マルチターゲット型
新規去勢抵抗性前立腺がん治療薬の開発岐阜薬科大学
生命薬学大講座 生化学研究室
准教授 遠藤 智史
1

去勢抵抗性前立腺がん (CRPC)【前立腺がん】• 2017年以降、本邦で男性で最も高い罹患率を示す。
• 進行が比較的遅く、10年生存率が95％を超える予後の良いがんである。
〈想定されているCRPCへの移行機序〉• 副腎でのアンドロゲン産生亢進
• Androgen Receptor (AR) の遺伝子増幅• AR遺伝子変異によるフルタミドなどの第一世代抗アンドロゲン薬のアゴニストへの変換• 増殖シグナルの亢進によるARリン酸化• 多くの症例でホルモン療法不応性の去勢抵抗性前立腺がん (CRPC) に移行する。
DHEA

精巣

副腎

Testosterone
AKR1C3
DHT

AR
遺伝子増幅

成長因子

抗アンドロゲン薬

AR

AR

変異ARAKTMAPKPARAR↑PSA ↑Growth ↑Survival

PARAR

2
Satoshi Endo, PhD, Gifu Pharmaceutical University

前立腺におけるアンドロゲン合成

Cholesterol
TestisAdrenal gland

DHEA-S

5%

DHEA
route

Traditional
pathway95%

Denmeade SR, et al.: Nat Rev Cancer 2: 389-396, 2002.,
Mostaghel E, et al.: Best Pract Res Clin Endocrinol Metab 22: 243-258, 2008. modified.

Testosterone

Back-door
pathway

5α-Reductase

Testosterone

DHT

5α-Androstane-3α,17β-diol

P

Glucuronate
conjugation

PARAR

↑PSA

↑Growth↑Survival

Prostate
3
Satoshi Endo, PhD, Gifu Pharmaceutical University前立腺がん細胞におけるアンドロゲン合成

Cholesterol
TestisAdrenal gland

DHEA-S
DHEA
route

Traditional
pathway

Denmeade SR, et al.: Nat Rev Cancer 2: 389-396, 2002.,
Mostaghel E, et al.: Best Pract Res Clin Endocrinol Metab 22: 243-258, 2008. modified.

Testosterone

Back-door
pathway

5α-Reductase

Cholesterol

Testosterone

DHT

5α-Androstane-3α,17β-diol

P

Glucuronate
conjugation

PARAR

↑PSA

↑Growth↑Survival

Prostate cancer
4
Satoshi Endo, PhD, Gifu Pharmaceutical UniversityCRPC治療薬の作用機序

Cholesterol
TestisAdrenal gland

DHEA-S
DHEA
route

Traditional
pathway

Denmeade SR, et al.: Nat Rev Cancer 2: 389-396, 2002.,
Mostaghel E, et al.: Best Pract Res Clin Endocrinol Metab 22: 243-258, 2008. modified.

Testosterone

Abiraterone(CYP17A1inhibitor)

Back-door
pathway

5α-Reductase

Cholesterol

Testosterone

DHT

5α-Androstane-3α,17β-diol

PP
AR

Enzalutamide(AR antagonist)

Glucuronate
conjugation

AR

↑PSA

↑Growth↑Survival

Prostate cancer
5Satoshi Endo, PhD, Gifu Pharmaceutical University

CRPC治療薬の作用機序
feedback
Cholesterol
ACTH
21α-Hydroxylase

HSD
3B1,2

Pregnenolone
CYP17A1

17α-Hydroxy-pregnenolone
CYP17A1

Progesterone
HSD
3B1,2

HSD
3B1,2

DHEA
AKR1C35-Androstene-3β,17β-diol

CYP17A1

17α-Hydroxy-progesterone
CYP17A1

Androstene-dioneAKR1C3HSD
3B1,2

Testosterone

11β-Hydroxylase

11-Deoxycorticosterone
21α-Hydroxylase

Corticosterone

11β-Hydroxylase

11-Deoxycortisol

CortisolAbiraterone(CYP17A1 innibitor)
Side effects of abiraterone(Hypokalemia, fulminant hepatitis,liver failure)
6
Satoshi Endo, PhD, Gifu Pharmaceutical University

