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  • 1. History of Patupilone: From Bench to Clinic Paul M.J. McSheehy, PhD Novartis Pharma AG Basel, Switzerland
  • 2. Epothilones A and B 16-membered macrolide-lactones from myxobacteria Sorangium cellulosum So ce90
    • Identified by Reichenbach et al as anti-fungal agent, 1993
    • Paclitaxel-like mechanism shown by Bollag et al , 1995
    • More soluble and more lipophilic than taxanes
    R = H: Epothilone A R = CH 3 : Epothilone B (Patupilone)
  • 3. Tubulin / microtubules: Effective targets for anticancer therapy
    • Tubulin:
    • heterodimer of 55 kDa α - and β -tubulin
    • Microtubules:
    • hollow fibers with 22-24 nm diameter
    • 12-13 protofilaments / microtubule
    • Polymerization inhibitors
    • Colchicine
    • Vincristine, Vinblastine
    • Many others
    Microtubule Dynamics
    • Polymerization enhancers
    • Taxol, Taxotere
    • Epothilones
    • Discodermolide
    • Eleutherobine, Sarcodyctin
    • 2 GTP:
    • hydrolysis required for tubulin addition
    (+) end (-) end
  • 4. Tubulin: The binding site β -tubulin 274 α -tubulin
    • Paclitaxel binding site ( β -tubulin) similar; overlapping but not identical
    • Patupilone has a higher affinity (Buey et al , 2004)
    β -tubulin
  • 5. Consequences of interference with microtubule function
    • Disruption of cell cycle
      • reduced proliferation,
      • increased cell death (apoptosis / mitotic catastrophe)
    • Disruption of protein movement and therefore function
      • affects gene expression
      • affects enzyme function (e.g. HIF-1),
    • Disruption of cell movement and shape
      • affects metastasis, endothelial cell permeability
  • 6. Effect of in vitro incubation time on anti-proliferative activity of Patupilone vs. Paclitaxel In contrast to paclitaxel, short exposure times of patupilone suffice to produce potent anti-proliferative effects
  • 7. Comparison of activity on Paclitaxel-sensitive human tumour cell lines in vitro Patupilone shows increased potency in vitro (median of 14-fold) against a broad panel of human cancer cell lines IC 50 (nM) 4.40 0.25 T-24 Bladder 12.9 1.10 U87MG Glioma 5.59 0.23 HepG2 Liver 1.66 0.26 A-431 “ 2.31 0.19 KB-31 Epidermoid 3.60 0.64 ZR-75-1 “ 1.43 0.13 MDA-MB-231 Breast 4.77 0.52 PC-3M “ 4.14 0.31 Du-145 Prostate 2.29 0.34 HCT-15 “ 2.79 0.32 HCT-116 Colon 5.65 0.29 NCI-H460 “ 3.19 0.23 A549 Lung 5.03 0.35 SK-OV-3 “ 4.42 0.65 1A9 Ovarian Paclitaxel Patupilone Cell Line Histotype IC 50 median (nM) 4.4 0.31 Paclitaxel Patupilone
  • 8. Patupilone is selectively cytotoxic towards proliferating cells YO-PRO-1 fluorescent dye: detects apoptosed (permeable) cells yielding an EC 25 Proliferating human PBLs Resting human PBLs Similar observations made on human tumour cells: a) leukaemia: MTT assay, b) colon: 3 H-Thd Drug concentration [nM] Drug concentration [nM]
  • 9. Comparison of activity on Paclitaxel-resistant human cell lines in vitro Patupilone retains activity in paclitaxel-resistant cell lines over-expressing either P-gp or harbouring ß-tubulin mutations 1971 6 P-gp CCRF-CEM/VBL (Vinblastine) Leukaemic 1250 3 P-gp SW620AD-300 Colon Median : P-gp and others P-gp ß-tubulin Ala364Thr ß-tubulin Phe270Val Drug Resistance mechanism RF: (subline IC 50 /parental IC 50 ) 230 2.8 5020 16 MCF-7/ADR (Doxorubicin) Mammary 230 1 KB-8511 (Colcemid) Epidermoid 24 1.4 1A9/PTX22 (Paclitaxel) Ovarian 24 2.8 1A9/PTX10 (Paclitaxel) Ovarian Paclitaxel Patupilone Cell Line (Selecting agent) Histotype
