Th histone
The
The histone
hi t
d
deacetylase
tyl
i hibit
inhibitor
inhibitor SB939 acts synergistically with Sorafenib
hibi SB939 acts
t synergistically
y gi tii lly with
i h Sorafenib
ith
S f ib in an i an
in
orthotopic
orthotopic model of hepatocellular
th
h t pi model
d l off hepatocellular
h p t ll l carcinoma carcinoma
i
Veronica
Veronica Novotny
i Novotny‐Diermayr*,
Novotny Diermayr
Diermayr*
i
*, Babita
Babita
bi Madan, Yung Kiang Loh, Lai Chun Ong, Mohammed K. Pasha, Li Jun Ding, Ramesh
Madan
Madan,
d
Yung Kiang
i
Loh
Loh,
h Laii Chun
h Ong,
Ong Mohammed
h
d K.
K Pasha,
Pasha
h Lii Jun Ding,
Ding
i
Ramesh
h Jayaraman, Kantharaj
Jayaraman Kantharaj
Jayaraman,
h j Ethirajulu, Jeanette M. Wood. Ethirajulu
Ethirajulu,
hi j l Jeanette M.
M Wood.
Wood
d
SS*BIO
BIO Pte
Pte Ltd, 1 Science Park Rd
Ltd 1 Science Park Rd, #05
#05‐09
09 The Capricorn Science Park II SINGAPORE 117528 Tel:
09 The Capricorn, Science Park II, SINGAPORE 117528, Tel: +65 6827 5000, www.sbio.com
Tel +65 6827 5000 www sbio com , * veronica_diermayr@sbio.com
veronica diermayr@sbio com
80
Sorafenib 60 mg/kg
g g b.i.d.+
b i d + SB939 125 mg/kg
g gq
q.o.d.
od
Sorafenib 30 mg/kg
g g b.i.d.+
b i d + SB939 125 mg/kg
g gq
q.o.d.
od
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 16
Day
y
12
Time(h)
16
20
24
*
*
*
*
1
Figg 5: Western blot analysis
Fig. 5: Western blot analysis of tumor lysates treated with 45 mg/kg Sorafenib and 125 mg/kg SB939
y of tumor lysates
y
treated with 45 mg/kg
g/ g Sorafenib and 125 mg/kg
g/ g SB939
post-SB939 dosing
0
post-SB939 dosing
VEGF % iincrease over base
VEGF % increase over base level on d1
b
level
l l on d
d1
d2
d
21-1
18
8h
2
acH3
VEGF
pMAPK
p
β-actin
β
actin
ti
40
35
30
25
20
15
10
5
0
pre‐d
p
d
0 5h
0.5h
1h
2h
6h
18 h
18 h
post‐SB939
post
SB939 dosing
SB939 dosing (
d i (+6h
( 6h for post
6h for
f post‐
Sorafenib
Sorafenib dosing) f bd
dosing))
(averages from 3 mice per time point)
(averages from 3 mice per time point)
kg
+
SB
g/
g/
10
m
So
ra
fe
ni
b
SB
93
9
12
5m
m
45
93
9
kg
kg
g/
kg
g/
m
b
• Tumor
T
VEGF was increased
i
d compared
d to
t base
b
l l 6.5
levels
6 5 h after
ft Sorafenib
S f ib treatment,
t t
treatment
t probably
b bl as
a consequence off its
it anti
anti‐angiogenic
ti angiogenic
i
i effects
ff t (known
(k
effect
ff t off VEGF receptor
t tyrosine
t
i kinase
ki
i hibi
inhibitors
as a response
p
to reduced
d d tumor p
perfusion
f i and
d increased
i
d hypoxia).
hyp i ) The
hypoxia)
Th increase
i
i VEGF
in
was reduced
d d to some extent by
by the
h addition
ddi i off SB939.
SB939
ra
fe
ni
3
10
• pMAPK
p
was not inhibited
h b d in tumor tissue after
f
treatment with
h the
h highest
h gh
d
dose
off Sorafenib,
Sorafenib
f b,
indicatingg no effect on the Raf signalling
g
gp
pathwayy
pathway.
