Liver-targeting with the novel nucleotide prodrug
MIV-818 designed for the treatment of liver cancers
R BETHELL, K TUNBLAD, B RIZOSKA, A LINDQVIST, S JURIC, S SEDIG, F ÖBERG, B CLASSON, A ENEROTH, J ÖHD AND M ALBERTELLA
Medivir AB, Huddinge, SWEDEN
MIV-818 delivers high concentrations of active triphosphate to liver
RESULTS
MIV-818 is a novel nucleotide prodrug of troxacitabinemonophosphate (TRX-MP), that has been designed to deliver high
levels of the chain-terminating nucleotide troxacitabine-triphosphate
(TRX-TP) to the liver after oral dosing while minimizing systemic
exposure – offering multiple advantages over troxacitabine itself:
• Oral bioavailability and increased permeability
Troxacitabine
Hep3B cell line, mean EC50
0.029 µM
0.24 µM
HepG2 cell line, mean EC50
0.017 µM
HUH-7 cell line, mean EC50
0.043 µM
Human hepatocytes, mean EC50
Human intestinal S9 fraction (µL/min/mg)
>100 µM
7
Stable
Human liver S9 fraction (µL/min/mg)
42
Due to the instability of MIV-818 in mouse blood (see Table) the efficacy studies were
performed with troxacitabine.
The aim was to establish exposures to TRX-TP required for effective tumor growth
suppression in different HCC in vivo models in order to define target therapeutic
concentrations in the clinic.
Dose-response effects of troxacitabine on tumour growth inhibition (TGI) were
demonstrated in the Hep3B (below), Huh-7 and HepG2 (not shown) xenograft models.
Stable
T r o x a c it a b in e 2 .5 m g / k g
Human hepatocytes CLint (µL/min/10-6 cells)
58
Stable
Stable (<2)
1000
Stable
Very unstable
(>150)
Stable
Ester 1
T r o x a c it a b in e 1 0 m g / k g
800
600
400
R TV x4
0
The combination of MIV-818 and sorafenib is synergistic
20
20
10
5
2,5
1,25
0,62
0,31
0
0,00076 0,0022 0,0068
0,02
0,062
0,185
0,55
1,66
100,41 100,23 100,23 100,55
99,76
100,41 100,46 100,18 100,09 100,09
99,62
99,9
100,18 100,18
99,9
100,13 100,23
99,81
99,71
99,62
66,86
59,97
67,37
76,26
80,26
76,54
79,33
75,52
82,03
82,68
25,54
32,14
38,24
47,64
52,02
57,41
67,98
74,49
78,26
79,61
10,6
16,46
26,7
36,38
42,99
50,15
64,86
72,58
77,24
78,17
0
1,48
15,48
29,77
36,8
43,45
61,46
70,21
76,82
77,75
0
0
0
19,44
31,91
38,47
59,32
68,63
75,38
77,66
0
0
0
5,34
17,25
33,96
57,23
70,12
75,38
76,07
25
30
35
40
45
50
55
60
6
12
18
24
1
0 .1
LOQ - Limit of
quantitation TP
0
6
Liver
1.0
Troxacitabine (25 mg/kg)
101%
~26d
62 µMxh
Troxacitabine (10 mg/kg)
81%
~16d
51 µMxh
Troxacitabine (2.5 mg/kg)
70%
~11d
8 µMxh
• Consistent effect – seen in other cell lines
The effect of the different exposures to TRX-TP on DNA damage (pH2AX) and
proliferation (BrdU) was examined in Hep3B tumours, excised after the treatment
period (bidx5d ip).
