rock2 selective inhibitors for the treatment of fibrosis · rock2 is a nodal point in cell...
TRANSCRIPT
(AIM:REDX)
ROCK2 selective inhibition is an exciting innovative approach to treat fibrotic diseases. Redx are advancing towards the clinic
ROCK2 selective inhibitors for the
treatment of fibrosis
3rd Annual Anti-Fibrotic Drug Development Summit18-20 November | Boston
• Members of the AGC (protein kinase A, G and C, PKA/PKG/PKC) family of serine/threonine kinase family
• Originally identified as downstream effectors of RhoA
• ROCKs facilitate RhoA-induced stress fiber formation and focal adhesion assembly
• ROCKs are expressed in both invertebrates and vertebrates
• RhoA/ROCK signalling is closely involved in regulating cell morphology, growth, migration, and apoptosis
• Activated GTP-bound RhoA can further activate ROCK to phosphorylate several substrates
− Substrates involved in autophagy, cell survival & apoptosis, vesicle dynamics, cytoskeleton regulation, cell growth & regeneration, cell shape & motility
2
Rho-associated coiled-coil containing kinases (ROCKs)
ROCK2 selective inhibitors for the treatment of fibrosis
Jiao et al. Curr Mol Pharmacol. 2017;10(2):115-140; Hartmann et al. Front Pharmacol. 2015 Nov 20;6:276
• Two isoforms of ROCK, ROCK1 (ROCKβ) and ROCK2 (ROCKα)
• Selective ROCK isoform inhibition is challenging because of high degree of sequence homology
− 65% amino acid sequences in common
− 92% homology within their kinase domains
• ROCK1 and ROCK2 widely expressed in tissues of embryos and adults
• ROCK1 and ROCK2 are ubiquitously expressed in different tissues.
− ROCK1 mRNA is highly expressed in the lungs, liver, spleen, kidneys, and testis
− ROCK2 mRNA is enhanced in the brain, heart, lung and skeletal muscle
− Both isoforms expressed in endothelial cells (ECs)
• ROCK1 mainly distributed in the plasma membrane
• ROCK2 localized in cytoplasm
• Simultaneous inhibition of ROCK1 and ROCK2 induce hypotension
• ROCK2 is overexpressed in key diseases
3
Rho-associated coiled-coil containing kinases (ROCKs) isoforms
ROCK2 selective inhibitors for the treatment of fibrosis
Pelosi et al. Mol Cell Biol. 2007 Sep;27(17):6163-76.Koch et al. Pharmacol Ther. 2018 Sep;189:1-21
4
ROCK2 is a nodal point in cell signalling pathways associated with fibrotic diseases
ROCK2 selective inhibitors for the treatment of fibrosis
Metabolic disease
Lung fibrosis
Other organs
Inflammation
ROCK2❖ ↑ROCK2 in acute and chronic
inflammation
❖ ROCK2 inhibitors shown to be anti-inflammatory in vivo
❖ ROCK2 inhibition protects from inflammatory damage in IBD models
❖ ↑ROCK2 in liver fibrosis and diabetic kidney models
❖ ROCK2 inhibition = ↓ liver fibrosis
❖ ROCK2 inhibition = ↓ kidney fibrosis
❖ ROCK2 haplotype KO = ↓ fibrosis in UUO model
❖ ROCK2 inhibitor PoC in human IPF trial
❖ Clinical response in lung scores in cGvHD
❖ ROCK2+/- protected lung fibrosis
ROCK2 inhibition could target many diseases, highlighted by clinical validation across multiple organs in cGvHD
❖ ROCK2 inhibitor = efficacy across multiple organs in cGVHD clinical trial
❖ ROCK2 inhibitors = ↓ fibrosis in skin (SSc) model
❖ ROCK2 conditional KO = ↓ hypertension, hypertrophy & atherosclerosis
5
Selective ROCK2 inhibitors can inhibit fibrosis without inducing hypotension associated with ROCK1/2 inhibitors
ROCK2 selective inhibitors for the treatment of fibrosis
Hypotension is induced by a ROCK1/2 inhibitor in rats Redx ROCK2i has no impact on mean blood pressure in rats
Effect of a single oral treatment of azaindole 1 (0, 3, 10 mg/kg) on mean arterial blood pressure in normotensive rats. N=6, data are % change from baseline. British Journal of Pharmacology (2007) 152, 1070–1080.
