September 13, 2017
Ohara Pharmaceutical Co., Ltd.

A Cooperative Research Study with the National Cancer Center Japan
Ohara Pharmaceutical Co., Ltd. (Head Office: Shiga, CEO: Dr. Seiji Ohara; hereinafter referredto as “Ohara Pharma”) and the Division of Epigenomics, National Cancer Center ResearchInstitute (Chief: Dr. Toshikazu Ushijima) are currently conducting a cooperative research study
on drug development aiming to improve epigenetic abnormalities.
Epigenetics is widely known as a mechanism for regulating the expression of genetic
information in a base sequence-independent manner, with DNA methylation being the firstepigenetic factor to be discovered. Methylation is usually controlled by DNA methyltransferase(DNMT). However, in cancer patients, aberrant DNA methylation inactivates key tumor
suppressors, indicating that epigenetic abnormalities also play a role in oncogenictransformation. In particular, many hematologic malignancies are considered to be associatedwith such abnormalities, and DNMT inhibitors are clinically applied to patients withmyelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). In Japan, azacytidine(Trade name: Vidaza®) has been available as an injectable drug for MDS since 2011. In othercountries throughout the world including the U.S., decitabine (Trade name: Dacogen® forinjection) is used, as well as azacytidine. In addition, several new DNMT-related drugs andtechnologies are currently under development, as follows.
Recently, Dr. Ushijima and his colleagues announced the successful development of a newsystem to assess DNMT inhibitors (Epigenetics, 2016 Dec 9:0. doi: 10. 1080/15592294. 2016.
1267887. [Epub ahead of print]). This assessment system was used to compare a candidate
compound for DNMT inhibition developed by Ohara Pharma with existing DNMT inhibitors
used in a clinical setting, and comparative data have been published (E0RTC-NCI-AACR 2016,
Munich: See the attached materials).
Dr. Ushijima and his group commented that, “Regarding aberrant DNA methylation, we place
great expectations on a new drug which is orally available and more highly stable in blood level
than decitabine. Epigenomic drugs actually have great potential, although their indications arelimited to blood cancers, at present. Seeing the latest trend in the development of new DNMT
inhibitors, we can find some clinical developments for solid cancers, including brain tumors,nasal and laryngeal cancer, and head and neck cancer. They also retain the potential to broaden
their indications to other diseases in which epigenetic abnormalities are confirmed, such as
diabetes mellitus, immune diseases, Alzheimer's disease, and schizophrenia. We are waiting forthe arrival of a new oral product whose dose and regimen can be easily adjusted in order tooptimize treatment.”
Ohara Pharma is making full use of its know-how regarding the synthesis and formulation ofnucleic acid medicines to develop a novel DNMT inhibitor. On the other hand, regarding anoral preparation, an optimal preparation to be introduced into a clinical setting is underdevelopment through a collaborative research study undertaken not only with Dr. Ushijima’s

group, but also with the Department of Drug Discovery and Biomedical Sciences at SagaUniversity, where we are promoting discussions to select an effective dose and regimen.
We will attempt to develop both a cancer diagnostic method and an optimum drug/treatment
for aberrant DNA methylation, and carry out continuous research so that they can be applied ina clinical setting, as soon as possible.
For more information, please visit the website of the National Cancer Center Japan using thefollowing link:
http://www.ncc.go.jp/jp/ri/division/epigenomics/project/020/20170906143626.html
Contact details for inquiries regarding this issue:
Hideo Tonomura,Ph.D. Director, Member of the Board
Person in charge of Orphan drug and Drug Discovery BusinessOhara Pharmaceutical Co., Ltd.
TEL: 03-6740-7701
FAX: 03-6740-7702
Yoshitaka Nakata,Ph.D. Executive Officer
R&D Center
Ohara Pharmaceutical Co., Ltd.
TEL: 0748-88-2200
FAX: 0748-88-2300

257 : Development of Novel DNA Demethylating Agents with Greater Stability and Less Toxicity
Naoko Hattori1, Magoichi Sako2, Kana Kimura1, Akiko Mori1, Eriko Okochi-Takada1, Hideo Tonomura3, Toshikazu Ushijima1
1Div. of Epigenomics, National Cancer Center Research Institute, Tokyo, JAPAN; 2Drug Development Laboratories, OHARA Pharmaceutical Co., Ltd., Shiga, JAPAN;
3Orphan Drug and New Business Development, OHARA Pharmaceutical Co., Ltd., Tokyo, JAPANResults and moderate apoptosis
Induction of genome-wide DNA demethylation

To develop novel and stable DNA
demethylating agents

Backgrounds is expanding from hematological Epigenetic therapy

ResultIdentification of twoscompounds as novel andO
HOH2CNOHOOH
NH2NNO
HOH2COHODNA methylation (%)

OP-21

10.0

1.0

OP-120.1

 Luciferase assay
 Cell viability

Plating

OP-12

OP-12 (10 µM)

OP-21 (1 µM)(A) Demethylation
ofmarker genes wasobserved in the cells
treated with OP-12 or
OP-21.
(B) Genome-wide
DNAdemethylation
wasinduced by treatmentof OP-12 or OP-21.
(C) Apoptosis
wasinduced by SGI-110 or
OP-21 but not by OP12 in TF-1 cells.

