History of Patupilone:
From Bench to Clinic
Paul M.J. McSheehy, PhD
Novartis Pharma AG
Basel, Switzerland
Epothilones A and B
16-membered macrolide-lactones from myxobacteria
Sorangium cellulosum So ce90
• Identified by Reichenb...
Tubulin / microtubules:
Effective targets for anticancer therapy
Tubulin:
− heterodimer of 55 kDa
α- and β-tubulin
Microtu...
Tubulin: The binding site
β-tubulin
274
α-tubulin
• Paclitaxel binding site (β-tubulin) similar; overlapping but not ident...
Consequences of interference with microtubule
function
• Disruption of cell cycle
– reduced proliferation,
– increased cel...
Effect of in vitro incubation time on anti-proliferative
activity of Patupilone vs. Paclitaxel
In contrast to paclitaxel, ...
Comparison of activity on Paclitaxel-sensitive
human tumour cell lines in vitro
IC50 (nM)
Histotype Cell Line Patupilone P...
Patupilone is selectively cytotoxic towards
proliferating cells
YO-PRO-1 fluorescent dye: detects apoptosed (permeable) ce...
Comparison of activity on Paclitaxel-resistant
human cell lines in vitro
RF: (subline IC50/parental IC50)
Histotype Cell L...
Prolonged retention of Patupilone in rodents
including brain & tumours after a single iv dose
4 mg/kg in nude mouse 1.5 mg...
Tissue half-lives of Patupilone vs. Taxanes
following single-dose administration to mice
a
Estimates based on Blum et al.,...
Patupilone access and activity is unaltered
in large poorly vascularised tumours
Parameter Sm HT29 tumours Lg HT29 tumours...
Comparison of activity on Taxol-sensitive
human tumour xenografts in vivo
Histotype Tumour
iv dose Response Survival
Patup...
Patupilone retains activity against Taxol-resistant
human tumour xenografts in vivo
Histotype Tumour
iv dose Response Surv...
Overview of in vivo activity of Patupilone:
human tumour s.c. xenograft models in nude mice
• Drug-sensitive tumour models...
Patupilone activity against a human lung tumour
(H460-Luc) growing in mouse brain
Days post cell injection
(treatment on d...
Anti-metastatic activity in orthotopic models
Patupilone showed strong activity in these models –
whether this involves pr...
Anti-vascular activity detectable after 2 days
before a change in rat breast tumour size
DCE-MRI: blood volume maps
Vehicl...
Drug transporters: Patupilone
• Weak/no substrate for 7 different drug transporters:
– P-gp
– BCRP
– MRP-1, -2, -3, -4, -5...
Simultaneous administration of Patupilone with
carboplatin provides synergistic tumour cell kill in vitro
Lung (A549) cell...
Patupilone shows strong activity in an orthotopic human
lung tumour model and synergises with RAD001
• Human H460-Luc cell...
Combination with ionising radiation (IR) in human
SW480 tumour xenografts
Patupilone 2 mg/kg, day 0; IR (4 x 3 Gy) on days...
Patupilone combinations:
Conclusions to date (Aug-2006)
In vitro
• Scheduling important…Why?
• Positive interactions where...
Patupilone: Summary
(a potent microtubule stabiliser)
• More soluble than Taxanes
• Binds β-tubulin with a very high affin...