Data are plotted LS mean±SEM n=6 animals. Statistical effect of treatment analysed by one way ANOVA with Fisher’s LSD post test, compared to vehicle treated animals, *p<0.05.
• ROCK1/2 inhibitors deliver an anti-fibrotic effect in preclinical studies but induce hypotension, limiting further clinical development
• Selective ROCK2 inhibitors can inhibit fibrosis without inducing hypotension
• No cardiovascular events highlighted from PhI or PhII clinical trials with selective ROCK2i KD025
0 2 4 6 8 10 12 14 16 18 20 22 24-50
-40
-30
-20
-10
0
10
20
Time (h)
Me
an
blo
od
pre
ss
ure
ch
an
ge (
%)
*
Vehicle
REDX10178 (100 mg/kg)
dark cycle
10 mg/kg
3 mg/kg
vehicle
6
Redx ROCK2 inhibitors are potent and have greater than 100-fold selectivity over ROCK1 in biochemical assays
ROCK2 selective inhibitors for the treatment of fibrosis
10 -12 10 -10 10 -8 10 -6 10 -4
0
20
40
60
80
100
120
[REDX10843] (M)
Pe
rce
nta
ge
in
hib
itio
n (
%) ROCK1
ROCK2
10 -12 10 -10 10 -8 10 -6 10 -4
0
20
40
60
80
100
120
[REDX10178] (M)
Pe
rce
nta
ge
in
hib
itio
n (
%) ROCK1
ROCK2
Assay REDX10178 IC50 (µM) REDX10616 IC50 (µM) REDX10843 IC50 (µM) REDX10842 IC50 (µM)
Biochemical Activity ROCK2 [ATP 20 µM] 0.002 0.004 0.017 0.006
Biochemical Activity ROCK1 [ATP 20 µM] 0.2 3.0 2.5 2.2
Fold selectivity ROCK2/ROCK1 90-fold 730-fold 150-fold > 300-fold
10 -12 10 -10 10 -8 10 -6 10 -4
0
20
40
60
80
100
120
[REDX10616] (M)
Pe
rce
nta
ge
in
hib
itio
n (
%) ROCK1
ROCK2
Note: data are all from n≥2
10-12 10-10 10-8 10-6 10-4
0
20
40
60
80
100
120
[REDX10842] (M)
Pe
rce
nta
ge
in
hib
itio
n (
%) ROCK1
ROCK2
7
Redx ROCK2 inhibitors are potent and highly selective in cellular mechanistic assays
ROCK2 selective inhibitors for the treatment of fibrosis
• MCF7 cell line expresses both ROCK1 and ROCK2 isoforms (parental line)
• ROCK1 or ROCK2 stably knocked down using shRNA to develop cell lines selective for each ROCK isoform
• ROCK inhibition in cells analysed by the inhibition of pMYPT1, downstream of ROCK signalling
ROCK cellular signalling
AssayREDX10178 IC50
(µM)REDX10616 IC50
(µM)REDX10843 IC50
(µM)REDX10842 IC50
(µM)
Cellular activity ROCK2 0.9 1.0 2.4 1.3
Cellular activity ROCK1 20 > 30 26 > 30
GAPDH
ROCK2
GAPDH
ROCK1
par
enta
l
RO
CK
1 K
D
par
enta
l
RO
CK
2 K
D
ROCK expression in MCF7 cell lines
8
Redx ROCK2 inhibitors reduce pro-fibrotic and pro-inflammatory activity of kidney mesangial cells cultured in high glucose
ROCK2 selective inhibitors for the treatment of fibrosis
• Protein expression of secreted detected in the culture media
• High glucose stimulates a profibrotic phenotype in kidney mesangial cells
• Cells secrete growth factors and cytokines into the supernatant e.g. CTGF, PDGF-BB, TIMP-1 and MCP-1
Assay REDX10178 IC50 (µM) REDX10616 IC50 (µM) REDX10843 IC50 (µM) REDX10842 IC50 (µM)
Phenotypic activity TIMP-1 Mouse mesangial cells 0.2 0.9 2.8 0.9
Phenotypic activity PDGF-BB Mouse mesangial cells 0.2 0.4 1.4 0.8
