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Pharminox SAB - 23 March 2009

Pharminox SAB - 23 March 2009. PHORTRESS. Phortress – Agenda. Introduction Malcolm Stevens Biology Tracey Bradshaw Development Sue Barrowcliffe SAB Review Additional data needs Development strategy: indications, monotherapy vs combination

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Pharminox SAB - 23 March 2009

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  1. Pharminox SAB - 23 March 2009 PHORTRESS

  2. Phortress – Agenda • Introduction Malcolm Stevens • Biology Tracey Bradshaw • Development Sue Barrowcliffe • SAB Review • Additional data needs • Development strategy: indications, monotherapy vs combination • At what point should Pharminox exercise its option

  3. Phortress ▪Novel structure ▪ Synthetically accessible ▪ Pharmaceutically robust ▪ Potent, selective antitumour activity ▪ Distinct mechanism of action ▪Phase I Clinical Evaluation Strictly confidential

  4. * * * * * * NCI GI50 mean graphs 5F 203 Phortress Lung Ovarian Renal Breast Colon cancer Ovarian cancer Renal cancer Breast cancer Strictly confidential

  5. MCF-7MDA-MB-435 Control ● ○ 5F 203 (d0-3) ■ □ In vivoantitumour activity of 5F 203 5 mg/kg 5F 203 ip d 0 – 3. Strictly confidential

  6. Optimum T/C Phortress 25* 48* 12*/12* 47* 67 62 39*+/42*+ 49* 65* Doxorubicin 12* 42* 9* 36* 81 61 79 10* 46* Phortress: in vivoantitumour activity Breast Xenograft 4296 4151 4134 3366 3366/Tam 4586 MCF-7 4898 4000 Evaluation = = = = = = ++ - - CYP1A1 ↑ ↑ — n/e ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ Strictly confidential Fichtner I et al, Br Can Res & Tr 2004

  7. Depletion of 5F 203 from nutrient media Strictly confidential

  8. Aryl Hydrocarbon Receptor Pathway L ligand binding L AhR H s p 90 H s p 90 L L AhR AhR ARNT release and translocation XRE DNA binding L cytosol AhR AhR nucleus H s p 90 H s p 90 ARNT AhR dimerisation ARNT transcription Strictly confidential

  9. MCF-7 MDA-MB-435 Control 5F 203 1μM AhR Translocation: Sensitive vs resistant cells Strictly confidential

  10. * Effect of PhortressIn vitro: Induction of CYP1A1 Strictly confidential

  11. Generation of DNA adducts in vitro:10 μM Phortress MCF-7 MDA-MB-435 IGROV-1 Strictly confidential

  12. MCF-7 MDA-MB-435 Formation of DNA SSB and DSB Alkaline (SSB) Neutral (DSB) Strictly confidential

  13. Generation of DNA adducts in xenografts of MCF-7-bearing mice 24 h post treatment (20 mg/kg Phortress i.p). MCF-7 MDA-MB-435 IGROV-1 - + - + - + • Selective induction of 52 Kda CYP 1A1 Pharmacodynamic assessments in vivo Strictly confidential

  14. 4 h 24 h Pharmacodynamic assessments in vivo Tail moment histograms of comets derived from MCF-7 xenografts 4 h and 24 h after treatment of NMRI mice (i.p.) with 20 mg/kg Phortress or saline vehicle. Strictly confidential

  15. AhR AhR 5F-203 AhR Phortress: understood mechanism of action Phortress Phortress HSP90 HSP90 Arylhydrocarbon receptor nucleus 5F-203 ARNT 5F-203 Binding to promoter of CYP1A1 gene Cytosolic AhR translocates to nucleus Increased cyp1a1 mRNA 5F-203 cytoplasm 5F203* DNA Adducts Increased CYP1A1 cell membrane Drug metabolising enzyme Double Strand Breaks Metabolism to bioreactive species Cell death Strictly confidential

  16. Phortress – Mechanism of Action Additional Considerations

  17. Molecular genetics of hereditary and sporadic RCC • ▪VHL • VHL tumour suppressor gene • pVHL regulates transcription and production of growth factors • Binds HIF, targetting HIF-1α for degradation through the ubiquitin • proteolytic pathway. ▪c-Met Met oncogene encodes transmembrane receptor tyrosine kinase c-Met. Met activation promotes cell proliferation, enhanced motility, inhibits apoptosis. Strictly confidential

  18. Von Hippel-Lindau disease HIF-1 transcription factor HIF-1α HIF-1β Aka ARNT Strictly confidential

  19. Von Hippel-Lindau disease HIF-1 transcription factor under aerobic conditions: pVHL targets HIF-1α for ubiquitination and degradation HIF-1α HIF-1β Aka ARNT Strictly confidential

  20. Von Hippel-Lindau disease HIF-1 transcription factor under hypoxic conditions: HIF-1 binds to HRE in promoter of HIF-dependent genes driving transcription of proteins promoting: angiogenesis (VEGF) glycolysis (GLUT 1) HIF-1α HIF-1β Aka ARNT Strictly confidential