CRPC治療薬の作用機序

Cholesterol
TestisAdrenal gland

DHEA-S
DHEA
route

Traditional
pathway

AKR1C3
inhibitors

Testosterone

Abiraterone(CYP17A1inhibitor)

Back-door
pathway

5α-Reductase

Cholesterol

Testosterone

DHT

5α-Androstane-3α,17β-diolPPAR

Enzalutamide(AR antagonist)

Glucuronate
conjugation

AR↑PSA↑Growth↑Survival

Prostate cancer
7
Satoshi Endo, PhD, Gifu Pharmaceutical University

アルドケト還元酵素 (AKR) 1C3
1.

AKR1C3 highly expresses in CRPC tissues

The overexpression of AKR1C3 has been suggested to depend on activation of Nrf2-keap1
signaling via oxidative stress.

【Expression levels ofsteroidogenic enzymes(CRPC / PC patients)】
GeneFoldchange

CYP17A1

16.1

3BHSD1

8.5

3BHSD2

7.5

AKR1C1

2.7

AKR1C2

1.1

AKR1C38.0

SRD5A12.63

SRD5A2-9.4

Montgomery et al. Cancer Res., 2008;68: 4447-4454.

【AKR1C3 expression in clinical samples】

Primary PCa

CRPC-strong

CRPC-medium

CRPC-week

Hamid et al., Mol. Med., 2012;18: 1449-1455.

8
Satoshi Endo, PhD, Gifu Pharmaceutical Universityアルドケト還元酵素 (AKR) 1C31.

2.

AKR1C3 highly expresses in CRPC tissues

The overexpression of AKR1C3 has been suggested to depend on activation of Nrf2-keap1
signaling via oxidative stress.

AKR1C3 participates in proliferation and survival of prostate cancer cells•

Androgen synthesis (Androstendione → Testosterone)•Suppression of synthesis of anti-proliferative prostaglandin (PGD₂ → 9α,11β-PGF2)•Detoxification of endogenous reactive aldehydes (4-Hydroxynonenal → 4-Hydroxynonenol)【Tumor sizes in LNCaP-bearing miceexpressing AKR1C3 or vector alone】

Yepuru et al., Clin. Cancer Res., 2013;19: 5613-23.

9Satoshi Endo, PhD, Gifu Pharmaceutical University

AKR1C3阻害剤の臨床開発
1. ASP9521 (Astellas): Discontinuation of clinical trial;

Phase I/II, Multi-center, Open Label, Study to Assess the Safety, Tolerability,
Pharmacokinetics, Pharmacodynamics and Anti Tumor Activity of ASP9521 inPatients With Metastatic Castrate-resistant Prostate Cancer
CWR22R xenograft mice (castrated)
ASP9521

1hAndrostenedione
1hEIA

Testosterone concentration

AKR1C3-expressing LNCaP cells
SeedingAndrostenedione + ASP9521
6 daysELISA / Viability assay

PSA concentration

Cell proliferation

Kikuchi et al., Invest New Drugs 2014;32: 995-1004.

10
Satoshi Endo, PhD, Gifu Pharmaceutical University

AKR1C3阻害剤の臨床開発
1. ASP9521 (Astellas): Discontinuation of clinical trial;

Phase I/II, Multi-center, Open Label, Study to Assess the Safety, Tolerability,Pharmacokinetics, Pharmacodynamics and Anti Tumor Activity of ASP9521 inPatients With Metastatic Castrate-resistant Prostate Cancer
PSA concentration (at week 13)[Discussion in the paper]
There was no evidence of clinical activity of ASP9521 in thisstudy. ....... We might hypothesis that AKR1C3 expression wasinsufficient to observe the effects of inhibition by ASP9521
in these patients. To text this hypothesis, AKR1C3 expression
levels should be measured in the target tissue.

Loriot et al., Invest New Drugs 2014;32: 995-1004.

