tesmilifene: mode of action -...
TRANSCRIPT
Tesmilifene: Mode of ActionMark Vincent MD, FRCPC, MRCP(UK)
Research & Development DayWednesday April 5, 2006
Harvard ClubNew York City
Agenda
1. Review Clinical Experience
2. Mode of Action Hypothesis• Selectivity for Multidrug Resistant (MDR) cancer cells• Broad potentiation of anthracyclines, taxanes and vinca alkaloids• Activation, not inhibition, of ATP-binding cassette (ABC) transporters• Targets cellular energetics
3. Implications for Future Clinical Development
1
2
3
Tesmilifene
ON
CH3
CH3
.HCl
ON
CH3
CH3
.HCl
N
CH3
CH3
.HCl
Potent Small Molecule Chemopotentiator
Clinical ExperienceN,N-diethyl-2-[4-phenylmethyl)phenoxy]-ethanamine monochloride
Phase Cancer # patients Design DosePhase I/II Advanced
refractory (various)48 Single arm tesmilifene with various antineoplastic
agents
Phase II Metastatic breast 98 (3 studies)
Single arm tesmilifene + ( anthracycline +/- taxane)
Phase II Breast / ovarian 70 Single arm tesmilifene + anthracycline + taxaneor tesmilifene + taxane
Phase II Hormone refractory prostate
29 Single arm tesmilifene + mitoxantrone + prednisone
Phase III (MA.19)Completed
Metastatic breast 305 Randomized tesmilifene + doxorubicin versus doxorubicin
Phase III (DEC)(pivotal)
Metastatic breast 723(fully enrolled)
Randomized(SPA)
tesmilifene + epirubicin + cyclophosphamideversus epirubicin + cyclophosphamide
MA.19 Phase III Study Conducted by NCIC
Reyno et al. (2004) J. Clin. Oncology, 22:269-276
Advanced / Metastatic Breast CancerN = 305 patients
tesmilifene+
doxorubicinN = 153
doxorubicinN = 152
Primary endpoints:Progression-Free Survival (PFS)Tumour Response (TR) (interim analysis)
Secondary endpoints:Overall Survival (OS) Quality of Life (QoL)
MA.19 Phase III Achieved a Large Overall Survival Benefit
Final Data Analysis
Primary Endpoint : Progression free survival/response rate– No significant difference
Secondary Endpoint : Overall survival
Tesmilifene / Doxorubicin : 23.6 monthsDoxorubicin = 15.6 months
50% increase in survival (p<0.03)J. Clin. Oncology (2004) 22: 269 -276
MA.19: Tesmilifene Most Effective in Rapidly Progressing Disease
Figure 1.1Survival Time (months) by Treatment
Duration of Disease : <=36 month
Source: /project/ym07teq/Aug02/g05.sas Programmer: hchen Run(23/09/2002 16:44)
Censored observations are indicated by dots
p=0.0016
Treatment :
DPPE/DOX (N= 91 Median= 29.7)DOX (N=100 Median= 12.2)
Per
cent
age
of P
atie
nts
(%)
0
20
40
60
80
100
Survival Time (months) 0 6 12 18 24 30 36
Survival Time (months)
Perc
enta
ge o
f Pat
ient
s (%
)Figure 1.2
Survival Time (months) by Treatment Duration of Disease : >36 month
Source: /project/ym07teq/Aug02/g05.sas Programmer: hchen Run(23/09/2002 16:44)
Censored observations are indicated by dots
p=0.7485
Treatment :
DPPE/DOX (N= 62 Median= 19.8)DOX (N= 50 Median= 28.6)
Perc
enta
ge o
f Pat
ient
s (%
)
0
20
40
60
80
100
Survival Time (months) 0 6 12 18 24 30 36
Survival Time (months)
Survival Time (months)
Perc
enta
ge o
f Pat
ient
s (%
)Pe
rcen
tage
of P
atie
nts
(%)
All patients (n = 305) Disease free interval ≥ 36 months (n = 112)
Disease free interval ≤ 36 months (n=191)
Patients with ≤ 36 months disease free interval
Overall survival:tesmilifene/doxorubicin = 29.7 months (n=91)
