1. RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
NUC050 was synthesized at NuChem Therapeutics. Growth inhibitory (GI) effects
of gemcitabine and VEPNPs were evaluated in 96-well plates by exposing cells to
drug for 72 hours. To determine whether VEPNPs could bypass nucleoside
transport downregulation, control wells did not include dipyridamole (DP) and
treated wells included a final DP concentration of 1 μM. To determine whether
NUC050 delivered a nucleoside monophosphate (MP) intracellularly, testing was
performed at Southern Research Institute (SRI) in CCRF-CEM cells either
deoxycytidine kinase (dCK) wild type (WT) or deficient (-). CEREP performed an
in vitro ADME panel on NUC050.
Mouse PK were performed by Eurofin Panlabs. Mice were administered 2 mg/kg
IV of NUC050, blood drawn on 3 mice at 8 different time points, and plasma
analyzed by LC/MS/MS. A pilot efficacy study was performed at SRI in nude mice
implanted with the LoVo cell line (colon cancer). NUC050 was administered IV
weekly at a dose of 50 mg/kg.
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
VEPNPs have shown promise in early development as exemplified by results
obtained with NUC050, one of the VEPNPs of gemcitabine.
1. The delta isoform of VE appears to be the preferred carrier moiety because of
its antiproliferative activity and efficient release of the nucleoside-MP.
2. NUC050 has demonstrated that it gets into cells by a nucleoside transport
independent mechanism and that it delivers a nucleoside-MP to the cell,
bypassing the two major mechanisms of tumor resistance to nucleosides.
• VEPNPs are most likely catabolized to VE and nucleoside-MP by a
phosphatase12 or a phosphodiesterase.
3. NUC050 has demonstrated substantial improvements in pharmacokinetics
when compared to gemcitabine, most notably a circulating half-life that is 13.9-
fold longer in mice.
• This half-life compares favorably to that of one of the lipid based prodrugs
of gemcitabine, CP-4126, reported to be 3-4 minutes in dogs.13
4. Evaluation of NUC050 in vivo has been adversely affected by its formation of
micelles. This issue is currently being addressed by a nanoemulsion
formulation that will reduce or eliminate the surfactant properties of NUC050
and will concentrate the drug at the tumor site by enhanced permeability and
retention.
5. NUC050 has demonstrated evidence of efficacy that needs to be confirmed in
a larger study.
6. NUC050 appears to be relatively more active in vivo than in vitro.
7. Efforts are underway to attempt to synthesize the VEPNP of other analogs,
including NUC013.
REFERENCES
1. Nutrients. 2011; 3(11):962-86.
2. J Am Coll Nutr. 2010; 29(3 Suppl):324S-333S.
3. Cell Prolif. 2011; 44(6):516-26.
4. Cell Prolif. 2013; 46(2):203-13.
5. Nutr Cancer. 2011; 63(5):763-70.
6. Microsc Microanal. 2012; 18(3):462-9.
7. Br J Pharmacol. 2011; 163(2):283-98.
8. J Nutr Biochem. 2009; 20(8):607-13.
9. Cancer Res. 2010; 70(21):8695-8705
10. Br J Cancer. 2008; 99(11):1832-41.
11. Drug Metab Dispos. 1992; 20(6):849-55
12. IUBMB Life. 2005; 57(1):23-25.
13. Invest New Drugs. 2011; 29:456-466.
Richard Daifuku MD-PhD
Epigenetics Pharma, Mercer Island, Washington, USA
Vitamin E phosphate (VEP) nucleoside prodrugs: a platform for intracellular delivery of
monophosphorylated (MP) nucleosides
D-α-tocopheryl phosphate-5'-gemcitabine triethylammonium salt (NUC017)
D-δ-tocopheryl phosphate-5'-gemcitabine triethylammonium salt (NUC050)
D-γ-tocotrienyl phosphate-5'-gemcitabine triethylammonium salt (NUC024)
Figure 2: Synthesized isoforms of vitamin E phosphate gemcitabine.
Compound
Breast
MDA-MB-
231
(µM)
Non-Small
Cell Lung
NCI-H460
(µM)
Colon
HCT-116
(µM)
Gemcitabine 0.11 0.02 0.01
α-tocopheryl
phosphate
23.40 52.24 46.86
NUC017 22.70 23.75 26.13
δ-tocopheryl
phosphate
29.56 69.67 70.58
NUC050 5.08 1.69 3.67
γ-tocotrienyl
phosphate
26.42 69.14 55.71
NUC024 4.90 4.75 4.01
GI50 (µM)
MDA Breast H460 Lung H116 Colon
Compound DP (-) DP (1 µM ) DP (-) DP (1 µM ) DP (-) DP (1 µM )
Gemcitabine 3.08 56.77 0.02 0.82 0.03 2.39
NUC050 17.16 23.30 2.14 1.47 3.07 6.74
NUC024 30.34 27.77 7.16 15.98 5.55 12.61
Table 1: Comparison of in vitro GI50 between gemcitabine,
vitamin E phosphate isoforms and VEP gemcitabine prodrugs
NUC017, NUC050 and NUC024
Table 2: Comparison of GI50 of gemcitabine, NUC050 and NUC024 in the
presence or absence of DP.