C e ll c o n t r o l H e p a t o c y t e s
2 .0
1 .5
1 .0
0 .5
0 .0
2 .5 m g /k g
1 0 m g /k g
2 5 m g /k g
20
2.5 mg/kg
10
v e h ic le
2 .5 m g /k g
1 0 m g /k g
R esponse
Dose
20000
Compound
BrdU
(% of vehicle)
10000
CC50 (µM)
1° Hepatocyte
(2d)
Hep 3B
(5d)
Window
MIV-818
>100
0.024
>4,000
Sorafenib
40
2.6
<20
0
-1 0
-8
-6
-4
lo g (I)µ M
• Prodrugs show high selectivity for HCC cell
lines relative to primary human hepatocytes
compared to sorafenib in viability assays
2 5 m g /k g
10 mg/kg
30000
Liver
CES1, CES2, Cath A
• Large selective index in terms of DNA-damage
response observed between HepG2 and fresh
human hepatocytes (24h)
• Dramatic induction of DNA damage a potential
surrogate biomarker of clinical efficacy
Tissue
Cmax
µM
AUC
µM*hr
5.4
Troxacitabine
Plasma
42
67
10
Troxacitabine triphosphate
Liver
<0.05
<1.2
1.9
TPliver/troxplasma ratio
<0.018
• Single dose oral administration of MIV-818 to rat gave significant liver exposure to the TRX-TP
• Low systemic exposure to troxacitabine was seen, consistent with aim of reduced systemic toxicity
• In contrast, troxacitabine administration did not yield quantifiable TRX-TP in liver, and gave high
systemic exposure to troxacitabine, which is known to cause significant toxicity
• >100-fold increased delivery of TRX-TP to liver from MIV-818 compared to troxacitabine
• Efficacious liver exposure to troxacitabine triphosphate from MIV-818 but not troxacitabine
•
Potent inhibition of HCC cell line growth and selective induction of DNA
damage relative to primary human hepatocytes
•
Increased conversion to the active metabolite, troxacitabine triphosphate,
TRX-TP
•
Orally bioavailable and targeted for metabolism and activation in the liver
MIV-818 is synergistic with sorafenib in vitro, suggesting that it might prove
particularly efficacious in combination treatment
DNA damage
0
v e h ic le
TPliver/troxplasma ratio
Troxacitabine ip (80
µmol/kg)
30
(m e a n S D )
LIVER-TARGETING CONCEPT
40000
% tis s u e p H 2 A X -p o s it iv e
C e ll c o n t r o l H e p G 2
Proliferation
(m e a n S D )
50000
24
MIV-818 is a novel phosphoramidate prodrug of troxacitabine that shows
greatly improved in vitro properties compared to the parent nucleoside,
including
Vehicle
% t is s u e B r d U - p o s it iv e
GADD 153 induction
18
CONCLUSIONS
Synergy plot (MacSynergy II software)
Hills indicate synergistic interactions
HepG2 vs. primary human hepatocytes
12
T im e ( h )
Troxacitabine triphosphate
TP tumour (AUC0-t)
2 .5
Fast and complete hydrolysis in
liver - conversion to TP
Trapped inside cells due to
charge
0
%TGI = (1-[Tt/T0] / [Ct/C0] / 1[C0/Ct]) x 100; where Tt and T0
are TV of treated mouse X at day
t or 0. Ct and C0 are the mean
TV of the control group at day t
and 0.
Tumour growth delay (TGD) is
calculated as T-C where T and C
are times in days for mean TV in
the treated and control groups to
reach 4x the initial TV.