doxorubicin = 12.2 months (n=100)
>140% increase in overall survival (p=0.0016)
Tes / DoxDox
N.S. (p = 0.7485)
Tes / Dox (23.6 months)Dox (15.6 months)
> 50% increase (p<0.03)
Phase I PK Study Tesmilifene has no impact on doxorubicin metabolism
No Effect of Tesmilifene on PK of DoxorubicinPlasma Concentration of Doxorubicin With and Without Tesmilifene (n=5)
Preliminary Results
1
10
100
1000
10000
0 100 200 300 400 500 600 700 800 900 1000
Time (min)
Con
cent
ratio
n (n
g/m
L)
Treatment A Treatment B
Tesmilifene’s mode of action must accommodate the decoupling of early
and late endpoints
Cellular Hypothesis: Tesmilifene targets minority aggressive clones
Tesmilifene sensitizes “minority aggressive clones” to chemotherapy resulting in a decoupling of early and late endpoints in aggressive tumors
Drug sensitive tumor cellsDrug resistant minority clones (“aggressive”)
Chemotherapy Regrowth
RegrowthChemotherapy
Chemotherapy
+ TesmilifeneRegrowth
Similar “Apparent”Tumor Response
Differences in Overall Survival
Finding 1:Tesmilifene Selectively PotentiatesMultidrug Resistant (MDR) Cells to Anthracyclines, Taxanes, Vinca Alkaloids
Tesmilifene: Selectively Potentiates Drug Resistant Cells
Drug resistant cell lines = 13
Tesmilifene-mediated potentiationobserved in 12 cell lines
(92%)
Drug sensitive cell lines = 19
Tesmilifene-mediated potentiationObserved in only 3 cell lines
(15%)
32 cancer cell lines screenedTesmilifene + (anthracyclines, or
taxanes, or vinca alkaloids)
Potentiation observed in 15 cell lines(47%)
Tesmilifene : Selective Potentiation of Chemotherapy in MDR Cells
Broad Spectrum Potentiation of Natural Product Cytotoxics
MCF-7taxMCF-7XX
BreastDoxorubicin
Paclitaxel
HN5a/V15eHN5aXXXX
Head and NeckDoxorubicin
EpirubicinDocetaxel
Vinorelbine
BHK/pgp+BHKXXX
KidneyDoxorubicin
PaclitaxelVincristine
HCT116/mdr+HCT116XXX
ColonDoxorubicin
PaclitaxelVinblastine
Drug ResistantDrug SensitiveTissue Cytotoxic
MCF-7taxMCF-7XX
BreastDoxorubicin
Paclitaxel
HN5a/V15eHN5aXXXX
Head and NeckDoxorubicin
EpirubicinDocetaxel
BHK/pgp+BHKXXX
KidneyDoxorubicin
PaclitaxelVincristine
HCT116/mdr+HCT116XXX
ColonDoxorubicin
PaclitaxelVinblastine
Drug ResistantDrug SensitiveTissue Cytotoxic
X = no potentiation; = potentiation
Dose Dependent Potentiation ONLY in MDR+ Cells
0
20
40
60
80
100
120
0 0.2 0.4 0.6 0.8 1 1.2
Taxotere (nM)
Rel
ativ
e Pr
olife
ratio
n (%
con
trol
)
0 tesmilifene 2.5 µM tesmilifene 7.5 µM tesmilifene
0
20
40
60
80
100
120
0 0.5 1 1.5
Taxotere (nM)
Rel
ativ
e Pr
olife
ratio
n (%
con
trol
)
0 tesmilifene
2.5 uM tesmilifene
7.5 uM tesmilifene
Drug Sensitive HN-5a Cells(no effect)
Drug Resistant HN-5a/V15e Cells(potentiation)
0
20
40
60
80
100
120
0 10 20 30
Taxotere (nM)
Rel
ativ
e Pr
olife
ratio
n (%
con
trol
)
Tesmilifene : Broad Spectrum Chemopotentiation7.5 µM Tesmilifene is not cytotoxic but potentiates multiple chemotherapies
across various multidrug resistant cell lines
0
50
100
Control 7.5 uM tesmilifene 20 nM docetaxel 20 nM docetaxel +7.5 uM tesmilifene
Rel
ativ
e C
ell D
ensi
ty (%
of C
ontr
ol)
0
50
100
Control 7.5 uM tesmilifene 50 nM docetaxel 50 nM docetaxel +7.5 uM tesmilifene
Rel
ativ
e C
ell D
ensi
ty (%
of C
ontr
ol)
0
50
100
