1. RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
NUC013 was synthesized at NuChem Therapeutics. DNMTI was measured
colorimetrically in a 96-well microplate kit. In vitro testing of NUC013 for activity
was performed in the NCI 60 panel at 10 µM in 57 cell lines of different tissues of
origin: breast, colon, CNS, lung, melanoma, ovarian, prostate, renal and
hematogenous.
Pharmacokinetics were performed at Eurofin Panlabs. Mice were administered 10
mg/kg of NUC013 IV and plasma samples measured on 3 mice at 8 time points by
LC/MS/MS. Maximum tolerated dose (MTD) studies were performed at Southern
Research Institute (SRI), as were tumor xenograft models in nude mice implanted
with HL-60 (human leukemia, p53 null) or LoVo (colon cancer, p53 WT) cell lines.
Drug or saline were administered IV for three consecutive days a week for three
weeks (n = 10/group).
INTRODUCTION
OBJECTIVES
There is a growing interest in epigenetic therapy, i.e., in treatments that result in the
expression of aberrantly repressed genes in cancer cells. Since epigenetic change
in cancer cells is reversible, it presents an interesting target for chemotherapeutic
intervention. DNA methyl transferase inhibitors (DNMTIs) result in a decrease in
the DNA methylation of cancer cells and the expression of genes that control the
cell cycle or apoptosis, including the tumor suppressor gene TP53.
NUC013 is a novel DNMTI being developed by Epigenetics Pharma. It has been
shown to be both more effective and safer than decitabine in vitro and in vivo.
NUC013 has demonstrated activity against cell lines of most major tissue types.
Furthermore, while at least as active as decitabine against TP53 null cancer cell
lines, it is significantly more active against TP53 wild type (WT) cell lines.
MATERIALS AND METHODS
Figure 2: Comparison of DNA methyl transferase inhibition in
colon cancer cells (HCT116) of NUC013 and 5-azacytidine.
RESULTS
CONCLUSIONS
1. NUC013 is a DNA methyl transferase inhibitor.
2. NUC013 has been shown to have a GI50 < 10 µM against hematogenous
tumors and at least one cell line from each solid tumor tissue tested, including
breast, colon, CNS, renal, lung, melanoma, ovarian and prostate.
3. In vitro activity has translated into in vivo activity against both a hematogenous
tumor (TP53 null) and a solid tumor (TP53 WT) in xenograft models.
• Longer duration of treatment may have resulted in even better outcomes.
4. NUC013 has been shown to be superior to decitabine both in vitro and in vivo.
• While NUC013 is at least as effective as decitabine against TP53 null cell
lines, it is significantly more effective against TP53 WT cell lines.
o NUC013 most likely derepresses TP53 and inhibits p53R2, resulting in self-
potentiation.
o Decitabine derepresses TP53 and induces p53R2, most likely resulting in
“self antagonism.”
‒ p53R2 generates deoxycytidine diphosphate competing as a
triphosphate (TP) with decitabine-TP for incorporation.
– Induction of p53R2 has been associated with cancer progression and
resistance to therapy.2-4
– Decitabine has been shown to induce more apoptosis in TP53 null cells
than in TP53 WT cells.5-7
5. NUC013 has been shown to be safer than decitabine in mice. The MTD of
decitabine is around 5 mg/kg while that of NUC013 is over 120 mg/kg
administered on the same schedule, suggesting that the therapeutic index may
be in the range of 100-150 fold better than decitabine.
REFERENCES
1. Modern Drug Synthesis. Li JJ, Johnson DS (eds.). Wiley. 2010.
2. DNA Repair (Amst). 2014; 22:24-9
3. Mol Cell Biochem. 2015; 399(1-2):179-88
4. J Dermatol Sci. 2012; 68(1):19-24
5. Oncogene. 2004; 23(3):735-43
6. Proc Natl Acad Sci U S A. 2013; 110(1):E89-98
7. Oncotarget. 2014; 5(19):8924-36
To develop a safer and more effective DNMTI, than the currently approved 5-
azacytidines, that can treat hematogenous and solid tumors.
Figure 4: Comparison of growth inhibition by decitabine or NUC013 of TP53
WT colon cancer cell line LoVo (GI50 decitabine > 50 µM vs. NUC013 3.0 µM).
Richard Daifuku MD-PhD
Epigenetics Pharma, Mercer Island, Washington, USA
In vitro and in vivo testing of a novel DNA methyl transferase inhibitor (DNMTI) with activity against
hematogenous and solid tumors whether p53 null or wild type (WT)
NCI 60 Cell Panel Decitabine NUC013
< 50% growth at 10 µM 6 24
≥ 50% growth at 10 µM 49 31
Table 1: Allocation of cell lines of NCI 60 cell panel treated with decitabine or
NUC013 by % growth inhibition at 10 µM. Fisher’s exact two-tail, p = 0.0002.