11Satoshi Endo, PhD, Gifu Pharmaceutical University

AKR1C3阻害剤の臨床開発
1. ASP9521 (Astellas): Discontinuation of clinical trial;

Phase I/II, Multi-center, Open Label, Study to Assess the Safety, Tolerability,
Pharmacokinetics, Pharmacodynamics and Anti Tumor Activity of ASP9521 inPatients With Metastatic Castrate-resistant Prostate Cancer

We decided to withdraw the development of ASP9521
from the results of the prostate cancer Phase I clinical
trial and business strategy. (in the website of Astellas)

2. BAY1128688 (Bayer) : Discontinuation of clinical trial;

A Randomized, Placebo-controlled, Double-blind, Parallel-group, Multi-center,
Exploratory Dose-response Study to Assess the Efficacy and Safety of Different
Oral Doses of BAY1128688 in Women With Symptomatic Endometriosis Over a
12-week Treatment Period

Recently, the phase II clinical trial was terminated due tohepatotoxicity, which might have resulted from thenonspecific actions of BAY1128688, possibly due toinhibition of AKR1D1 and consequent bile acid deficiency.
(Rizner et al., Pharmacol. Res., 2020;152:104446)
12Satoshi Endo, PhD, Gifu Pharmaceutical Universityクロメン誘導体によるAKR1C3阻害活性

We previously discovered compoundswith a chromene moiety showing
inhibitory potency for AKR1C3. Novelderivatives based on it were synthesized.

65417328Compounds

R1IC50 (nM)

4d8-OH

25 ± 2.055-OH

370 ±1066-OH> 10,000

77-OH> 10,000

88-OMe> 10,000

The hydroxyl group at position 8 of the chromene ringis important for the inhibitory activity

13

Satoshi Endo, PhD, Gifu Pharmaceutical University

８-ヒドロキシクロメン誘導体
Compounds
4a4b4c4d4e4f4g4h4i4j4k4l4m4n4o4p4q4r4s4t4u4v4w4xR2H
2-Fluoro
3-Fluoro
4-Fluoro
2-Chloro
3-Chloro
4-Chloro
2-Methyl
3-Methyl
4-Methyl
2-Ethyl
3-Ethyl
4-Ethyl2-Hydroxy
3-Hydroxy
4-Hydroxy
3,4-Difluoro
3,4,5-Trifluoro
2-Trifluoromethyl
3-Trifluoromethyl
4-Trifluoromethyl
2,4-Ditrifluoromethyl
2-Isopropyl
3-Isopropyl

IC50 (nM)
36 ± 0.564 ± 529 ± 325 ± 256 ± 7
46 ± 0.752 ± 452 ± 5.555 ± 0.9
27 ± 0.8
21 ± 0.7
29 ± 0.545 ± 0.5>10,000
60 ± 0>10,000
220 ± 3047 ± 0.5
20 ± 0.1
50 ± 0.3
24 ± 0.1
58 ± 0.1
30 ± 2.2
47 ± 0.3

R2Ser308

84j

Trp227

Tyr317

Phe306

Tyr319NADP+Phe311

4j

Tyr24

Met1202.8Å

3.0Å

Asn167
Tyr55

Trp86

Leu54
His117〔Putative interactions〕• A hydrogen bond between 8-OH groupof 4j and catalytic residues of AKR1C3(Tyr55 and His117)• A π-π stacking interactions between abenzene ring of R2 moiety and Phe306
14
Satoshi Endo, PhD, Gifu Pharmaceutical University

８-ヒドロキシクロメン誘導体
Compounds
4a4b4c4d4e4f4g4h4i4j4k4l4m4n4o4p4q4r4s4t4u4v4w
4xR2H
2-Fluoro
3-Fluoro
4-Fluoro
2-Chloro
3-Chloro
4-Chloro
2-Methyl
3-Methyl
4-Methyl
2-Ethyl
3-Ethyl
4-Ethyl2-Hydroxy
3-Hydroxy
4-Hydroxy
3,4-Difluoro
3,4,5-Trifluoro
2-Trifluoromethyl
3-Trifluoromethyl
4-Trifluoromethyl
2,4-Ditrifluoromethyl
2-Isopropyl
3-Isopropyl