So
4
0
00
0.0
e
5
Biomarkers of Target Efficacy and Tumor Response: (Fig. 5)
Biomarkers
of Target Efficacy and Tumor Response: (Fig.
(Fig 5)
• Acetylation of histone H3 (acH3) was induced in tumor tissue by SB939 treatment and remained
upregulated
l t d for
f up to
t 18 h,
h indicating
i di ti effective
ff ti target
t
t inhibition
i hibiti by
b SB939.
SB939
2
b
Fig 3: Mean diseased Liver weights per treatment group
Fig.
Fig. 3: Mean diseased Liver weights per treatment group
02
0.2
** p < 0.05
0 05 and
d
*** p < 0.001
0 001
using
i ANOVA with
ih
Dunnet's
Dunnet
's post test
**
cl
*SB939 dose was reduced from 125 to 100 mg/kg q.o.d. from day 14 to 21
*SB939
d
dose
was reduced
d d from
f
125 to 100 mg/kg
/k q.o.d.
d ffrom day
d 14 to 21
4
Ve
hi
217
ra
fe
ni
380
6
So
18
%K
%
Ki6
67
7-p
po
os
siitiiv
ve c
ce
ellls
AUClast ((h*μg/mL)
μg/ )
04
0.4
93
9
5207
20
SB
969
9697
+
99
kg
72
2
kg
Clast
( / )
(ng/mL)
l
P lif ti index
Proliferation
i d (d21)
8
***
06
0.6
g/
24
4
g/
24
4
10
m
18
8
12
5m
18
8
b
Tlast (h)
kg
22
9
32
g/
4
93
16
Apoptotic index (d21)
ra
fe
ni
Cmax (μg/mL)
7
1
m
2
SB
0 17
0.17
So
05
0.5
45
Tmax (h)
Fig
Fig. 7: Automated analysis of apoptotic‐
ig 7: Automated
d analysis
ly i off apoptotic‐
p p i and proliferation index in Hep3B tumors on d21
and
d proliferation
p lif
i iindex
d iin Hep3B
p tumors on d21
d
b
-100
SSorafenib 45 mg/kg
Sorafenib
f ib
45 mg/kg
/k
Dayy 1
Day 1
Dayy 21
Day 21
g/
kg
-60
60
125 mg/kg
125
mg/kg
/k
Dayy 21*
Day 21*
m
SB939
Dayy 1
Day 1
ra
fe
ni
Tumor weight
Tumor weight only
g onlyy
Sorafenib
S
f ib (10
( mg/kg)
g/kg))
+ SB939 #44
• The
h effects
ff
off SB939 on tumor morphology
ph l gy are stillll under
d investigation,
investigation
g
, but
b p
preliminary
l
y analysis
ly
suggests
gg
that SB939 leads to increased defects in mitotic spindle
p
formation
10
8
So
4
e
0
18
b
16
cl
14
hi
-40
Sorafenib
S
f ib (10
( mg/kg
g/kg )
#11b
• SB939 alone or in combination slightly reduced the proliferation index at an effective anti
anti‐tumor
tumor
dose (Fig.
(Fig 7),
7) but did not increase apoptosis,
apoptosis and in combination therapy,
therapy reduced the apoptotic
effects
ff t off Sorafenib.
S f ib
Ve
% in
nh
hib
biitiio
on
n
Di
Diseased
Diseased liver weight
d liver
li
weight
i ht
90
85
8
12
d21-1
d2
1h
d2
d
21-1
1h
d2
d
21-2
2h
d2
d
21-6
6h
95
8
10
Time(h)
d1 pr
pre
e-d
do
os
se
e
d2
d
21-0
0..5
5h
100
-20
20
d1
d
1-0
0..5
5h
105
6
PK Parameter
PK Parameter
So
ra
ve
fe
hi
ni
cl
b
e
So
(1
0
ra
m
f
e
SB
g/
ni
kg
b
93
)
(4
9
5
So
(1
m
25
ra
g
fe
/k
m
g)
ni
g
/k
b
So
g)
10
ra
q.
m
o.
fe
g
/k
ni
d.
g
b
+
45
SB
m
93
g/
kg
9
+
SB
93
9
%B
Bo
od
dy W
We
eig
gh
htt (M
Me
ea
an
nS
S..E
E..M
M.))
