Testosterone ODM-201 ORM-15341
Bicalutamide Enzalutamide ARN-509
Background
Materials and Methods
Conclusions
ResultsActivation of androgen receptor (AR) signaling is crucial for prostate cancer growth at all stages of the disease. Also castration resistant prostate cancer (CRPC) is still dependent on AR and is often characterized by AR overexpression and resistance to conventional antiandrogens such as bicalutamide. ODM-201 is a novel, new generation antiandrogen that has unique properties and superior efficacy in preclinical models.
•
•
•
•
•
ODM-201 is a new generation AR antagonist with
Superior affinity and activity to wild-type AR Inhibition of
androgen-mediated nuclear translocation of AR
Excellent antiandrogenic as well as antitumor activity both in
the in vitro and in vivo models of CRPC
No brain entry or testosterone elevation in preclinical models -
low seizure risk
Low potential for CYP mediated drug-drug interactions
The studies were sponsored by Orion Corporation,
Orion Pharma and Endo Pharmaceuticals.
AR binding affinity: Binding affinity to wild type AR was determined in cytosolic lysates obtained from ventral prostates of castrated rats using a competition binding assay.
Antagonism of ODM-201: Functional activity and potency to hAR were determined in HEK293 cells stably transfected with full-length hAR and androgen-responsive luciferase reporter gene constructs. The cells were treated with test compound and 0.45 nM testosterone in steroid depleted media for 24 h and luciferase activity was measured. AR agonism and antagonism in hAR overexpressing cells: Functional activity of the test compounds was determined in HEK293 cells stably overexpressing hAR and androgen-responsive luciferase reporter gene construct. To measure agonism, cells were cultured in steroid-depleted media treated with test compounds (0.3 µM) for 24 h and luciferase activity was measured. To measure antagonism, the cells were treated with test compounds (1 uM) and testosterone (0.3 nM).
VCaP proliferation assay: Androgen-sensitive VCaP prostate cancer cells containing endogenous AR gene amplification were treated with sub-maximal concentration (0.1 nM) of mibolerone in steroid depleted growth medium. Cell growth was measured using WST-1 Cell Proliferation Assay (Roche) according to manufacturer's instructions.
AR nuclear translocation: AR overexpressing HEK293 cells were treated with 1 µM test compounds together with 0.3 nM testosterone in steroid depleted medium. AR subcellular localisation was studied by immunolabeling of AR with polyclonal AR ab (N-20) (Santa Cruz). DNA was labeled with DAPI. Cells were either imaged with Cellomics Arrayscan VTI (Thermo) and analyzed with NucTrans.V3 Assay Algorithm (Thermo), or imaged with Zeiss LSM780 confocal microscope.
Castration resistant VCaP xenograft: Tumors were established by subcutaneous injection of VCaP cells into male nude mice. After initial
3tumor growth, when the average tumor volume reached ~200 mm , mice were castrated. Treatment was initiated upon tumor regrowth. Mean tumor volumes were calculated for each treatment group.
Testosterone analyses: Serum testosterone levels were measured from intact nude male mice with VCaP orthotopic tumors after 3 week treatment of test compounds using RIA (radioimmune assay) method.
Brain/plasma ratios: ODM-201 and ORM-15341 concentrations were studied in mouse plasma and brain homogenates after 6-day oral dosing of ODM-201 25-100 mg/kg BID. Enzalutamide concentrations were studied after 6-day oral dosing of 10 mg/kg QD and ARN-509 concentrations after single oral dose of 10 mg/kg QD. AUC values for 0-24
plasma and brain were determined and brain/plasma ratio was calculated.
Effect on GABA receptors: Whole-cell patch-clamp recordings from A
acutely isolated rat striatal neurones were performed at room temperature with currents evoked at a set interval by brief exposure to 100 µM GABA.
CYP3A4 induction: HepaRG cells were treated with 10 µM concentration of test compounds with rifampicin as a positive control. The levels of CYP3A4 mRNA were used for evaluation of CYP induction potential.