IC50 (nM)
36 ± 0.564 ± 529 ± 325 ± 256 ± 7
46 ± 0.752 ± 452 ± 5.555 ± 0.9
27 ± 0.8
21 ± 0.7
29 ± 0.545 ± 0.5>10,00060 ± 0>10,000
220 ± 3047 ± 0.5
20 ± 0.1
50 ± 0.3
24 ± 0.1
58 ± 0.1
30 ± 2.2
47 ± 0.3

R2Ser308

84j

Trp227

Tyr317

Phe306

Tyr319NADP+Phe311

4j

Tyr24

Met1202.8Å

3.0Å

Asn167
Tyr55

Trp86

Leu54
His117〔Putative interactions〕• A hydrogen bond between 8-OH groupof 4j and catalytic residues of AKR1C3(Tyr55 and His117)• A π-π stacking interactions between abenzene ring of R2 moiety and Phe306
15
Satoshi Endo, PhD, Gifu Pharmaceutical University

８-ヒドロキシクロメン誘導体
Compounds
4a4b4c4d4e4f4g4h4i4j4k4l4m4n4o4p4q4r4s4t4u4v4w
4xR2H
2-Fluoro
3-Fluoro
4-Fluoro
2-Chloro
3-Chloro
4-Chloro
2-Methyl
3-Methyl
4-Methyl
2-Ethyl
3-Ethyl
4-Ethyl2-Hydroxy
3-Hydroxy
4-Hydroxy
3,4-Difluoro
3,4,5-Trifluoro
2-Trifluoromethyl
3-Trifluoromethyl
4-Trifluoromethyl
2,4-Ditrifluoromethyl
2-Isopropyl
3-Isopropyl

IC50 (nM)
36 ± 0.564 ± 529 ± 325 ± 256 ± 7
46 ± 0.752 ± 452 ± 5.555 ± 0.9
27 ± 0.821 ± 0.7
29 ± 0.545 ± 0.5>10,000
60 ± 0>10,000
220 ± 3047 ± 0.5
20 ± 0.1
50 ± 0.3
24 ± 0.1
58 ± 0.1
30 ± 2.2
47 ± 0.3

R2Ser308

84j

Trp227

Tyr317

Phe306

Tyr319NADP+

Phe311

4j

Tyr24

Met1202.8Å

3.0Å

Asn167
Tyr55

Trp86

Leu54
His117〔Putative interactions〕• A hydrogen bond between 8-OH groupof 4j and catalytic residues of AKR1C3(Tyr55 and His117)• A π-π stacking interactions between abenzene ring of R2 moiety and Phe306
16
Satoshi Endo, PhD, Gifu Pharmaceutical University

８-ヒドロキシクロメン誘導体
Compounds
4a4b4c4d4e4f4g4h4i4j4k4l4m4n4o4p4q4r4s4t4u4v4w
4xR2H
2-Fluoro
3-Fluoro
4-Fluoro
2-Chloro
3-Chloro
4-Chloro
2-Methyl
3-Methyl
4-Methyl
2-Ethyl
3-Ethyl
4-Ethyl2-Hydroxy
3-Hydroxy
4-Hydroxy
3,4-Difluoro
3,4,5-Trifluoro
2-Trifluoromethyl
3-Trifluoromethyl
4-Trifluoromethyl
2,4-Ditrifluoromethyl
2-Isopropyl
3-Isopropyl

IC50 (nM)
36 ± 0.564 ± 529 ± 325 ± 256 ± 746 ± 0.752 ± 4
52 ± 5.555 ± 0.9
27 ± 0.8
21 ± 0.7
29 ± 0.545 ± 0.5>10,000
60 ± 0>10,000
220 ± 3047 ± 0.5
20 ± 0.1
50 ± 0.3
24 ± 0.1
58 ± 0.1
30 ± 2.2
47 ± 0.3