Control 7.5 uM tesmilifene 20 nM navelbine 20 nM navelbine +7.5 uM tesmilifene
Rel
ativ
e C
ell D
ensi
ty (%
of C
ontr
ol)
HN-5a/V15e Cells HN-5a/V200a Cells
MCF-7/V25a Cells
0
50
100
Control 7.5 uM tesmilifene 500 nM Epirubicin 500 nM Epirubicin +7.5 uM tesmilifene
Rel
ativ
e C
ell D
ensi
ty (%
of C
ontr
ol)
MCF-7/V25a Cells
Potentiation of Anthracyclines and Taxanes in P-gp Expressing Cells
Tesmilifene Mediated Potentiation in pgp+ BHK cells
0 100 200 300 400 500 600
Taxol
Doxorubicin
IC(50) nM
no Tes5 um Tes
5-fold
8-fold
Tesmilifene Mediated Potentiation in P-gp+ BHK Cells(Cells transfected to overexpress P-gp)
0 100 200 300 400 500 600
Taxol
Doxorubicin
IC(50) nM
no Tes5 µM Tes
5-fold
8-fold
Finding 2:Tesmilifene-mediated potentiation is associated with increased apoptosis
Tesmilifene : Enhances Cell Death via Apoptosis
Tesmilifene Decreases Cell Proliferationin Combination with Doxetaxel
Tesmilifene Increases Apoptotic Deathin Combination with Docetaxel
HN5a/V15e drug resistant cells
0
20
40
60
80
100
120
Rel
ativ
e C
ell D
ensi
ty (%
of C
ontr
ol)
0
5
10
15
20
25
30
35
40
% o
f Cel
ls w
ith A
nnex
inV
and
PI S
tain
ing
Docetaxel - - 7.5 nM 7.5 nM 20 nM 20 nMTesmilifene - 7.5 µM - 7.5 µM - 7.5 µM
Finding 3:Tesmilifene potentiates chemotherapy but DOES NOT INHIBIT efflux transporters such as P-glycoprotein
Tesmilifene : Does Not Enhance Daunorubicin Uptake
Daunorubicin uptake in drug resistant CEM/VLB 1000 (MDR+) cells is NOT enhanced by tesmilifene
Brandes et al (1994) J. Clin. Oncol. 12:1281-1290
daunorubicin (1 µg/mL) daunorubicin (1 µg/mL)+
verapamil (20 µmol/L)
Increased intracellular uptake
daunorubicin (1 µg/mL)+
tesmilifene (20 µmol/L)
no enhancement
Tesmilifene : Potentiation of Resistance to Doxorubicin
MCF-7 Breast Cancer Cells
MCF-7tax (P-gp overexpression)
MCF-7dox (MRP-1 overexpression)Chemotherapy
Drug SensitiveResistance Factor=
IC50 (MDR+ cells)
IC50 (sensitive cells)
0 10 20 30 40 50
Doxorubicin Resistance Factor
MCF-7dox
MCF-7tax
0 tesmilifene3 µM tesmilifene
BUT Tesmilifene Does Not Enhance Intracellular Doxorubicin Uptake
ParentalDrug Sensitive
Cell LineDrug Resistant Clones
0%
20%
40%
60%
80%
100%
120%
MCF-7 MCF-7dox MCF-7taxCel
lula
r Upt
ake
of D
oxor
ubic
in (%
con
trol
)
0 tesmilifene3 uM tesmilifene
Tesmilifene : Potentiation of Resistance to Paclitaxel
Resistance to Paclitaxel observed only in P-gp expressing MCF-7tax cell line
Tesmilifene potentiates paclitaxel in resistance MCF-7tax cell line
0 50 100 150 200
Paclitaxel Resistance Factor
MCF-7dox
MCF-7tax
0 tesmilifene3 µM tesmilifene
Tesmilifene does not Enhance Intracellular Paclitaxel uptake
0%
20%
40%
60%
80%
100%
120%
140%
MCF-7 MCF-7dox MCF-7tax
Cel
lula
r Pac
litax
elU
ptak
e (%
Con
trol
)
0 tesmilifene
3 µM tesmilifene
2 µM P-gp inhibitor
Finding 5:MDR cells, especially P-gp expressing cells, are “hypersensitive” to tesmilifene
Tesmilifene : Enhanced Cytotoxicity against Drug Resistant Cells
MDR+ Sensitivity to Tesmilifene
0
25
50
75
100
125
0 20 40 60 80
Tesmilifene concentration (uM)
Rel
ativ
e Pr
olife
ratio
n (a
vera
ge %
con
trol
)
HN5a (sensitive) HN-5a/V15e (resistant)line HN5a line HN5a/V13e
Tesmilifene is more cytotoxic to drug resistant HN-5a/V15e cells compared with drug sensitive HN-5a parental cells.
