History of Patupilone:From Bench to Clinic

Paul M.J. McSheehy, PhD

Novartis Pharma AG

Basel, Switzerland

Epothilones A and B16-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 AR = CH3: Epothilone B

(Patupilone)

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

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

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

Effect of in vitro incubation time on anti-proliferativeactivity of Patupilone vs. Paclitaxel

In contrast to paclitaxel, short exposure times of patupilonesuffice to produce potent anti-proliferative effects

Comparison of activity on Paclitaxel-sensitivehuman tumour cell lines in vitro

IC50 (nM)Histotype Cell Line Patupilone PaclitaxelOvarian 1A9 0.65 4.42

“ SK-OV-3 0.35 5.03Lung A549 0.23 3.19

“ NCI-H460 0.29 5.65Colon HCT-116 0.32 2.79

“ HCT-15 0.34 2.29Prostate Du-145 0.31 4.14

“ PC-3M 0.52 4.77Breast MDA-MB-231 0.13 1.43

“ ZR-75-1 0.64 3.60Epidermoid KB-31 0.19 2.31

“ A-431 0.26 1.66Liver HepG2 0.23 5.59

Glioma U87MG 1.10 12.9Bladder T-24 0.25 4.40

IC50 median (nM)

Patupilone Paclitaxel0.31 4.4

Patupilone shows increased potency in vitro (median of 14-fold) against a broad panel of human cancer cell lines

Patupilone is selectively cytotoxic towardsproliferating 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: 3H-Thd

Drug concentration [nM] Drug concentration [nM]

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-tubulinPhe270Val

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 linesover-expressing either P-gp or harbouring ß-tubulin mutations

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

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

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

Patupilone access and activity is unalteredin 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

Tu

mo

ur

volu

me

(mm

3)

Days post treatment

Comparison of activity on Taxol-sensitivehuman tumour xenografts in vivo

Histotype Tumouriv dose Response Survival

Patupilone Taxol Patupilone Taxol Patupilone Taxol

Colon HCT-1163 mg/kg

1/week

20 mg/kg

3/week

T/C

3%

Regression

-24%8/8 7/8

Breast MDA-MB-4688 mg/kg

once

20 mg/kg

qd2 * 5

Regression

- 47%

Regression

-98%6/8 5/8

Prostate PC-3M6 mg/kg

once

20 mg/kg

3/week

Regression

- 50%

Regression

-26%8/8 8/8

Patupilone shows equivalent activity in Taxol-sensitive tumoursat comparable tolerability

Patupilone retains activity against Taxol-resistanthuman tumour xenografts in vivo

Histotype Tumouriv dose Response Survival

Patupilone Taxol Patupilone Taxol Patupilone Taxol

Colon HCT-154 mg/kg

once20 mg/kg qd2 * 5

Regression

- 61%

T/C

50%8/8 7/8

Epidermoid KB-85114 mg/kg

1/week20 mg/kg qd2 * 5

Regression

- 98%

T/C

100%8/8 8/8

Lung A5494 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

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 activitycomparableto standard drugs

Patupilone activitysuperior to Taxol

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

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

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

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

Simultaneous administration of Patupilone withcarboplatin provides synergistic tumour cell kill in vitro

Lung (A549) cell line Colon (HCT116) cell line

Simult

aneo

us

Carbo

platin

first

Patup

ilone

first

Simult

aneo

us

Carbo

platin

first

Patup

ilone

first

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

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(P=0.0004)

• BW loss unchanged

• Effect independent of scheduling

Hofstetter et al, Clin Cancer Res,11:1558, 2005

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

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