CYP inhibition: Inhibition of CYP enzyme activities was determined by following CYP isoform specific marker reactions with LC/MS.
ODM-201 ORM-15341Main Metabolite
* Partial agonism
Compound
ODM-201
Main metabolite of ODM-(ORM-15341)
201
Bicalutamide
Enzalutamide
ARN-509
AR affinityKi (nM)
9
8
12
39
53
AntagonismWT AR
IC50 (nM)
65
25
150
155
168 c) Effect of ODM-201, its metabolite and enzalutamide on GABA A
receptors
* Seizures reported
*Refs. Clegg et al, Cancer Research 2012; Forster et al, Prostate 2011**Rat autoradiography (QWBA confirms brain/plasma ratio of 14C-ODM-201 related radioactivity was 0.04-0.06, indicating negligible penetration to the brain
ODM-201 + Mainmetabolite 3% **Enzalutamide 19%*
ARN-509 29%*
b) Brain/plasma ratio of ODM-201, its metabolite and other antiandrogens in mouseODM-201 has low brain/plasma ratio
7. ODM-201 has very low potential for CYP - mediated drug-drug interactions
a) ODM-201 does not increase serum testosterone levels in mouseSerum testosterone levels in mice with VCaP orthotopic tumors after 3-wk dosing (mean ± SD)
a) Inhibition of AR nuclear translocation by ODM-201, its metabolite and other antiandrogens in AR overexpressing cells
c) AR expression in cell lines
Comparison of AR binding affinity and antagonism to other antiandrogens
Inhibition of androgen-induced VCaP cell proliferation by ODM-201, its metabolite and other antiandrogens
a) Agonism of ODM-201, its metabolite and other antiandrogens for hAR in AR overexpressing cells
b) Antagonism of ODM-201, its metabolite and other antiandrogens for hAR in AR overexpressing cells
b) Representative confocal microscopic images of AR overexpressing cells treated with or without testosterone combined with indicated antiandrogens.
0.00
10.00
20.00
30.00
40.00
50.00
60.00 S -Testosterone (nmol/l)
Vehicle (MPG) Enzalutamide 20 mg/kg qd p.o. ODM-201 50 mg/kg bid p.o.
* In CRPC patients, enzalutamide reported to increase bone marrow testosterone (Ref. Efstathiou et al., 2011 J Clin Oncol abstract)
6.Low or negligible seizure risk with ODM-201
5. Superior inhibition of tumor growth by ODM-201 in castration resistant mouse VCaP xenograft model
4. Inhibition of AR nuclear translocation by ODM-201
1. Superior potency of ODM-201 and its active metabolite to AR
2. Potent activity of ODM-201 in VCaP prostate cancer cells
3. Potency of ODM-201 in AR overexpressing cells
0
200
400
600
800
1000
1200
1400
1600
14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119
Tumor size (mm3, mean ± SEM)
***
*
SHAM
ORX
ORX + Enzalutamide 20 mg/kg qd
ORX + ODM-201 50 mg/kg qd
ORX ODM-201 + 50 mg/kg bid
SHAM / ORX
Treatment
S.C. inoculation of VCaP prostate ca cells
Castration / ORX Start of oral Treatment Euthanasia
p=0.0245enzalutamide vs ODM-201 50 bid
~8 wks ~4wks ~37days
***: p<0.001
** : p<0.01
* : p<0.005
vs ORX over the treatment time
Days after Inoculation
Testosterone ODM-201 ORM-15341
Bicalutamide Enzalutamide ARN-509
0
20
40
60
80
100
120
AR
nu
cle
ar
loc
ali
sa
tio
n (
% o
f c
on
tro
l)
DMSO
Bic+T ODM-201 + T
Testosterone
b) ODM-201 shows no CYP inhibition