NCI 60 Cell Panel TP53 (null) TP53 (WT)
< 50% growth at 10 µM 3 2
≥ 50% growth at 10 µM 31 17
Table 2: Allocation of cell lines of NCI 60 cell panel treated with decitabine by %
growth inhibition at 10 µM and TP53 status. Fisher’s exact two-tail, p = 1.00.
NCI 60 Cell Panel TP53 (null) TP53 (WT)
< 50% growth at 10 µM 10 14
≥ 50% growth at 10 µM 24 5
Table 3: Allocation of cell lines in NCI 60 cell panel treated with NUC013 by %
growth inhibition at 10 µM and p53 status. Fisher’s exact two-tail, p =0.0035.
Figure 5: Mean concentration-time profile of NUC013 after
IV administration in mice
Table 4: Pharmacokinetic parameters after IV administration in mice.
Dose of NUC013
% tumor reduction
compared to saline
control
P-value
5.8 mg/kg 24.3 0.048
20 mg/kg 50.9 <0.0001
40 mg/kg 53.7 <0.0001
NUC013 Toxicology
Figure 1: 5-aza-2’,2’-diflurorodeoxycytidine [NUC0013].
Figure 3: Testing of NUC013 in NCI 60 cell line panel : % growth
inhibition at 10 µM.
Figure 6: Survival proportion in HL-60 xenografts. Saline control
compared to NUC013 (20 mg/kg). [MS saline control = 26.5 days, MS
NUC013 = 32 days (HR = 0.26, p = 0.032)].
Figure 5: Survival proportions in HL-60 (human leukemia) xenografts.
Saline control compared to decitabine (5 mg/kg) and NUC013 (5.8 mg/kg).
[Saline median survival (MS) = 25 days, decitabine MS = 26 days (hazard
ratio (HR) = 0.92), NUC013 MS = 28.5 days (HR = 0.59, p = 0.19)].
Figure 7: Survival proportion in LoVo xenografts. Saline control compared
to decitabine (5 mg/kg) and NUC013 (5.8 mg/kg). [MS saline control > 60
days, MS decitabine = 31 days (HR = 26.89, p <0.0001)].
Figure 8: Survival proportion in LoVo xenografts. Saline control compared
to 20 mg/kg and 40 mg/kg NUC013.
NUC013 is more active than decitabine in vitro
NUC013 pharmacokinetics
NUC013 is more active and safer
than decitabine in vivo
NUC013 is a DNA methyl transferase inhibitor
Table 5: Percent tumor reduction in mice with LoVo xenografts treated with
NUC013 compared to saline control 8 days following last dose of study drug.
NUC013 has been tested in mice at doses of up to 120 mg/kg for three consecutive
days per week for three weeks without evidence of weight loss or death over the time
period of observation.
2’,2’-difluoro-5-azadeoxycytidine (NUC013) is a novel cytidine analog comprised of
the 5-azacytosine base of 5-azacytidine or decitabine conjugated with the
difluorinated sugar of gemcitabine.
0 2 0 4 0 6 0 8 0
0
5 0
1 0 0
D a y s
Percentsurvival
S a lin e c o n tro l
N U C 0 13
D e c ita b in e
0 1 0 2 0 3 0 4 0
0
5 0
1 0 0
D a y s
Percentsurvival
s a lin e c o n tro l
N U C 0 13
D e c ita b in e
0 2 0 4 0 6 0 8 0
0
5 0
1 0 0
D a y s
Percentsurvival
S a lin e c o n tro l
2 0 m g /kg N U C 0 1 3
4 0 m g /kg N U C 0 1 3
0 1 0 2 0 3 0 4 0 5 0
0
5 0
1 0 0
T im e (d a y s )
Percentsurvival
C o n tro l
N U C 0 13
Because of changes in the composition of the NCI 60 panel over time, only 55 of
these cell lines were tested for both NUC013 and decitabine, while only 53 of
those had published TP53 status. Comparisons of performance of NUC013 vs
decitabine show: NUC013 is significantly more active than decitabine in the NCI
60 panel (Table 1), decitabine is not more active in TP53 WT than TP53 null cell
lines (Table 2) and NUC013 is significantly more active in TP53 WT than TP53
null cell lines (Table 3).
The half-life of NUC013 in mice is comparable to that reported for decitabine.1
1/
Animal
T1/2
(h)
C0
(ng/mL)
AUClast
(h*ng/mL)
AUCInf
(h*ng/mL)
AUC Extr
(%)
VSS
(L/kg)
CL
(mL/min/kg)
MRT
(h)
Last time point for
AUClast (h)
IV-Mouse 0.335 36941 11082 11086 0.04 0.26 9.10 0.292 4