R2Ser308

84j

Trp227

Tyr317

Phe306

Tyr319NADP+

Phe311

4j

Tyr24

Met1202.8Å

3.0Å

Asn167
Tyr55

Trp86

Leu54
His117〔Putative interactions〕• A hydrogen bond between 8-OH groupof 4j and catalytic residues of AKR1C3(Tyr55 and His117)• A π-π stacking interactions between abenzene ring of R2 moiety and Phe306
17
Satoshi Endo, PhD, Gifu Pharmaceutical University

AKR1C3-NADP+-4j 複合体の共結晶構造

Whole structure

4j-binding mode

PDB:7C7G
18
Satoshi Endo, PhD, Gifu Pharmaceutical University

ドッキングモデルと結晶構造の比較⚫ 4j-docking model (Glide)
AKR1C3: Light yellow
4j: purple⚫ 4j-binding crystal structure
AKR1C3: Light blue
4j: magenta

H 2Oflip

19
Satoshi Endo, PhD, Gifu Pharmaceutical University

ドッキングモデルと結晶構造の比較
A H-bond Interaction with Ser129

via a water molecule

4jSer129

Apo structure: White
4j-binding structure: Light blue
20
Satoshi Endo, PhD, Gifu Pharmaceutical University前立腺がん細胞増殖阻害
MTT assay (72 h-culture)

7020 nM Pregnenolone (P)140120100**

80**

60**

400******

**

20

#60

#**

Cell population (%)

Cell numbers (%)

Flow cytometry****

Vehicle20 nM P
20 nM P + 20 μM 4d
20 nM P + 20 μM 4j

504030****20##10* **

000

10 10 10 10 20 10 20 10 20 (μM)

Flu Ab En4d4j

Flu : Flutamide (Androgen receptor antagonist)
Ab : Abiraterone (CYP17A1 inhibitor)
En : Enzalutamide (Androgen receptor antagonist)

4l

G0/G1
##S

G2/M

#p < 0.01, p < 0.05 vs Vehicle** p < 0.01, * p < 0.05 vs P alone

21Satoshi Endo, PhD, Gifu Pharmaceutical University核内AR発現の抑制
Nuclear AR levels (CWR22Rv1 cells)
10 nM Androstenedione

Nuclear
AR00104d4j0010(μM)

4l

10 μM Androstenedione

250

Nuclear AR expression (%)

1010 nM Androstenedione##200**

150
10050000101010 (μM)

4d4j

4l

**p < 0.01, *p < 0.05 vs Androstenedione alone

T : Testosterone22
Satoshi Endo, PhD, Gifu Pharmaceutical University

AR下流遺伝子の発現抑制
PSA mRNA (CWR22Rv1 cells)
20 nM Pregnenolone

Relative mRNA expression of PSA

500

##400

**

300
200
10000010 10 10 10 20 10 20 10 20 (μM)
Flu Ab En

4d4j4l

T : Testosterone

23
Satoshi Endo, PhD, Gifu Pharmaceutical University

ARアンタゴニスト活性(A) AR agonist activity (SC-3 cells)

Compounds

3.5
3No activity

2.5

21.5
1
0.50DMSODHT

Cell numbers (Ratio)

4(B) AR antagonist activity

4a 4b 4c 4d 4e 4f 4g 4j 4k 4l 4m 4n 4o 4p 4q 4rIC50 (μM)
Hydroxyflutamide
0.234b4.44c3.34d6.54e
3.34j2.44k8.14l5.84m
3.624
Satoshi Endo, PhD, Gifu Pharmaceutical Universityアンドロゲン非依存的細胞増殖抑制

PGD₂

PGH₂

AKR1C39α,11β-PGF2

PGF receptor

Activation ofMAPK, PI3K cascades

Proliferation

Cell numbers (×104)

PGF2α

MTT assay(72 h-culture, androgen-independent PC3 cells)
1010108886**** 64420Vehicle
10 μM 4d20 μM 4d0 h 24 h 72 h20**
**