Fig 1:: % Body
Fig. 1: % Body weight during the MTD study in SCID mice
Fig.
ody weight during the MTD
MT study in SCID
SCI mice
4
1 000
1,000
Table 1: PK p
Table 1: PK parameters for SB939 and Sorafenib
parameters for SB939 and Sorafenib on d1 and d21 after co‐administration
on d1 and d21 after co administration
d1 pr
pre
e-d
do
os
se
e
MTD ((Fig.
(Figg. 1):
):
• In non
non‐tumor
tumor bearing mice the combination of Sorafenib (30 or 60 mg/kg q.d.)
q d ) and
SB939 (125 mg/kg
/k q.o.d.)
d ) was wellll tolerated
t l t d (max.
(
BW loss
l
off 3.6%
3 6% and
d 8.7%
8 7%
respectively)
ti l ) (Fig.
(Fi 1),
1) but
b t less
l
wellll tolerated
t l t d in
i tumor
t
b i mice.
bearing
mice
i The
Th dose
d
off SB939
was reduced
d d to
t 100 mg/kg
/k q.o.d
q o d from
f
d
days
14‐20
14
20 in
i the
th efficacy
ffi
study
t d (Figs
(Fi 2 &3)
2
Day 1(45 mg/kg)
D
/k )
Day 21 (45 mg/kg)
ra
fe
ni
0
-80
80
Diis
D
se
ea
as
se
ed
e
d liv
ve
err we
e
eig
gh
g
htt ((g
g))  S
S.E
E..M
M..
M
In vitro proliferation assay:
• Hep3B
H 3B cells
ll do
d nott have
h
a B
B‐Raf
R f mutation
Raf
t ti
and
d were relatively
l ti l resistant
i t t to
t anti
anti‐
ti
proliferative
lif ti effects
ff t off SB939 or Sorafenib
S f ib (IC50 off 1.23
1 23 μM
M and
d 3.66
3 66 μM
M for
f SB939 and
d
S f ib respectively)
Sorafenib
p i ly)
Day 1(125 mg/kg)
D y 21 (100
Day
(
mg/kg)
g/kg))
10
20
SB939 ((125 mg/kg)
g/kg))
#32
#32a
• Interestingly
IInterestingly,
t
ti l after
ft treatment
t t
t with
ith SB939 alone
l
or addition
dditi off SB939 to
t the
th low
l
d
dose
off Sorafenib
S f ib
(hi h dose
(high
d
S f ib + SB939 nott analyzed)
Sorafenib
l d) no necrosis
i or fibrosis
fib i could
ld be
b detected,
d t t d despite
detected
d it the
th
significant
ig ifi
reduction
d i in
i diseased
di
d liver
li
weight
weight.
igh
So
100
10 000
10,000
% TU
UN
NE
EL
L--p
po
ositiv
ve
e nu
nuc
cllei
1,000
,
0
R l
Results:
Mean
M
n con
c nc
c, ng
ng/m
mL
L( S.E
S E.M
M.))
Figg 2: % Inhibition of diseased Liver and Tumor weight
Fig. 2: % Inhibition of diseased Liver‐
and Tumor weight
g
10 000
10,000
d1
d
1-18
8h
•
vehicle
hi l
#9b
Sorafenib
SB939
d1-1 h
d1
d1
d
1-2 h
d1
d
1-6 h
•
•
Fig
Fig. 6: Representative pictures from H&E stained tumor sections on d21
ig 6: Representative
p
i p
pictures
i
from
f
H&E
& stained
i d tumor sections
i
on d21
d
100,000
100 000
100,000
Meaan co
M
on
nc,, ng/
n /m
mL(( S..E..M
M.)
