History of Patupilone: From Bench to Clinic

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History of PatupiloneFrom Bench to Clinic

• Paul M.J. McSheehy, PhD
• Novartis Pharma AG
• Basel, Switzerland

2
Epothilones A and B16-membered
macrolide-lactones from myxobacteria
R H Epothilone A R CH3 Epothilone
B (Patupilone)
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

3
Tubulin / microtubulesEffective targets for
anticancer therapy

• Tubulin
• heterodimer of 55 kDa a- and ß-tubulin

• Microtubules
• hollow fibers with 22-24 nm diameter
• 12-13 protofilaments / microtubule

• Polymerization inhibitors
• Colchicine
• Vincristine, Vinblastine
• Many others

() end
Microtubule Dynamics

• 2 GTP
• hydrolysis required for tubulin addition

• Polymerization enhancers
• Taxol, Taxotere
• Epothilones
• Discodermolide
• Eleutherobine, Sarcodyctin

(-) end
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Tubulin The binding site
ß-tubulin
ß-tubulin
274
a-tubulin

• Paclitaxel binding site (ß-tubulin) similar
  overlapping but not identical
• Patupilone has a higher affinity (Buey et al,
  2004)

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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

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Effect of in vitro incubation time on
anti-proliferativeactivity of Patupilone vs.
Paclitaxel
In contrast to paclitaxel, short exposure times
of patupilone suffice to produce potent
anti-proliferative effects
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Comparison of activity on Paclitaxel-sensitivehum
an tumour cell lines in vitro
Patupilone shows increased potency in vitro
(median of 14-fold) against a broad panel of
human cancer cell lines
8
Patupilone is selectively cytotoxic
towardsproliferating cells
YO-PRO-1 fluorescent dye detects apoptosed
(permeable) cells yielding an EC25
Proliferating human PBLs
Resting human PBLs
Drug concentration nM
Drug concentration nM
Similar observations made on human tumour
cells a) leukaemia MTT assay, b) colon 3H-Thd
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Comparison of activity on Paclitaxel-resistant
human cell lines in vitro
Patupilone retains activity in paclitaxel-resistan
t cell lines over-expressing either P-gp or
harbouring ß-tubulin mutations
10
Prolonged retention of Patupilone in
rodentsincluding 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
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Tissue half-lives of Patupilone vs. Taxanes
following single-dose administration to mice
aEstimates based on Blum et al., Novartis Release
Ready Report 1999, RD-1999-03642 bData from
Fujita et al., Jpn J Cancer Chemother 1994, 21
659-664 cData from Bissery et al., Anticancer
Drugs, 1995, 6 339-355
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Patupilone access and activity is unalteredin
large poorly vascularised tumours
2.5 mg/kg i.v. weekly
Tumour volume (mm3)
Days post treatment
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Comparison of activity on Taxol-sensitivehuman
tumour xenografts in vivo
Patupilone shows equivalent activity in
Taxol-sensitive tumours at comparable tolerability
14
Patupilone retains activity against
Taxol-resistanthuman tumour xenografts in vivo
Patupilone is active in Taxol - resistant tumours
at tolerated doses
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Overview of in vivo activity of Patupilonehuman
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 activitycomparableto standard drugs
Patupilone activitysuperior to Taxol
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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
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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

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Anti-vascular activity detectable after 2
daysbefore a change in rat breast tumour size
DCE-MRI blood volume maps
Vehicle Day 0
Day 2
Patupilone Day 0
Day 2
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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

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Simultaneous administration of Patupilone
withcarboplatin provides synergistic tumour cell
kill in vitro
Lung (A549) cell line
Colon (HCT116) cell line
Simultaneous
Simultaneous
Patupilone first
Patupilone first
Carboplatin first
Carboplatin first
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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

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Combination with ionising radiation (IR) in
humanSW480 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(P0.0004)
• BW loss unchanged
• Effect independent of scheduling

Hofstetter et al, Clin Cancer Res,111558, 2005
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Patupilone combinationsConclusions to date
(Aug-2006)

• In vitro
• Scheduling importantWhy?
• 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

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Patupilone Summary(a potent microtubule
stabiliser)

• More soluble than Taxanes
• Binds ß-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 ß-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