Non-treated cells

OP-21

Low in vivo toxicity of two novel agents

DrugMaterials & Methods

10.05

SGI-110

1.03

SGI-110

0.1

1

10.0

Day 01.0

B(µM)

0.1

*Compounds showing theluciferase activity equal toor more than that of Aza or
one-tenth of that of DAC

NMock This rapid elimination in plasma makes itchallenging to determine the maximum biologicaldose for individual patients.

NOP-21

Relative activityof Caspase-3/7

NH2

OP-12

10.0
3.0
1.0
0.3
0.1
10.0
3.0
1.0
0.3
0.1
10.03.01.0
0.3
0.1
Mock (DMSO)DAC 0.1
Mock(PBS)

CN

SGI-110 (1 µM)

SGI-110AB

OSR2(µM)

stable demethylating agents

 Two approved DNA demethylating agents, 5azacytidine(azacitidine,AZA)
and
5-aza-2'deoxycytidine (decitabine, DAC), suffer from rapid
deamination by cytidine deaminase and hydrolytic
ring-cleavage of the base moiety.

AB

OP-12

OP-21

Days after treatment
7-week-old 1 2 3 4 5 6 7 8

SGI-110 OP-22

OP-21

OP-23

OP-25

OP-26

OP-11

OP-12

OP-24

Measurement of body weight

DDNA methylation and apoptosis analyses

Identification of novel agents
 Cell viability assay : Cell Count Reagent SF (WST-8)
 SGI-110 : second generation demethylating agent that isresistant to cytidine deaminase

In vivo drug tolerability
 Female athymic nu/nu mouse Evaluation of body weight, morbidity, leukocyte count, andhematocrit

100
10
1.0
0.1
0.0
1
Mock
100
10
1.0
0.1
0.0
1
Mock
100
10
1.0
0.1
0.0
1
Mock
100
10
1.0
0.1
0.01
Mock
100
10
1.0
0.1
0.01
Mock
100
10
1.0
0.1
0.01Mock100101.00.1
0.01Mock100
101.00.1
0.01
Mock
1.00.1Mock qMSP and Illumina Infinium HumanMethylation 450K
 TF-1 : a cell lines of AML developed from MDS
 Apoptosis assay : Caspase-Glo® 3/7 assay

CSGI-110

OP-22

OP-21

OP-23

OP-24

OP-25

OP-26

OP-11

OP-12

(A) Fifty-nine derivatives of AZA and DAC were synthesized as
potentially more resistant to the deamination by cytidine
deaminase and/or hydrolytic ring-cleavage. Nine-teen compounds
among the 59 were considered to have DNA demethylating activity.
(B) Schedule of drug treatment.
(C) Seven compounds showed luciferase activity equal to that of SGI110.
(D) OP-12 and OP-21 showed a moderate and a strong cell-growth
suppressive effect, respectively.

DHematocrit (%)

Secretory luciferases

Sampling (bloodand organs)

Number ofleukocytes (/µL)

EGF
P

Relative cell viability

Secretory EGFP PuroR
luciferase gene

2 days/wk, 3 cycles

N = 3 ip

OP-XX

Drug-free
2127DMSO

DrugDrug
Days after administration
SGI-110 :OP-12 :
OP-21 :
6.35 mg/kg
22.8 mg/kg
7.40 mg/kg

NALiverAza100101.00.10.01
Mock100
101.00.10.01
Mock100
101.00.10.01
Mock100
101.00.10.01
Mock100
101.00.10.01
Mock100
101.00.10.01
Mock100
101.00.10.01
Mock
100
10
1.00.10.01
Mock
100
10
1.00.10.01
Mock
1.00.1
Mock

DAC

100
101.00.10.01
Mock

(µM)

0.1
Mock

Demethylated promoter
DNA
demethylating CpG island of UCHL1agents(mg/kg)Bonemarrow[Okochi-Takada, Hattori et al., Epigenetics, Revision Submitted]*MTD of DAC(reference value)Luciferase activity

 Quantification of DNA demethylation by luciferase activity
 Isolation of optimal subclones with high S/N ratio Suitable system for high throughput screening

Relative body weight(average)

CScreening system

Methylated promoter CpG
island of UCHL1

UCHL1-MetLuc

10.0
3.0
1.0
0.3
0.1
10.0
3.0
1.0
0.3
0.1
10.0
3.0
1.0
0.3
0.1
Mock (DMSO)DAC 0.1
Mock(PBS)

tumor to solid tumor.

A

Treated cells

Two novel DNA demethylating agents withstrong demethylation activity and less toxicity
were developed.

All CpG islands(n= 63,379)

Objective

TSS200 and CpG
island (n= 7,384)

Conclusion(mg/kg)100 µm
SGI-110OP-12(A) Administration doses (upperpanel) and schedule (lowerpanel) of SGI-110, OP-12,and OP-21.
(B) Severe body weight loss
and increase of mortality
were observed in 6.35 mg/kg
of SGI-110 and high-dosesof OP-12 and OP-21 groups.
(C) Leukopenia was absent inmice with OP-12 and OP-21
without body weight loss.
Doses with severe bodyweight loss are marked with
red rectangles.
(D) Myelosuppression
andhepatotoxicity were absent
in mice with OP-12 and OP21 without body weight loss.

OP-21

Summary & Discussion
 OP-12 and OP-21 showed strong demethylating activities with less toxicity than SGI-110 and DAC.
 OP-12 might have a distinct mode of demethylating action.
 Anti-tumor activities of OP-12 and OP-21 are evaluating using xenografts of a colorectal cancer cell line (HCT116),
and preliminary assessment showed that OP-12 and OP-21 were equally effective to DAC.