****

64Vehicle
10 μM 4j
20 μM 4j0 h 24 h72 h

20Vehicle
10 μM 4l
20 μM 4l

0 h 24 h72 h** p < 0.01 vs vehicle

25
Satoshi Endo, PhD, Gifu Pharmaceutical University

in vivo抗がん活性

Xenograft mouse model

No influence on morphology of normal tissue
Lung x40

Liver x40

Kidney x40

Cancer x40

Tumor volume (cm3)200Control
150

100
##
##

50
###
##012

3

#

##

4

5

6

##4l4j

7weeks**p < 0.01, *p < 0.05 versus Control

Necrosis（Approximately 40%）
26
Satoshi Endo, PhD, Gifu Pharmaceutical University

アパルタミド耐性前立腺がん細胞
AKR1C3 upregulation【RT-PCR】

Acquisition of cross-resistance【Western blotting (WB)】【Sensitivity to CRPC drugs】

27
Satoshi Endo, PhD, Gifu Pharmaceutical University

アパルタミド耐性細胞における感受性回復効果

28
Satoshi Endo, PhD, Gifu Pharmaceutical University

アパルタミド耐性細胞における感受性回復効果【22Rv1】[Combination index (CI)]

80

CBR49

*

60

**

40**

**

0 μM
1 μM
5 μM

10 μM

20**

0050

Apa (μM)
2050
1.21 1.08
1.06 0.88
0.45 0.44

1000.80
0.74
0.37

Fa-CI plot

CompuSynソフトウェアを用いた併用係数 (CI) の算出[Fa-CI plot]

22.0

阻害効果 (CI>1)

1.5CI100

CI

Viable cells (%)

120

CBR49(μM)
1012.0951.14
100.881.00.5100

相乗効果 (CI<1)

0.0

Apa (μM)

0.0 0.2 0.4 0.6 0.8 1.00Fa【22Rv1-R】

100

CBR490 μM

80* 1 μM**40
205 μM****10 μM
**

0050

100

[Fa-CI plot]•2.01.5CI

Viable cells (%)

120

CBR49
Apa (μM)
(μM)
1020
50
100
10.954 0.954 0.427 0.117
51.488 0.989 0.205 0.014
100.482 0.331 0.032 0.007•

1.00.500.5Fa[Combination index (CI)]60

相加効果 (CI=1)

11Fa; fraction affected(≒ Viability)

CI値が1.0以下となり、CBR49はアパルタミドの抗癌活性を相乗的に高めることが示唆された。
AKR1C3発現量の高いアパルタミド耐性細胞ではより高い相乗効果が認められた。

0.0Apa (μM)

0.0 0.2 0.4 0.6 0.8 1.0** p < 0.01, * p < 0.05 vs Control

Fa

29
Satoshi Endo, PhD, Gifu Pharmaceutical University

AKR1C3阻害剤の想定される用途• 研究試薬としてのAKR1C3阻害剤• AKR1C3を高発現する前立腺がんに対する治療薬• 既存薬との併用によって、作用増強および耐性化抑制効果を示すがんアジュバンド薬• 既存薬に対して抵抗性を獲得したがん細胞に対する抗がん剤耐性克服薬

30
Satoshi Endo, PhD, Gifu Pharmaceutical University

本技術に関する知的財産権

••
•
•

発明の名称
出願番号
出願人
発明者

：AKR1C3選択的阻害剤及びその用途：特願2018-055182：岐阜市、富山大学、産業医科大学：遠藤智史、松永俊之、五十里彰、豊岡尚樹、藤本直浩

31
Satoshi Endo, PhD, Gifu Pharmaceutical University

お問い合わせ先岐阜薬科大学◎生命薬学大講座 生化学研究室 遠藤 智史
TEL058-230-8100 (内線3669)
FAX
058-230-8105
e-mail sendo＠gifu-pu.ac.jp◎事務局 庶務会計課 中尾 真一郎
TEL058-230-8100
FAX
058-230-8200
e-mail syomuk＠gifu-pu.ac.jp
32
Satoshi Endo, PhD, Gifu Pharmaceutical University