Phenotypic activity MCP-1 Mouse mesangial cells 0.3 1.3 2.1 0.7
Phenotypic activity CTGF Mouse mesangial cells 0.4 0.4 1.5 ND
5 Day 0 2 4 6 8
Compound treatment
Glucose (25 mM) – refreshed on day 3 and 6
1 3 7 Harvest supernatant
9
Selective ROCK2 inhibitors reverse the myofibroblast phenotype of activated human hepatic stellate cell myofibroblasts
ROCK2 selective inhibitors for the treatment of fibrosis
• ROCK signalling central to the mechanosensing of the ECM tension
• HSC cell line (LX2) cultured for 2-3 weeks on plastic to induce differentiation into myofibroblasts (LX2-MF)− No exogenous stimuli; cells are activated by matrix stiffness and autocrine factors
− Phenotype and activation status confirmed by expression of α-SMA
• Selective ROCK2 inhibitors suppress α-SMA in the LX2-MF cells – suggesting a reversal of the myofibroblast like phenotype
• No toxicity was observed with compounds (up to 10 µM)Green: α-SMA; purple: nuclei (DRAQ5)
DMSO
REDX10843: 1 µM 10 3 1 0.4 0.1 0.04 0.01
0
20
40
60
80
100
REDX10843
REDX10843 concentration (µM)
S
MA
exp
ressio
n(%
of
DM
SO
co
ntr
ol)
10 3 1 0.4 0.1 0.04 0.01
0
20
40
60
80
100
120
REDX10616
REDX10616 concentration (µM)
S
MA
exp
ressio
n
(% o
f c
on
tro
l)
10 3 1 0.4 0.1 0.04 0.01
-50
0
50
100
150
200
REDX10178
REDX10178 concentration (µM)
S
MA
exp
ressio
n(%
of
DM
SO
co
ntr
ol)
Expression of α-SMA detected by immunocytochemistry
10
REDX10842 is highly selective when tested against 468 kinases at 10 µM
ROCK2 selective inhibitors for the treatment of fibrosis
• 20 targets inhibited with > 50% of control by 10 μMof REDX10842
• Kinases with IC50 values < 3 µM assayed at KM [ATP] marked with circles
• ROCK2, the most potently inhibited kinase, is denoted with a blue circle
• IC50 values of kinases inhibited > 50% by 10 μMof REDX10842 with IC50 < 30 µM are reported in the table
KinaseIC50 [ATP] = Km (µM)
Fold Selectivity
over ROCK2
ROCK2 0.004 1
MEK2 0.4 103
MST1/STK4 1.0 243
GLK/MAP4K3 1.1 258
ROCK1 1.1 263
CHK2 2.2 520
MEK1 2.4 568
MST2/STK3 4.2 997
YSK4/MAP3K19 4.8 1,153
MEKK3 7.5 1,801
MEKK2 8.8 2,113
LATS2 14 3,289
11
REDX10842 is highly selective when tested in a CEREP SafetyScreen44 panel at 10 µM
ROCK2 selective inhibitors for the treatment of fibrosis
• 7 targets inhibited with more than 25%
• Follow up IC50: PDE3A IC50 23 µM
Enzyme % inhibition
PDE3A (h) 60
dopamine transporter (h) (antagonist radioligand) 51
5-HT transporter (h) (antagonist radioligand) 51
COX2(h) 49
delta (DOP) (h) (agonist radioligand) 30
CCK1 (CCKA) (h) (agonist radioligand) 27
A2A (h) (agonist radioligand) 25
PDE3A
5-HT tr
ansporte
r
delta(D
OP)A2A
CB1CB2
BZD (c
entral)
M1
5-HT2A
acety
lcholin
estera
se
Na+ channel (
site 2
)D1
beta 1
V1a
alpha 2
AM
3
beta 2
Lck k
inase
KV channel
ETA
norepin
ephrine tr
ansporte
r
COX1-50
0
50
100
% in
hib
itio
n
• REDX10843 dosed therapeutically in the murine bleomycin induced lung fibrosis model at 50 mg/kg BID
• Pirfenidone used as positive control and dosed at 100 mg/kg BID