GI50 (µM)
Cell line Gemcitabine NUC050
CEM WT 0.002 0.59
CEM dCK(-) 124.5 19.2
Table 3: Comparison of GI50 of gemcitabine and NUC050 in
CEM cells with and without dCK.
Figure 3: Mean concentration-time profile of NUC050 after IV
administration in mice.
Animal
t1/1/2
(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 3.9 42351 19028 19101 0.38 0.2 0.8 1.8 24
Table 4: Pharmacokinetic parameters after IV administration of 2 mg/kg of
NUC050 in mice.
Figure 1: Isoforms of tocopherols and tocotrienols.2
Figure 4: Survival proportion in LoVo (colon cancer) xenografts. Saline
control (n= 5) compared to NUC050 (n = 3).
VEP gemcitabine prodrugs were tested in the presence and absence of
dipyridamole (DP), an inhibitor of nucleoside transport, and compared to
gemcitabine. The GI50 of VEPNPs are relatively unaffected while that of
gemcitabine increased by 18 to 80-fold.
NUC050 was tested in vitro for activity against gemcitabine in wild type (WT)
leukemic CEM cells and CEM cells without deoxycytidine kinase (dCK(-)). In the
dCK (-) cells, gemcitabine is not phosphorylated to gemcitabine-monophosphate
(gemcitabine–MP), a precursor to the therapeutically active diphosphate (DP)
and triphosphate (TP). Gemcitabine IC50 went from 0.002 µM in dCK WT cells to
124.5 µM in dCK (-) cells, an increase of 62,250-fold. NUC050 IC50 went from
0.59 µM to 19.2 µM, an increase of only 32.5-fold, compatible with intracellular
delivery of gemcitbine-MP.
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 50
The vitamin E phosphate nucleoside prodrug (VEPNP) platform is potentially
applicable to any purine or pyrimidine and is designed to bypass two major
mechanisms of tumor resistance to nucleosides, nucleoside transport and kinase
downregulation.
There are two main forms of vitamin E: tocopherols and tocotrienols. In part, the
rationale for using VE as a carrier moiety is that some of the isoforms of
tocopherols1, and more so tocotrienols2, have been reported to have
antiproliferative activity. Indeed tocotrienols have shown activity against a number
of different cancers, including breast,3,4 leukemia,5,6 liver,7,8 pancreas9 and
prostate10 among others. Formal ranking of relative biopotency of tocopherols and
tocotrienols for suppression of cell growth and induction of cell death displays a
consistent relationship corresponding to δ-tocotrienol ≥ γ-tocotrienol > α-
tocotrienol > δ-tocopherol >> γ and α-tocopherol.2
OBJECTIVES
Develop a prodrug platform for nucleoside analogs designed to:
1. Bypass the two major mechanisms of resistance to nucleoside analogs,
whether constitutive or acquired, namely, nucleoside transport and kinase
downregulation.
2. Improve the pharmacology of nucleosides, e.g., such that cytidine analogs will
not be a substrate for cytidine deaminase.
3. Benefit from the antiproliferative activity of some of the isoforms of VE.
The activity of the above isoforms in vitro appears to be related, at least in part,
to the steric hindrance to enzymatic cleavage provided by methyl groups
proximal to the VE-phosphate bond. Alpha isoforms have two methyl groups in
close proximity to the bond, gamma one and delta none.
Structure of VEP gemcitabine prodrugs
VEPNP in vitro activity
• Intrinsic clearance by liver microsomes: Half-life > 60 minutes.
• > 99% protein bound in human plasma.
VEPNPs bypass nucleoside transport
and deoxycytidine kinase
NUC050 has a more prolonged circulating
half life than gemcitabine in mice
The half-life of unformulated NUC050 is 3.9 hours compared to 0.28 hours
reported for gemcitabine in mice (an increase of 13.9-fold).11
In a pilot study performed in a mouse xenograft model of colon cancer, NUC050
was administered weekly at 50 mg/kg for 3 weeks (equimolar to 18.1 mg/kg of
gemcitabine). One mouse died 5 days after starting treatment, most likely due
to drug toxicity, the two remaining mice survived until the study conclusion and
had tumor weight reduction of 50.6% compared to saline controls 8 days
following the last dose of study drug.
NUC050 shows preliminary evidence of
activity in a mouse xenograft model
PlasmaConcentration
(ng/ml)
NUC050 in vitro pharmacology