  • 10. Prolonged retention of Patupilone in rodents including brain & tumours after a single iv dose 4 mg/kg in nude mouse 1.5 mg/kg in Lewis rat Time Post-Administration (h) Time Post-Administration (h) Patupilone crosses the BBB (P-gp Res ) and shows high retention
  • 11. Tissue half-lives of Patupilone vs. Taxanes following single-dose administration to mice a Estimates based on Blum et al ., Novartis Release Ready Report 1999, RD-1999-03642 b Data from: Fujita et al ., Jpn J Cancer Chemother 1994, 21: 659-664 c Data from Bissery et al ., Anticancer Drugs, 1995, 6: 339-355 t 1/2 (hr) 2-5 3.3 17 Liver 2-5 4.3 23 Muscle 22 12 89 Tumour Docetaxel c (37 mg/kg) Paclitaxel b (30 mg/kg) Patupilone a (4 mg/kg) Tissue
  • 12. Patupilone access and activity is unaltered in large poorly vascularised tumours 2.5 mg/kg i.v. weekly Tumour volume (mm 3 ) Days post treatment >0.15 0.53 ± 0.13 0.59 ± 0.10 [ Patupilone ] in tumour, (µM) >0.15 11.7 ± 4.4 5.2 ± 1.5 [ Patupilone ] in plasma, (nM) 0.024 0.06 ± 0.01* 0.24 ± 0.07 BFI 0.0009 0.22 ± 0.01** 0.33 ± 0.02 rBVol 0.00006 505 ± 66** 115 ± 11 Tumour Volume (mm 3 ) p-value Lg HT29 tumours Sm HT29 tumours Parameter Human colon HT29 - 14 ± 4 20.5 Sm - 12 ± 3 23.5 Lg Δ %BW T/C
  • 13. Comparison of activity on Taxol-sensitive human tumour xenografts in vivo Patupilone shows equivalent activity in Taxol-sensitive tumours at comparable tolerability Taxol Taxol Taxol 8/8 8/8 Regression -26% Regression - 50% 20 mg/kg 3/week 6 mg/kg once PC-3M Prostate 5/8 6/8 Regression -98% Regression - 47% 20 mg/kg qd2 * 5 8 mg/kg once MDA-MB-468 Breast 7/8 8/8 Regression -24% T/C 3% 20 mg/kg 3/week 3 mg/kg 1/week HCT-116 Colon Patupilone Patupilone Patupilone Tumour Histotype Survival Response iv dose
  • 14. Patupilone retains activity against Taxol-resistant human tumour xenografts in vivo Patupilone is active in Taxol - resistant tumours at tolerated doses Taxol Taxol Taxol 8/8 8/8 T/C 125% T/C 40% 15 mg/kg 3/week 4 mg/kg 1/week 1A9 PTX10 Ovarian 8/8 8/8 T/C 100% T/C 7% 20 mg/kg 3/week 4 mg/kg 1/week A549 Lung 8/8 8/8 T/C 100% Regression - 98% 20 mg/kg qd2 * 5 4 mg/kg 1/week KB-8511 Epidermoid 7/8 8/8 T/C 50% Regression - 61% 20 mg/kg qd2 * 5 4 mg/kg once HCT-15 Colon Patupilone Patupilone Patupilone Tumour Histotype Survival Response iv dose
  • 15. Overview of in vivo activity of Patupilone: human tumour s.c. xenograft models in nude mice
    • Drug- sensitive tumour models:
      • Breast: MDA-MB468 (regr)
      • Prostate: PC-3M (regr), Du-145 (regr)
      • Lung: NCI-H596 (regr), NCI-H460
      • Colon: HCT-116, HT-29
      • Ovarian: SKOV-3, 1A9
      • Glioma: U-373, U87MG
      • Cervical: HeLa, KB31
      • Lung colonies HT1080
    • Drug- resistant tumour models:
      • Lung: A549
      • Ovarian 1A9 PTX10
      • Colon: HCT-15
      • Cervical/Oral: KB-8511 (regr, cures)
    Patupilone activity comparable to standard drugs Patupilone activity superior to Taxol
  • 16. Patupilone activity against a human lung tumour (H460-Luc) growing in mouse brain Days post cell injection (treatment on day 5) Days post cell injection (treatment on day 7) %T/C (Patupilone) = 25.1 (D14) %T/C (Patupilone) = 10.0 (D16) Significantly less body-weight loss and increased survival
  • 17. Anti-metastatic activity in orthotopic models