• Oropharyngeal administration of 1.5 U/kg bleomycin on day 1, compound dosing initiated from day 7-21
12
Effect of ROCK2 selective inhibitors in a murine bleomycin-induced IPF model
ROCK2 selective inhibitors for the treatment of fibrosis
Murine bleomycin-induced IPF model
Murine bleomycin-induced IPF model
• REDX10842 dosed therapeutically in the murine bleomycin induced lung fibrosis model at 5, 20 and 50 mg/kg BID
• Pirfenidone used as positive control and dosed at 100 mg/kg BID
• Oropharyngeal administration of 1.5 U/kg bleomycin on day 1, compound dosing initiated from day 7-21
13
REDX10843 reduces fibrosis and collagen deposition in the lung in murine bleomycin-induced IPF model
ROCK2 selective inhibitors for the treatment of fibrosis
Ashcroft score Masson’s trichrome score
0
1
2
3
4
5
Ash
cro
ft s
co
re
**
No Bleomycin
Vehicle BID
REDX10843 50 mg/kg BID
Pirfenidone 100 mg/kg BID
0
1
2
3
4
5
Ash
cro
ft s
co
re
**
No Bleomycin
Vehicle BID
REDX10843 50 mg/kg BID
Pirfenidone 100 mg/kg BID
0
1
2
3
4
Masso
n's
tri
ch
rom
e s
co
re
**
0.00
0.05
0.10
0.15
0.20
Co
llag
en
(m
g/m
L)
****
BALF Collagen
14
REDX10843 suppresses pro-fibrotic and pro-inflammatory gene expression in lung and plasma in murine bleomycin-induced IPF model
ROCK2 selective inhibitors for the treatment of fibrosis
PD lung gene expression Plasma PD biomarkers
-100 -80 -60 -40 -20 0
MCP-1
IL-6
TGFß
CTGF
TIMP-1
MMP2
COL1A1
FN1
Reduction from vehicle (%)
Gen
e
REDX10843 50 mg/kg BID
Pirfenidone 100 mg/kg BID
**
**
***
****
*** **
*
**
***
**
-100 -80 -60 -40 -20 0 20
HGF
PDGF-BB
MMP3
MMP2
TIMP-1
S100A9
WISP1
ICAM-1
Reduction from vehicle (%)
Pla
sm
a b
iom
ark
er
REDX10843 50 mg/kg BID
Pirfenidone 100 mg/kg BID
**
***
***
**
**
**
-100 -80 -60 -40 -20 0 20
HGF
PDGF-BB
MMP3
MMP2
TIMP-1
S100A9
WISP1
ICAM-1
Reduction from vehicle (%)
Pla
sm
a b
iom
ark
er
REDX10843 50 mg/kg BID
Pirfenidone 100 mg/kg BID
**
***
***
**
**
**
15
REDX10842 reduces fibrosis and collagen deposition in the lung in murine bleomycin-induced IPF model
ROCK2 selective inhibitors for the treatment of fibrosis
0
1
2
3
4
5
Ash
cro
ft s
co
re
***
p=0.07
****
**
Ashcroft score
0
1
2
3
4
Masso
ns T
rich
rom
e
**
p=0.08
****
**
Masson's Trichrome
-100 -80 -60 -40 -20 0
Collagen
TIMP-1
MMP12
TIMP-1
MMP8
PAI-1
WISP-1
Reduction from vehicle (%)
REDX10842 50 mg/kg BID
REDX10842 5 mg/kg BID
BA
Lp
lasm
a
*
*
*
**
***
**
-100 -80 -60 -40 -20 0
Collagen I
Collagen III
SMA
CTGF
TIMP-1
WISP-1
PAI-1
Fibronectin
CXCL10
Reduction from vehicle (%)
Gen
e
REDX10842 50 mg/kg BID
****
REDX10842 5 mg/kg BID
****
**
****
*
***
******
***
***
******
**
****
******
*** **
*
16
REDX10842 suppresses pro-fibrotic and pro-inflammatory gene expression in lung, BAL and plasma in murine bleomycin-induced IPF model
ROCK2 selective inhibitors for the treatment of fibrosis
-100 -80 -60 -40 -20 0
Collagen I
Collagen III
SMA
CTGF
TIMP-1
WISP-1
PAI-1
Fibronectin
CXCL10
Reduction from vehicle (%)
Gen
e
REDX10842 50 mg/kg BID