•
C ll proliferation
Cell
lif ti assays: YO
YO‐PRO‐1
PRO 1 or CellTiter
C llTit Glo
CellTiter‐Glo
Gl assay
id, 9‐16
Mi Female
Mice:
F
l C.B‐17‐Igh1b‐Prkdc
C B‐17‐Igh1b‐Prkdc
B 17 Igh1b P kd scid
9 16 weeks
k
Animall model:
d l Hep3B,
Hep3B
p , orthotopic
h p liver
l
xenograft
g f (p
( f
(performed
d by
by VivoPharm)
h
)
Vehicles: Sorafenib tablets were crushed and formulated in NNP‐PEG300
NNP PEG300 ((1:9);
); SB939
was dissolved in 0.5%
0 5% MC,
MC 0.1%
0 1% Tween
Tween‐80
80
Doses: Sorafenib 10 or 45 mg/kg p.o.
p o once daily (q.d.).
(q d ) SB939 125 mg/kg p.o.
p o every
second
d day
d (q.o.d.).
(
d)
C bi ti schedule:
Combination
h d l Sorafenib
S f ib was dosed
d d first,
fi t followed
first
f ll
d by
b SB939 6 h later
l t
PK and
d PD analysis
ly i was p
performed
f
d as described
d
ib d p
previously
i ly (Novotny
(N
(Novotny‐Diermayr
y Diermayr
Di
y et al.
all Mol
M l
C
Cancer
Ther;
h ; 9(3)
( ) 2010,
20 0, p 642
2010
6 2 ff)
The Ariol
Ariol®Automated
Automated Image
g Analyzer
y was used to analyze
y immuno‐histochemistry
immuno histochemistryy slides
45 m
45
mg
g//k
kg
g
So
S
orra
affen
nib
b+
+S
SB
B9 3
39
9
•
•
•
•
• Similar effects were observed based on excised tumor wets weights
g
but onlyy
the effects of the high
g dose Sorafenib alone and in combination with SB939
were statistically significant.
significant H&E staining revealed that there was a lot of
li er tissue
liver
tiss e excised
e cised together with
ith the tumor.
t mor
10 m
mg
g//k
kg
g
So
S
orra
affen
nib
b+
+S
SB
B9 3
39
9
M th d
Methods:
Histological Analysis (Fig. 6 & 7): Histological
i l gi l Analysis
ly i ((Fig
ig 6 & 7):
)
• Histological
g
analysis
y of the tumor tissue at the end of the experiment
p
revealed that both doses of
Sorafenib
Sorafenib,
So
a e b, including
c ud g tthee dose tthat
at d
did
d not
ot reduce
educe d
diseased
seased liver
e weight,
weight
e g t, dec
decreased
eased tthee p
proliferation
o e at o
index (Ki67) but only 10 mg/kg Sorafenib increased apoptosis (TUNEL).
(TUNEL) The most striking effect seen
with
ith both doses of Sorafenib was
as an increase
in rease in necrosis
ne rosis and fibrosis.
fibrosis
The
h PK profiles
p f l off Sorafenib
f b and SB939 were not affected by co‐administration
and
d SB939 were not affected
ff
d by
by co administration
d
• The high dose of Sorafenib had a slightly greater effect (40%) and this was • The PK profiles of Sorafenib
f th enhanced
further
h
d in
i combination
bi ti
with
ith SB939 (54%) but
b t the
th difference
diff
Fi 4 PK profiles
Fig.4:
Fig.4: PK profiles for SB939 and Sorafenib
fil ffor SB939 and
d Sorafenib
S f ib on d1 and d21 after co
on d1 and
d d21 after
ft co‐administration
administration
d i i t ti
b t
between
th groups was nott statistically
the
t ti ti ll significant.
significant
i ifi t
12
25
5>
>1
10
00
0m
mg
g//k
kg
g
SB
S
B9
93
39
9
SB939 is
i an hydroxamic
h d
i acid
id based
b d class
l
I II and
I,
d IV HDAC inhibitor,
i hibit with
inhibitor
ith very favourable
f
bl
physicochemical
physicochemical,
p
hy i h i l p
pharmaceutical
h
i l and
d p
pharmacokinetic
h
ki i p
properties
properties,
p i
resulting
l i g in
i an
excellent
ll
b
bioavailability
l b l y and
d marked
k d accumulation
l
in tumor tissue.
tissue In clinical
l
l p
phase
h
I
studies with SB939 in solid tumors,
tumors, drugg exposure
p
was demonstrated to be higher
g
and
side effects milder/fewer compared to first generation HDAC inhibitors such as SAHA.