a) ODM-201 and its metabolite ORM-15341 show no CYP3A4 induction
*In SPC: Enzalutamide is a strong CYP3A4 inducer
ODM-201 – New generation antiandrogen with excellent antiandrogenic and antitumor activity in nonclinical models of CRPC
Anu Moilanen, Reetta Riikonen, Riikka Oksala, Laura Ravanti, Eija Aho, Gerd Wohlfahrt, Päivi Taavitsainen, Olli Törmäkangas, Pekka J. Kallio. Orion Corporation Orion Pharma, Finland
AR-HEK HS-HEK VCaP
AR
AR-HEK: HEK293 cells stably transfected with AR HS-HEK: HEK293 cells stably transfected to overexpress ARVCaP: Prostate cancer cell line derived from a bone metastasis of a patient with CRPC and containing endogenous AR gene amplification
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0
20
40
60
80
100
120
Testosterone ODM-201 ORM-15341
Bicalutamide Enzalutamide ARN-509
0
20
40
60
80
100
120
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ECC 2013 Abstract E17-2119
Poster presented at the European Cancer Congress, Amsterdam, 27 September – 1 October 2013
CYP 1A2 2A6 3A4 2B6 2C8 2C9 2C19 2D6 2E1
CYP inhibition by ODM-201 in human liver microsomes; IC50 (µM)
>100 >100 >100 >100 >100 30 64 82No
inhibition
Rifampicin ODM-201 ORM-15341 Enzalutamide ARN-509
CYP3A4induction inHepa RGcells at 10 µM
Yes No No Yes* Yes
Assay ODM-201 ORM-15341 Enzalutamide
GABA current
inhibition in rat striatal
neurons (IC50)
5.5 uM 3.0 uM 5.2 uM*
3%
3%
22%
62%
ODM-201
ORM-15341
Enzalutamide
ARN-509
Compound
ODM-201
ORM-15341
Bicalutamide*
Enzalutamide
ARN-509
Prolifer VCaPIC50 (µM)
0.5
0.6
1.2*
0.4
0.3
ODM-201 – New generation antiandrogen with excellent antiandrogenic and antitumor activity in nonclinical models of CRPC
Anu Moilanen, Reetta Riikonen, Riikka Oksala, Laura Ravanti, Eija Aho, Gerd Wohlfahrt, Päivi Taavitsainen, Olli Törmäkangas, Pekka J. Kallio. Orion Corporation Orion Pharma, Finland
Testosterone ODM-201 ORM-15341
Bicalutamide Enzalutamide ARN-509
Testosterone ODM-201 ORM-15341
Bicalutamide Enzalutamide ARN-509
Background
Materials and Methods
Conclusions
ResultsActivation of androgen receptor (AR) signaling is crucial for prostate cancer growth at all stages of the disease. Also castration resistant prostate cancer (CRPC) is still dependent on AR and is often characterized by AR overexpression and resistance to conventional antiandrogens such as bicalutamide. ODM-201 is a novel, new generation antiandrogen that has unique properties and superior efficacy in preclinical models.
•
•
•
•
•
ODM-201 is a new generation AR antagonist with
Superior affinity and activity to wild-type AR Inhibition of androgen-mediated nuclear translocation of AR
Excellent antiandrogenic as well as antitumor activity both in
the in vitro and in vivo models of CRPC
No brain entry or testosterone elevation in preclinical models - low seizure risk
Low potential for CYP mediated drug-drug interactions
The studies were sponsored by Orion Corporation,
Orion Pharma and Endo Pharmaceuticals.
AR binding affinity: Binding affinity to wild type AR was determined in cytosolic lysates obtained from ventral prostates of castrated rats using a competition binding assay.