****
REDX10842 5 mg/kg BID
****
**
****
*
***
******
***
***
******
**
****
******
*** **
*
PD lung gene expression PD biomarkers
17
Effect of ROCK2 selective inhibitors in a murine unilateral ureteral obstruction (UUO) model
ROCK2 selective inhibitors for the treatment of fibrosis
Murine unilateral ureteral obstruction (UUO) model
• REDX10843 dosed therapeutically in the unilateral ureteral obstruction (UUO) murine model at 50 mg/kg BID
• Surgery performed on day 0, compound dosing from day 6-11
18
REDX10843 reduces kidney tubular damage and atrophy in murine UUO model
ROCK2 selective inhibitors for the treatment of fibrosis
• Tubular autofluorescence loss correlates with the break down of energy supply and is a damage marker for tubuli.
• Masson’s trichrome measure indicates tubular atrophy
• Enhanced tubular cell survival and reduced damage as measured by auto-fluorescence
• REDX10843 significantly reduced tubular damage
sham vehicle
UUO vehicle
UUO REDX10843 d6-11
Tubular damage
40
50
60
70
80
% lo
ss o
f fl
uo
rescen
ce
**
2.2
2.4
2.6
2.8
3.0
3.2
Sir
ius R
ed
sco
re
** UUO vehicle
REDX10843 50 mg/kg BID
Masson’s trichrome
35
40
45
50
55
60
65
% t
ub
ula
r a
tro
ph
y
*
19
REDX10843 modulates collagen deposition, macrophage infiltration and markers of tissue injury, inflammation and fibrosis in murine UUO model
ROCK2 selective inhibitors for the treatment of fibrosis
• REDX10843 reduced medulla collagen deposition, shown by a reduction in Sirius Red
• REDX10843 also reduced the macrophage infiltration score in the kidney medulla
• REDX10843 modulates gene expression markers of tissue injury, inflammation and fibrosis
2.2
2.4
2.6
2.8
3.0
3.2
Sir
ius R
ed
sco
re
** UUO vehicle
REDX10843 50 mg/kg BID
2.2
2.4
2.6
2.8
3.0
3.2
Sir
ius R
ed
sco
re
**
Sirius Red score
1.6
2.0
2.4
2.8
3.2
Macro
ph
ag
e in
fitr
ati
on
sco
re *
Macrophage infiltration
-100 -50 0
MCP-1
IL-6
TNF
IL-1
Nephrin
MMP2
Reduction from vehicle (%)
Gen
e
REDX10843 50 mg/kg BID
*
*
***
• REDX10843 dosed therapeutically in the murine STAM NASH model at 50 mg/kg BID or 50 mg/kg QD
• Telmisartan used as positive control and dosed at 10 mg/kg QD
• STZ administration at day 2, HFD induced from week 4, compounds dosed weeks 6-9
20
Effect of ROCK2 selective inhibitors in a murine liver models
ROCK2 selective inhibitors for the treatment of fibrosis
Murine CCl4-induced liver model Murine STAM NASH liver model
• REDX10842 dosed therapeutically in the murine CCl4-induced liver model at 5, 20 and 50 mg/kg BID
• CCl4 administered twice weekly IP
• Compounds dosed therapeutically from week 2 to 6
21
REDX10843 dosed QD or BID significantly reduces fibrosis area in a murine STAM NASH model
ROCK2 selective inhibitors for the treatment of fibrosis
contr
ol
Vehic
le Q
D
Vehic
le B
ID
REDX10
843
50 m
g/kg Q
D
REDX10
843
50 m
g/kg B
ID
Telim
ista
rtan
0.0
0.5
1.0
1.5
2.0
Fibrosis area (Sirius Red)
Sir
ius
Re
d p
os
itiv
e a
rea (
%)
**
***
**
22
