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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Testosterone ODM-201 ORM-15341

Bicalutamide Enzalutamide ARN-509

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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

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ntr

ol

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60

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ECC 2013 Abstract E17-2119