Antagonism of ODM-201: Functional activity and potency to hAR were determined in HEK293 cells stably transfected with full-length hAR and androgen-responsive luciferase reporter gene constructs. The cells were treated with test compound and 0.45 nM testosterone in steroid depleted media for 24 h and luciferase activity was measured. AR agonism and antagonism in hAR overexpressing cells: Functional activity of the test compounds was determined in HEK293 cells stably overexpressing hAR and androgen-responsive luciferase reporter gene construct. To measure agonism, cells were cultured in steroid-depleted media treated with test compounds (0.3 µM) for 24 h and luciferase activity was measured. To measure antagonism, the cells were treated with test compounds (1 uM) and testosterone (0.3 nM).
VCaP proliferation assay: Androgen-sensitive VCaP prostate cancer cells containing endogenous AR gene amplification were treated with sub-maximal concentration (0.1 nM) of mibolerone in steroid depleted growth medium. Cell growth was measured using WST-1 Cell Proliferation Assay (Roche) according to manufacturer's instructions.
AR nuclear translocation: AR overexpressing HEK293 cells were treated with 1 µM test compounds together with 0.3 nM testosterone in steroid depleted medium. AR subcellular localisation was studied by immunolabeling of AR with polyclonal AR ab (N-20) (Santa Cruz). DNA was labeled with DAPI. Cells were either imaged with Cellomics Arrayscan VTI (Thermo) and analyzed with NucTrans.V3 Assay Algorithm (Thermo), or imaged with Zeiss LSM780 confocal microscope.
Castration resistant VCaP xenograft: Tumors were established by subcutaneous injection of VCaP cells into male nude mice. After initial
3tumor growth, when the average tumor volume reached ~200 mm , mice were castrated. Treatment was initiated upon tumor regrowth. Mean tumor volumes were calculated for each treatment group.
Testosterone analyses: Serum testosterone levels were measured from intact nude male mice with VCaP orthotopic tumors after 3 week treatment of test compounds using RIA (radioimmune assay) method.
Brain/plasma ratios: ODM-201 and ORM-15341 concentrations were studied in mouse plasma and brain homogenates after 6-day oral dosing of ODM-201 25-100 mg/kg BID. Enzalutamide concentrations were studied after 6-day oral dosing of 10 mg/kg QD and ARN-509 concentrations after single oral dose of 10 mg/kg QD. AUC values for 0-24
plasma and brain were determined and brain/plasma ratio was calculated.
Effect on GABA receptors: Whole-cell patch-clamp recordings from A
acutely isolated rat striatal neurones were performed at room temperature with currents evoked at a set interval by brief exposure to 100 µM GABA.
CYP3A4 induction: HepaRG cells were treated with 10 µM concentration of test compounds with rifampicin as a positive control. The levels of CYP3A4 mRNA were used for evaluation of CYP induction potential.
CYP inhibition: Inhibition of CYP enzyme activities was determined by following CYP isoform specific marker reactions with LC/MS.
Compound
ODM-201
Main metabolite of ODM-(ORM-15341)
201
Bicalutamide
Enzalutamide
ARN-509
AR affinityKi (nM)
9
8
12
39
53
AntagonismWT AR
IC50 (nM)
65
25
150
155
168
ODM-201 ORM-15341Main Metabolite
Compound
ODM-201
ORM-15341
Bicalutamide*
Enzalutamide
ARN-509
Prolifer VCaPIC50 (µM)
0.5
0.6
1.2*
0.4
0.3
* Partial agonism
Assay ODM-201 ORM-15341 Enzalutamide
GABA current
inhibition in rat striatal
neurons (IC50)
5.5 uM 3.0 uM 5.2 uM*
c) Effect of ODM-201, its metabolite and enzalutamide on GABA A
receptors
* Seizures reported
*Refs. Clegg et al, Cancer Research 2012; Forster et al, Prostate 2011**Rat autoradiography (QWBA confirms brain/plasma ratio of 14C-ODM-201 related radioactivity was 0.04-0.06, indicating negligible penetration to the brain
ODM-201 + Mainmetabolite 3% **Enzalutamide 19%*
ARN-509 29%*
b) Brain/plasma ratio of ODM-201, its metabolite and other antiandrogens in mouseODM-201 has low brain/plasma ratio
3%
3%
22%
62%
ODM-201
ORM-15341
Enzalutamide
ARN-509
7. ODM-201 has very low potential for CYP - mediated drug-drug interactions
a) ODM-201 does not increase serum testosterone levels in mouseSerum testosterone levels in mice with VCaP orthotopic tumors after 3-wk dosing (mean ± SD)
a) Inhibition of AR nuclear translocation by ODM-201, its metabolite and other antiandrogens in AR overexpressing cells
c) AR expression in cell lines
Comparison of AR binding affinity and antagonism to other antiandrogens
Inhibition of androgen-induced VCaP cell proliferation by ODM-201, its metabolite and other antiandrogens
a) Agonism of ODM-201, its metabolite and other antiandrogens for hAR in AR overexpressing cells
b) Antagonism of ODM-201, its metabolite and other antiandrogens for hAR in AR overexpressing cells
b) Representative confocal microscopic images of AR overexpressing cells treated with or without testosterone combined with indicated antiandrogens.