REDX10843, dosed QD or BID, reduces profibrotic fibroblasts in a murine STAM NASH model
ROCK2 selective inhibitors for the treatment of fibrosis
contr
ol
Vehic
le Q
D
Vehic
le B
ID
REDX10
843
50 m
g/kg Q
D
REDX10
843
50 m
g/kg B
ID
Telim
ista
rtan
0
2
4
6
Reticular Fibroblasts (ER-TR7)
Po
sit
ive
are
a (
%)
**
*****
Original magnifications, x200
23
REDX10842 reduces fibrosis and collagen deposition in the liver in murine CCl4-induced liver model
ROCK2 selective inhibitors for the treatment of fibrosis
0
100
200
300
400
500
Hyd
roxyp
rolin
e (
g/l
iver)
p=0.06
***
Hydroxyproline
0
1
2
3
4
5
6
7
Co
lla
gen
Are
a F
racti
on
(%
)
***
Sirius Red (collagen I&III)
0
1
2
3
4
5
Ish
ak G
rad
ing
***
Ishak Score
Vehicle BID REDX10842 5 mg/kg BID REDX10842 20 mg/kg BID REDX10842 50 mg/kg BIDNo CCl4
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Liv
er
Ind
ex
no CCl4
vehicle
REDX10842 5 mg/kg BID
REDX10842 20 mg/kg BID
REDX10842 50 mg/kg BID
p=0.08**
24
REDX10842 reduces pro-fibrotic and pro-inflammatory gene expression in the liver in murine CCl4-induced liver model
ROCK2 selective inhibitors for the treatment of fibrosis
PD liver gene expression
-100 -80 -60 -40 -20 0
TIMP-1
MMP9
MMP8
S100A9
ALT
Reduction from vehicle (%)
Pla
sm
a p
rote
in
*
***
-125 -100 -75 -50 -25 0
CTGF
WISP-1
IL-6
CXCL10
PDGF-BB
Bcl2
TNF
Reduction from vehicle (%)
Gen
e
***
*
*
**
**
***
*
**
PD plasma gene expression
• Selective inhibition of ROCK2 is an exciting approach to target fibrosis
• Redx series has a good preclinical profile
• Potent and highly selective ROCK2 inhibitors against ROCK1 and against a panel of kinases and other receptor targets
• Redx ROCK2 inhibitors suppress pathways associated with fibrosis in in vitro kidney and liver models
• Early PK/PD evidence of target engagement of physiologically relevant pathways for fibrosis
• Robust preclinical efficacy demonstrated with REDX10843 and REDX10842 in murine liver, kidney and lung fibrosis models
• No safety issues observed in preliminary in vitro studies (cardiotoxicity, genotoxicity, mutagenicity, CYP profile) and in vivo rodent toxicology studies (14-day and CV)
• Redx plan to select an orally administered ROCK2 selective development candidate in H2 2019 and aim to enter clinical trials in 2021
25
Redx ROCK2 inhibitor programme summary
ROCK2 selective inhibitors for the treatment of fibrosis
26
Acknowledgments
ROCK2 selective inhibitors for the treatment of fibrosis
Biology
• Katie Anderson
• Sara Ceccarelli
• Kay Eckersley
• Rebecca Holland
• Rosie Knowles
• Emily Offer
DMPK
• Stuart Best
• Amy Cooke
• Alison Hunter
• Philip MacFaul
• Andrew Taylor
• Rebecca Taylor
Chemistry
• Inder Bhamra
• Andrew Belfield
• Matthew Box
• Chiara Colletto
• Charles Crossland
• Gayle Douglas
• Camille Gignoux
• Neil Hawkins
• Jean Marc Henry
• Paula Jackson
• Steven Glossop
• Jean-Francois Margathe
• Marcin Odachowski
• Sam Smith
Management
• Richard Armer
• Peter Bunyard
• Clifford Jones
Thank You