HN-5a (sensitive)
HN-5a/V15e(resistant)
Tesmilifene : Enhanced Cytotoxicity against Drug Resistant Cells
Cell Line P-gp Expression Resistance to Chemotherapy
Tesmilifene IC50(µM)
AUXB1 - Sensitive 40 – 50
C5 ++ Moderate Resistance 5 – 8
B30 +++ High Resistance 2 – 4
I10 -C5 Revertant
(Sensitive)~ 40
C5/Bcl-2 ++ Resistant >20
Finding 6:Tesmilifene-mediated Collateral Sensitivity Appears to be Dependent on ATPase Activity
Tesmilifene : Hypersensitivity in MDR Cells is ATPase dependent
0
20
40
60
80
100
120
0.1 1 10 100
Tesmilifene (uM)%
Cel
l Sur
viva
l
0
20
40
60
80
100
120
0.1 1 10 100
Tesmilifene (uM)
% C
ell S
urvi
val
Drug Sensitive AUXB1Drug Resistant C5
0 µM PSC 833
2 µM PSC 833
Drug resistant cells are hypersensitive to
tesmilifene-mediated cytotoxicity
PSC 833, an potent P-gp inhibitor,eliminates hypersensitivity
Finding 7:Tesmilifene activates the ATPase of P-glycoprotein in an in vitro model
Tesmilifene : Mediates Activation of ATPase in P-gp
Tesmilifene enhances ATPase activitytrans-(z)- Flupentixol (P-gp inhibitor) inhibits ATPase activity
ATP ADP + PiP-gp : lipid mixture+/- test drug
0
2
4
6
8
10
12
14
16
Control (no drug)
3 mMcolchicine
1 mMtesmilifene
3 mMcolchicine
+ 1 mMtesmilifene
3 mMcolchicine
+ 0.5 mMT-flupentixol
ATP
ase
Act
ivity
(F
old
Stim
ulat
ion
Rel
ativ
e to
Con
trol
)
Finding 8:Tesmilifene exposure induces elevation in reactive oxygen species (ROS) more readily in MDR+ cells
Tesmilifene: Induces ROS levels
Tesmilifene induces ROS levels in both cell lines.
However, in drug resistant tumor cells C5, this induction occurs at lower concentrations of tesmilifene.
0
5
10
15
20
25
30
35
0 5 10 25 50Concentration of Tesmilifene (µM)
% M
TT re
duct
ion
by O
2(r
elat
ive
to c
ontr
ol)
AuxB1C5
Overview of Cellular ATP Production
Tesmilifene: Mode of Action Summary
TesmilifeneSelectively targets MDR tumor cells, including P-gp expressing cells
Potentiates broad range of chemotherapies in various cancer cell types
Activates P-glycoprotein ATPase activity
Is not a P-gp inhibitor
Selectivity for multi-drug resistant cells is antagonized by P-glycoprotein
inhibitors
Increases intracellular reactive oxygen species levels
Induces cell death of multi-drug resistant cells via apoptosis
TesmilifeneClinical Opportunities and
Development Plans
Drug Resistance : The Limitation of Chemotherapy
Prevalence of P-gp Phenotype
% Patients with P-gp Phenotype Pre and Post ChemotherapyLeonard et al (2003) The Oncologist 8:411-424
0%
20%
40%
60%
80%
100%
AML Myeloma BreastCancer
Ovarian Bladder
Pre Treatment
Post Treatment
Targeting Multi-Drug Resistance: Efflux Pumps as Targets
• Search for cytotoxic drugs that are not substrates of efflux pumps– But promiscuity of efflux pumps, especially P-gp, has been difficult of
overcome
• Inhibitors of efflux pumps– In clinical studies, P-gp inhibitors have been associated with unacceptable
increases in toxicity and complicated pharmacokinetic interactions
• Drugs that specifically kill pump-expressing tumor cells by exploiting the Achilles heel of efflux pumps
– Collateral sensitivity of pump expressing cells – Impact of increase energy demands on tumor cells
Strategies
1
2
3
DEC Study: Pivotal Phase III (ongoing)The DEC StudyA Phase III study of Tesmilifene / Epirubicin / Cyclophosphamide versus Epirubicin / Cyclophosphamide as First-line Treatment in Metastatic/Recurrent Breast Cancer
Design (SPA)– Open label– Randomized– Sequential analysis design
Inclusion Criteria– Patients with rapidly progressing disease (DFI < 36 months)
Primary Endpoint– Overall survival
• Fully enrolled :– 723 patients randomized– 103 study centres in 18 countries– Last Patient In – September 2005
• Powered overall to detect a 33% increase in overall survival (trial may go positive earlier if higher differential survival emerges on pre-planned interim analyses).
Status
Tesmilifene Clinical Development Program
Completed, Ongoing, Planned Studies
Pre-Clinical Phase I Phase II Phase III Pivotal
-
Metastatic Breast (MA.19)-
Metastatic Breast (DEC)
Metastatic Breast (Taxotere)
HR Prostate – Ph II/III
Adjuvant Breast Cancer Ph III
Gastric – Ph II
Pre-
-
HR Prostate
Tesmilifene : Mode of Action
Cellular Hypothesis
Tesmilifene sensitizes “minority aggressive clones” to chemotherapy
Mechanism Hypothesis: 4 key elements1. Tesmilifene targets multidrug resistant (MDR) phenotypes
2. Mechanism does not involve inhibition of transporter, rather activation
3. Concommitant binding of chemotherapy and tesmilifene stimulates ATP comsumption
4. Dependence on Warburg-type metabolism makes cancer cells vulnerable to increased demand on ATP.
Tesmilifene: Mode of ActionQ&A