SAHA Only
6 7% Grade 3/4 fatigue (2/30 patients) or 6.9%
6.7%
6 9% (2/29 patients) were observed in two
i d
independent
d t Phase
Ph
I clinical
li i l studies
t di
making
ki
SB939 ideally
id ll suited
it d for
f combination
bi ti
th
therapies
therapies.
i The
Th aim
i off the
th presentt studies
t di was to
t explore
l
th potential
the
t ti l off SB939 for
f the
th
treatment off hepatocellular
h
ll l carcinoma
i
(HCC)
(HCC).
Ph
Pharmacokinetics
Pharmacokinetics (Fig. 4, Table 1):
ki i (Fig
(Fig 4 Table
T bl 1):
1)
Anti‐tumor
Anti
tumor Efficacyy ((Fig.
(Figg 2 & 3)) :
h p
plasma
l
AUC off Sorafenib
f ib and
d SB939 on d21
d were similar
i il to p
published
bli h d values
l
f both
for
b h
• In an orthotopic Hep3B xenograft model,
model the 10 mg/kg dose of Sorafenib • The
p
after chronic dosing.
dosingg The AUC was higher
g
on d1 which mayy be due to the diseased
was not efficacious on total liver weight (change in size + 0.7%).
0 7%) SB939 single compounds
g are mainlyy metabolised byy the liver ((CYP3A4).
(CYP3A4)) The lower AUC on d21
agentt treatment
t t
t decreased
d
d diseased
di
d liver
li
weight
i ht by
b 26 % and
d together
t th with
ith liver since both drugs
compared to d1 was observed with both drugs and may have been due to an improvement in liver
10 mg/kg
/k Sorafenib,
S f ib a further
Sorafenib
f th decrease
d
up to
t 37% was observed.
observed
b
d Analysis
A l i
function after the large reduction in tumor burden induced by the therapy.
therapy The lower AUC of
using
i g Clarke
Cl k ’s Synergism
Clarke’s
Sy gi
calculation
l l i
showed
h
d that
h there
h
was a synergistic
y gi i
SB939 on d21 was also
l due
d to
t the
th dose
d
reduction
reduction.
d ti
effect
ff
((CCI = ‐0.118).
‐0 118))
45 m
45
mg
g//k
kg
g
So
S
orra
affen
nib
b
I
Introduction:
d i
10 m
mg
g//k
kg
g
So
S
orra
affen
nib
b
Abstract #5436
Ab
Abstract
t t #5436
st
AACR 101
AACR:
AACR: 101
A
Annual
Annual Meeting
l Meeting
M ti g
C l i
Conclusions:
• SB939 is
i effective
ff ti as a single
i gl agent
g t therapy,
th py, despite
therapy
d pit Hep3B
H p3B cells
ll being
b i g resistant
i t t to
t
it in vitro
• At 10 mg/kg
g//kg Sorafenib
S f ib did nott reduce
d
t
tumor
size
i but
b t induced
i d d marked
k d necrosis
i
and fibrosis in the tumor tissue,
tissue which could be completely blocked in
combination
bi i with
i h SB939.
S 939
SB939
• The ineffective dose of 10 mg/kg Sorafenib showed synergistic anti
anti‐tumor
tumor activity
i combination
in
bi i with
i h SB939
• The mechanism of action for the combination of SB939 with Sorafenib warrants
f h investigation
further
i
ig i
and
d mayy p
provide
id a rationale
i
l for
f a Phase
Ph
II trial
i l for
f a
combination
bi ti
off SB939 with
ith VEGF receptor
pt tyrosine
ty i
ki
kinase
i hibit
inhibitors
such
h as
Sorafenib in HCC.
HCC