  21. Von Hippel-Lindau disease In VHL disease, mutant pVHL fails to target HIF-1 transcription factor for degradation. HIF-1 dependent transcription of proteins promoting: angiogenesis (VEGF) glycolysis (GLUT 1) driving renal carcinogenesis and metastasis HIF-1α HIF-1β Aka ARNT Strictly confidential

  22. AhR Effect of Phortress: a hypothesis Phortress 5F 203 HIF-1α HIF-1β Aka ARNT Strictly confidential

  23. AhR Effect of Phortress: a hypothesis 5F 203 HIF-1α HIF-1β Aka ARNT Dimerisation of ligand bound AhR with ARNT (aka HIF-1β) depletes HIF-1, may diminish HIF-driven transcription and signal transduction, inhibiting the pathway driving renal carcinogenesis. Strictly confidential

  24. AhR Effect of Phortress: a hypothesis 5F 203 HIF-1α HIF-1β Aka ARNT Ref: Rankin et al, 2000, Inactivation of ARNT suppresses VHL disease-associated tumours in mice. Mollecular Cell Biol 3163. Strictly confidential

  25. In collaboration with Kinexus Bioinformatics Corporation: Protein was isolated from MCF-7 and MDA 468 breast carcinoma cells treated for 24 h with vehicle alone, 1 μM GW 610 or 1 μM 5F 203. Samples were subjected to antibody microarray which tracked 600 cell signalling proteins in duplicate for > 250 phospho-sites, 240 protein kinases and 110 additional cell signalling proteins that regulate cell proliferation, stress and apoptosis. Down regulation of c-Met: HGFR tyrosine kinase activity. ▪c-Met: Hepatocyte growth factor receptor (HGFR) ▪Ligands: Hepatocyte growth factor (HGF), scatter factor Strictly confidential

  26. Aberrant Met activation in hereditary and sporadic papillary renal cell carcinoma Refs:Cancer Res 65 (11) 4598, 2005 Morris et al; J Urol 163 (4) 1241, 2000; Nature Genetics 16 (1) 68, 1997. Schmidt et al; Clin Can Res 12, 3657, 2006 Peruzzi and Botaro. c-Met: Role in tumourigenesis and metastasis Aberrant Met activityHuman tumour chromosomal rearrangement gastric gene amplification gastric, oesophageal, NSCLC, liver mets from colon, medulloblastoma germline mutation papillary renal, gastric somatic mutation papillary renal, childhood hepatocellular trisomy 7 hereditary papillary renal transcriptional upregulation ovarian, pancreatic, renal, breast, colorectal, hepatocellular HGF-dependent autocrine loop breast, glioblastoma Strictly confidential

  27. c-Met: Role in tumourigenesis and metastasis Down-regulation of c-Met inhibits growth in the liver of human colorectal carcinoma cell. Matthew H. Herynk et al, Cancer Res 63, 2990-96, 2003. Overexpression of c-Met, the protein tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma…..a small reduction in c-Met protein levels leads to profound biological effects, and c-Met inhibitors may be of therapeutic value in treatment of colon cancer. ▪Met activation stimulates cell migration and angiogenesis, conveying mitogenic signals and protecting from apoptosis, thus acting as a promoter of aggressive, invasive growth. ▪Met-dependent signalling includes: RAS-RAF-MEK-ERK and PTEN-PI3K-AKT cascades. mediating c-Met-induced scattering, proliferation and branching morphogenesis ▪Met tyrosine kinase – a powerful expedient for cancer dissemination Strictly confidential

  28. 0 h 24 h 72 h Control 100 nM 5F 203 Down regulation of c-Met: HGFR tyrosine kinase activity. Inhibition of ERK, MAPK, MEK kinase activity following exposure of breast cells to 5F 203 or GW 610. Strictly confidential

  29. Signalling interactions, opportunities for combination therapy? HIF-1 transcription factor binds to the HRE of the Met gene Met transcription is activated by HIF-1 (VHL disease promote Met activation and HIF signalling) Morris et al report a correlation between inactivation of VHL and increased Met signalling Met AhR HIF-1 PI3K / MAPK Torisel – Temsirolomus (Rapamycin analogue) FDA approval and used in USA and Europe for treatment of metastatic RCC. A selective small molecule MET inhibitor PHA665752 co-operates with rapamycin. Clinical Cancer Research 2005 11 (6) 2312. Ma PC et al. Interactions between PTEN and the c-Met pathway in glioblastoma and implications for therapy. Mol Can Ther 2009 8 (2) 376. Li Y et al. Putative mTOR inhibition (Temsirolomus) and Phortress combination. Strictly confidential

  30. Signalling interactions and opportunities for combination therapy? HIF Oxygen transport Iron metabolism Angiogenesis Glycolysis/glucose transport Erythropoietin HO-1 Transferrin Angiopoietin-2 COX-2 HGF FGF VEGF VEGFR GLUT-1,3 glucose transporter 1,3 Transcription factors Metabolism Growth factors/cytokines HIF-1α HIF 2α JUN CA-IX Acetyl CoA thiolase IGF-2 PDGF Stress response ECM/cell adhesion Proteases Thioredoxin metalloproteinases α-integrin Phortress combinations to consider: Multikinase inhibitors Sunitinib, Sorafenib. Anti-VEGF Ab Avastin Strictly confidential