• TRX-TP exposures in tumour leading to significant tumour growth delay were defined
M IV - 8 1 8 H e p a t o c y t e s
No or low
hydrolysis in
blood
T P L iv e r
10
0 .0 1
0.35
• Dose-dependent effects on tumour growth inhibition (TGI) and tumour growth delay (TGD)
M IV - 8 1 8 H e p G 2
No or low
hydrolysis in
the intestine
0 .0 1
Plasma
TGD
(at RTVx4)
Low toxicity in human primary hepatocytes suggests potential for
tumour selectivity
No or low
systemic
exposure
0 .1
Troxacitabine
Dramatic induction of DNA damage in treated tumours
DNA Fragmentation
CES1
dependent
protide
1
AUC
µM*hr
• MIV-818 shows synergy in vitro with sorafenib and regorafenib (not shown) in Hep3B cells
MIV-818 in primary human hepatocytes
Intestine
CES2/Cath A
10
Cmax
µM
DNA damage (pH2AX)
Triphosphate
(TP)
T r o x a c ita b in e P la s m a
Tissue
Maximal TGI
(Day 34)
Group
5
Hep3B Percent growth inhibition for each
concentration combination (WST-8 assay, Kit8)
Variations in prodrug esters modulate multiple parameters, e.g. 1st step
rate, potency, phys chem properties, liver/intestinal metabolism
Diphosphate
(DP)
100
T P L iv e r
MIV-818 po (80 µmol/kg)
Stu d y D ay
Synergy
0
Ester 2
Monophosphate
(MP)
T ro x a c ita b in e P la s m a
100
Hep3B xenograft model
Dosing initiated at mean tumour
volumes of 200 mm3
T r o x a c it a b in e 2 5 m g / k g
200
Sorafenib concentration (µM)
Example prodrug
( i.p . B I D x 5 d )
V e h ic le
MIV-818 concentration (µM)
Troxacitabine
Troxacitabine ip (80 µmol/kg)
D o s in g
1200
Mouse, rat whole blood CLint (µL/min/mg)
Metabolic activation of troxacitabine prodrugs
MIV-818 po (80 µmol/kg)
T im e ( h )
• Directed delivery to the liver and reduced systemic toxicity
We compare MIV-818 and troxacitabine using in vitro and in vivo
models in order to demonstrate liver targeting and a superior anticancer profile.
0.46 µM
>100 µM
Human, dog, cyno whole blood CLint (µL/min/mg)
• Increased cancer cell killing
0.17 µM
Anti-tumour activity and exposure to troxacitabine triphosphate
C o n c e n t a r t io n ( µ M )
Troxacitabine was active in preclinical cancer models and in clinical
studies, but ultimately failed in the clinic due to systemic dose
limiting toxicities.
MIV-818
C o n c e n t r a t io n ( µ M )
Troxacitabine was developed as a dioxalane nucleoside which was
not subject for enzymes conferring resistance to other nucleoside
analogues such as cytidine deaminase.
Assay
M ean SEM
Many systemic chemotherapeutics have failed to show efficacy in
hepatocellular carcinoma (HCC), often because systemic toxicity
prevents efficacious liver levels of the drug from being reached.
Despite the instability of MIV-818 in rat blood (see Table) the liver delivery of MIV-818
was examined in vivo and compared to ip administration of troxacitabine.
R e l a t iv e T u m o u r V o lu m e ( % )
INTRODUCTION
SAT-123
pH2AX
(fold change vs.
vehicle)
2.5 mg/kg
19%
12-fold
10 mg/kg
4%
31-fold
25 mg/kg
5%
32-fold
Efficacious exposures to TRX-TP were defined in vivo, that demonstrate
dramatic induction of DNA damage defining target concentrations for future
clinical studies
Oral dosing of MIV-818 results in a >100-fold increased delivery of the
active metabolite, TRX-TP, to the liver compared to troxacitabine
MIV-818 is in preclinical development for the treatment of HCC and other
liver cancers
25 mg/kg
• Dose-dependent inhibition of proliferation (BrdU) and induction of DNA damage (pH2AX) following
troxacitabine treatment (2h after last dose)
• Note induction of DNA damage and inhibition of proliferation within hypoxic areas (green)
indicating good penetration and conversion to TP in these hard to treat regions
CONTACT INFORMATION
[email protected]
[email protected]
Acknowledgements to Andrew Minchinton and Alastair Kyle from Cabenda,
Vancouver, Canada for immuno-fluorescent imaging and CrownBio for in vivo tumour models