    • Patupilone showed strong activity in these models –
    • whether this involves prevention of formation is not yet clear:
      • Human H460 lung mets from mouse lung to brain
      • Murine B16/BL6 melanoma to mouse lymph-nodes
      • Rat BN472 mammary to rat lymph-nodes
      • Rat MTLn3 mammary to rat lymph-nodes
      • Human HT1080 fibrosarcoma colonising nude mouse lung
      • Human H460 lung cells injected into mouse brain
  • 18. Anti-vascular activity detectable after 2 days before a change in rat breast tumour size DCE-MRI: blood volume maps Vehicle: Day 0 Day 2 Patupilone: Day 0 Day 2
  • 19. Drug transporters: Patupilone
    • Weak/no substrate for 7 different drug transporters:
      • P-gp
      • BCRP
      • MRP-1, -2, -3, -4, -5
    • Patupilone did not influence activity of 6 different drug transporters (MRP-4 not tested)
    • This implies
    • Reduced basal and acquired drug resistance (rationale for activity in colon, hepatoma, brain where Taxol little activity)
    • Good combination partner with other drugs
  • 20. Simultaneous administration of Patupilone with carboplatin provides synergistic tumour cell kill in vitro Lung (A549) cell line Colon (HCT116) cell line Simultaneous Carboplatin first Patupilone first Simultaneous Carboplatin first Patupilone first
  • 21. Patupilone shows strong activity in an orthotopic human lung tumour model and synergises with RAD001
    • Human H460-Luc cells injected in lung day 0 and treatment begins day-5: Patupilone (3 mg/kg q2W), RAD001 (10 mg/kg q1D)
    • Untreated mice: brain tumors detectable day 10, culled day 17
  • 22. Combination with ionising radiation (IR) in human SW480 tumour xenografts Patupilone 2 mg/kg, day 0; IR (4 x 3 Gy) on days 1-4
    • A positive interaction in vitro
    • Positive interaction in vivo ( P =0.0004)
    • BW loss unchanged
    • Effect independent of scheduling
    Hofstetter et al , Clin Cancer Res,11:1558, 2005
  • 23. Patupilone combinations: Conclusions to date (Aug-2006)
    • In vitro
    • Scheduling important…Why?
    • Positive interactions where Patupilone precedes cytotoxic:
      • Carboplatin
      • Oxaliplatin
      • Gemcitabine
    • Negative in simultaneous for:
      • All of above
      • 5FU
      • Gemetecan (a camptothecin)
      • Doxorubicin
      • Many others
    • Positive in simultaneous for:
      • Vincristine, Cladribine, RAD001
    • In vivo
    • Scheduling important for the cytotoxics Gemcitabine, Alimta
    • Scheduling NOT important for IR, PTK787, STI571, RAD001
    • Positive interactions for
      • Ionising Radiation
      • PTK787
      • STI571 (imatinib)
      • RAD001
      • Gemcitabine (Patupilonefirst)
      • Alimta (Alimta first)
      • Carboplatin
      • Doxil
  • 24. Patupilone: Summary (a potent microtubule stabiliser)
    • More soluble than Taxanes
    • Binds β -tubulin with a very high affinity
    • Weak substrate for all drug-transporters (P-gp etc)
    • Large volume of distribution; retained by tumours
    • Crosses BBB; retained in brain
    • Potent inhibitor of tumour cell proliferation in vitro and in vivo including those expressing P-gp and some with β -tubulin mutations
    • Can inhibit tumour growth in brain
    • Inhibits metastatic growth
    • Good combination potential – scheduling may be important with other cytotoxics
    • Early-response biomarkers: MRI; FDG/FLT-PET; IFP