: Saline containing 10%v/v DMSO and 0.05%v/v Tween 80. Blood parameters determined on Day 10 (n = 5). a Day on which maximum response observed in parentheses. T = Size of tumours in treated mice; C = Size of tumours in non-treated, control mice. Responses in bold type attain statistical significance with respect to control group (p < 0.05). b Day(s) on which toxic deaths observed in parentheses. Strictly confidential

  45. PMX 2058: In Vivo Conversion to PMX 290 • Rapid conversion to PMX 290 in vivo (rat) – via mono-adduct (GW002057) • PMX 290 concentrations  0.05µM for entire 8h study period Strictly confidential

  46. PMX 2058 Efficacy: HCT116 Xenografts (s.c) • PMX 2058 efficacy following administration of 100mg/kg/day x 5 i.v - attained borderline significance: p  0.05 • Comparable to PMX 290 at 50mg/kg/day x 5 i.p • PMX 2058 Maximal T/C: 45%; Body weight loss: 5% (Control body weight loss: 2%) • PMX 290 Maximal T/C: 35%; Body weight loss: 15% Strictly confidential

  47. PMX 2058: Consideration of Clinical Space Strictly confidential

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