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02_MCSHEEHY.ppt

  1. 1. History of Patupilone: From Bench to Clinic Paul M.J. McSheehy, PhD Novartis Pharma AG Basel, Switzerland
  2. 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 = CH3: Epothilone B (Patupilone)
  3. 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. 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. 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. 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. 7. Comparison of activity on Paclitaxel-sensitive human tumour cell lines in vitro IC50 (nM) Histotype Cell Line Patupilone Paclitaxel Ovarian 1A9 0.65 4.42 “ SK-OV-3 0.35 5.03 Lung A549 0.23 3.19 “ NCI-H460 0.29 5.65 Colon HCT-116 0.32 2.79 “ HCT-15 0.34 2.29 Prostate Du-145 0.31 4.14 “ PC-3M 0.52 4.77 Breast MDA-MB-231 0.13 1.43 “ ZR-75-1 0.64 3.60 Epidermoid KB-31 0.19 2.31 “ A-431 0.26 1.66 Liver HepG2 0.23 5.59 Glioma U87MG 1.10 12.9 Bladder T-24 0.25 4.40 IC50 median (nM) Patupilone Paclitaxel 0.31 4.4 Patupilone shows increased potency in vitro (median of 14-fold) against a broad panel of human cancer cell lines
  8. 8. Patupilone is selectively cytotoxic towards proliferating cells YO-PRO-1 fluorescent dye: detects apoptosed (permeable) cells yielding an EC25 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. 9. Comparison of activity on Paclitaxel-resistant human cell lines in vitro RF: (subline IC50/parental IC50) Histotype Cell Line (Selecting agent) Drug Resistance mechanism Patupilone Paclitaxel Ovarian 1A9/PTX10 (Paclitaxel) ß-tubulin Phe270Val 2.8 24 Ovarian 1A9/PTX22 (Paclitaxel) ß-tubulin Ala364Thr 1.4 24 Epidermoid KB-8511 (Colcemid) P-gp 1 230 Mammary MCF-7/ADR (Doxorubicin) P-gp and others 16 5020 Colon SW620AD-300 P-gp 3 1250 Leukaemic CCRF-CEM/VBL (Vinblastine) P-gp 6 1971 Median : 2.8 230 Patupilone retains activity in paclitaxel-resistant cell lines over-expressing either P-gp or harbouring ß-tubulin mutations
  10. 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-gpRes ) and shows high retention
  11. 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 Tissue t1/2 (hr) Patupilonea (4 mg/kg) Paclitaxelb (30 mg/kg) Docetaxelc (37 mg/kg) Tumour 89 12 22 Muscle 23 4.3 2-5 Liver 17 3.3 2-5
  12. 12. Patupilone access and activity is unaltered in large poorly vascularised tumours Parameter Sm HT29 tumours Lg HT29 tumours p-value Tumour Volume (mm3 ) 115 ± 11 505 ± 66** 0.00006 rBVol 0.33 ± 0.02 0.22 ± 0.01** 0.0009 BFI 0.24 ± 0.07 0.06 ± 0.01* 0.024 [Patupilone] in tumour, (µM) 0.59 ± 0.10 0.53 ± 0.13 >0.15 [Patupilone] in plasma, (nM) 5.2 ± 1.5 11.7 ± 4.4 >0.15 2.5 mg/kg i.v. weekly Human colon HT29 T/C Δ%BW Sm 20.5 - 14 ± 4 Lg 23.5 - 12 ± 3 Tumourvolume(mm3 ) Days post treatment
  13. 13. Comparison of activity on Taxol-sensitive human tumour xenografts in vivo Histotype Tumour iv dose Response Survival Patupilone Taxol Patupilone Taxol Patupilone Taxol Colon HCT-116 3 mg/kg 1/week 20 mg/kg 3/week T/C 3% Regression -24% 8/8 7/8 Breast MDA-MB-468 8 mg/kg once 20 mg/kg qd2 * 5 Regression - 47% Regression -98% 6/8 5/8 Prostate PC-3M 6 mg/kg once 20 mg/kg 3/week Regression - 50% Regression -26% 8/8 8/8 Patupilone shows equivalent activity in Taxol-sensitive tumours at comparable tolerability
  14. 14. Patupilone retains activity against Taxol-resistant human tumour xenografts in vivo Histotype Tumour iv dose Response Survival Patupilone Taxol Patupilone Taxol Patupilone Taxol Colon HCT-15 4 mg/kg once 20 mg/kg qd2 * 5 Regression - 61% T/C 50% 8/8 7/8 Epidermoid KB-8511 4 mg/kg 1/week 20 mg/kg qd2 * 5 Regression - 98% T/C 100% 8/8 8/8 Lung A549 4 mg/kg 1/week 20 mg/kg 3/week T/C 7% T/C 100% 8/8 8/8 Ovarian 1A9PTX10 4 mg/kg 1/week 15 mg/kg 3/week T/C 40% T/C 125% 8/8 8/8 Patupilone is active in Taxol - resistant tumours at tolerated doses
  15. 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 1A9PTX10 – Colon: HCT-15 – Cervical/Oral: KB-8511 (regr, cures) Patupilone activity comparable to standard drugs Patupilone activity superior to Taxol
  16. 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. 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. 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. 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. 20. Simultaneous administration of Patupilone with carboplatin provides synergistic tumour cell kill in vitro Lung (A549) cell line Colon (HCT116) cell line Sim ultaneousC arboplatin firstPatupilone first Sim ultaneousC arboplatin firstPatupilone first
  21. 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. 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. 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. 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

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