  31. Phortress – Development Overview • Preclinical Data • Key findings from tox studies • Potential issues moving forward • Clinical Data • Phase I Study Design – key issues • Update on Phase I trial status • AF Phase I trials • Potential Indications • Pros and Cons of options considered to date

  32. Phortress – Key Findings from Tox Studies • Mouse – Day 1 & 8 Dosing (1 cycle) – 2 dose levels • NOAEL not established in definitive study (10mg/kg lowest dose tested, only 2 dose groups) • 10mg/kg judged to be MTD, no significant tox findings • At 20mg/kg, increases in Liver enzymes, hepatocyte necrosis in 9/10 animals – some evidence of reversibility. • At both doses, DNA adducts detected in liver and lung. CYP1A1 induction in lung only. • Rat – Day 1 & 8 Dosing (1 cycle) – 2 dose levels • NOAEL 10mg/kg • Local injection site changes only at 20mg/kg • DNA adducts at top dose in liver & lung, CYP1A1 induction liver & lung both doses (note: adducts in 1 of 2 female controls)

  33. Phortress – Key Findings from Tox Studies • Dog – weekly x3, 1,5,8mg/kg, 4dogs/dose level • Emesis, low food consumption all doses • Increases in liver enzymes at 5 and 8mg/kg • Reversible histopathological changes in liver, gall bladder - same doses • Dog – single dose, 14.3mg/kg (1 or 24 hr infusion) • Evidence of significant liver and lung toxicity as well as myelotoxicity • Dog Study + B-Naphthoflavone – no change in toxicity • Monkey – single dose studies, 1 or 4h infusions • Reversible increase in transaminase at lowest dose 3.33mg/kg • 2 monkeys died at 6.67mg/kg both had evidence of significant pulmonary toxicity. • NCI publication suggested monitoring pulmonary function not an effective indicator of toxicity – hepatic toxicity possible surrogate, different to aminoflavone (Note: Pharminox do not have these data)

  34. Phortress – Issues with Preclinical Data • Adequacy of Data from Regulatory Perspective • No safety pharmacology studies (CVS, Resp, CNS) • No multiple cycle toxicity studies • Limited ADME data • No safety data on combination use (necessity depends on clinical indication) • Nature of the tox findings • Ability to monitor for lung toxicity • Are hepatic changes an appropriate surrogate (clinical experience would suggest this is the case) • Influence of existing CYP1A1 induction – use in smokers

  35. Phortress – Phase I Study Design • Stringent inclusion and exclusion criteria • Lung function in normal range • Liver function no worse than Grade 1 • No smokers • No Proton pump inhibitors or antifungals in prior 4 weeks (now PPI criteria relaxed to 2 wks) • Cautious Dose Escalation • 1.41 increments • 3 patients per dose level: Patient 1 completes 21 days before pt 2 entered, pt 3 minimum 7 days after pt 2 • Study Specific DLTs: 2-fold increase in any LFT, any pulmonary toxicity of any grade. • Dose Regime altered after liver toxicity in first cohort • First Cohort dosing days 1 and 8 at 3mg/m2 – LFT ↑ all pts • Revised dosing day 1 of 21 day cycle

  36. Phortress – Phase I Current Status

  37. Aminoflavone Phase I trials

  38. Phortress – Indication Options • Based on preclinical data • Breast • Ovarian • Based on Phase I & new mechanistic considerations • Renal • + other possibilities (Tracey) • Others – NCI screen showed lung cell lines sensitive • NSCLC • SCLC

  39. Indication Options – preliminary assessment

  40. Indication Options – preliminary assessment

  41. BACK-UPS

  42. Phortress 5F 203 Phortress 5F 203 Properties of amino acid prodrugs ▪ Prodrugs taken up readily by cells. ▪ Conversion to amine in culture with sensitive and insensitive cells. ▪ Sensitive cells only deplete amine from nutrient medium. Phortress 5F 203 Mouse: 18 mg/kg, iv Dog 14.3 mg/kg, iv Strictly confidential

  43. Phortress: in vivo antitumour activity IGROV-1 MCF-7 ◯ Control ■ 20 mg/kg d 0,7 ● 20 mg/kg, d 0 □ 4 mg/kg d 0-4 Strictly confidential

  44. AhR Translocation: Sensitive MCF-7 cells Control TBDD 10 nM DF 203 1μM 5F 203 1μM Strictly confidential

  45. Phortress – Patient 1 Liver Function Tests

  46. Phortress – The First 6 Patients

  47. Phortress – Exposure Data, Revised protocol In Mice at 20mg/kg, day 1 Phortress: AUC = 3.24 µmol/L.h 5F203: AUC = 5.58 µmol/L.h

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