0.00
10.00
20.00
30.00
40.00
50.00
60.00 S -Testosterone (nmol/l)
Vehicle (MPG) Enzalutamide 20 mg/kg qd p.o. ODM-201 50 mg/kg bid p.o.
* In CRPC patients, enzalutamide reported to increase bone marrow testosterone (Ref. Efstathiou et al., 2011 J Clin Oncol abstract)
6.Low or negligible seizure risk with ODM-201
5. Superior inhibition of tumor growth by ODM-201 in castration resistant mouse VCaP xenograft model
4. Inhibition of AR nuclear translocation by ODM-201
1. Superior potency of ODM-201 and its active metabolite to AR
2. Potent activity of ODM-201 in VCaP prostate cancer cells
3. Potency of ODM-201 in AR overexpressing cells
0
200
400
600
800
1000
1200
1400
1600
14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119
Tumor size (mm3, mean ± SEM)
***
*
SHAM
ORX
ORX + Enzalutamide 20 mg/kg qd
ORX + ODM-201 50 mg/kg qd
ORX ODM-201 + 50 mg/kg bid
SHAM / ORX
Treatment
S.C. inoculation of VCaP prostate ca cells
Castration / ORX Start of oral Treatment Euthanasia
p=0.0245enzalutamide vs ODM-201 50 bid
~8 wks ~4wks ~37days
***: p<0.001
** : p<0.01
* : p<0.005
vs ORX over the treatment time
Days after Inoculation
Testosterone ODM-201 ORM-15341
Bicalutamide Enzalutamide ARN-509
0
20
40
60
80
100
120
AR
nu
cle
ar
loc
ali
sa
tio
n (
% o
f c
on
tro
l)
DMSO
Bic+T ODM-201 + T
Testosterone
CYP 1A2 2A6 3A4 2B6 2C8 2C9 2C19 2D6 2E1
CYP inhibition by ODM-201 in human liver microsomes; IC50 (µM)
>100 >100 >100 >100 >100 30 64 82No
inhibition
b) ODM-201 shows no CYP inhibition
a) ODM-201 and its metabolite ORM-15341 show no CYP3A4 induction
Rifampicin ODM-201 ORM-15341 Enzalutamide ARN-509
CYP3A4induction inHepa RGcells at 10 µM
Yes No No Yes* Yes
*In SPC: Enzalutamide is a strong CYP3A4 inducer
AR-HEK HS-HEK VCaP
AR
AR-HEK: HEK293 cells stably transfected with AR HS-HEK: HEK293 cells stably transfected to overexpress ARVCaP: Prostate cancer cell line derived from a bone metastasis of a patient with CRPC and containing endogenous AR gene amplification
% o
f co
ntr
ol
0
20
40
60
80
100
120
0
20
40
60
80
100
120
% o
f co
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ol
ECC 2013 Abstract E17-2119