ROCK2 selective inhibitors for the treatment of fibrosis · ROCK2 is a nodal point in cell signalling pathways associated with fibrotic diseases ROCK2 selective inhibitors for the

(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


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


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


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

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-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


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


• 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


• 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


• 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


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


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


• 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


• 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


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


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



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


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



**

**

***

****

*** **

*

**

***

**

-100 -80 -60 -40 -20 0 20

HGF

PDGF-BB

MMP3

MMP2

TIMP-1

S100A9

WISP1

ICAM-1


Pla

sm

a b

iom

ark

er



**

***

***

**

**

**

-100 -80 -60 -40 -20 0 20

HGF

PDGF-BB

MMP3

MMP2

TIMP-1

S100A9

WISP1

ICAM-1


Pla

sm

a b

iom

ark

er



**

***

***

**

**

**

15

REDX10842 reduces fibrosis and collagen deposition in the lung in murine bleomycin-induced IPF model


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




BA

Lp

lasm

a

*

*

*

**

***

**

-100 -80 -60 -40 -20 0

Collagen I

Collagen III

SMA

CTGF

TIMP-1

WISP-1

PAI-1

Fibronectin

CXCL10


Gen

e


****


****

**

****

*

***

******

***

***

******

**

****

******

*** **

*

16

REDX10842 suppresses pro-fibrotic and pro-inflammatory gene expression in lung, BAL and plasma in murine bleomycin-induced IPF model


-100 -80 -60 -40 -20 0

Collagen I

Collagen III

SMA

CTGF

TIMP-1

WISP-1

PAI-1

Fibronectin

CXCL10


Gen

e


****


****

**

****

*

***

******

***

***

******

**

****

******

*** **

*

PD lung gene expression PD biomarkers

17

Effect of ROCK2 selective inhibitors in a murine unilateral ureteral obstruction (UUO) model


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


• 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


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


• 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


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


Gen

e


*

*

***

• 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


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


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


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


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




p=0.08**

24

REDX10842 reduces pro-fibrotic and pro-inflammatory gene expression in the liver in murine CCl4-induced liver model


PD liver gene expression

-100 -80 -60 -40 -20 0

TIMP-1

MMP9

MMP8

S100A9

ALT


Pla

sm

a p

rote

in

*

***

-125 -100 -75 -50 -25 0

CTGF

WISP-1

IL-6

CXCL10

PDGF-BB

Bcl2

TNF


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


26

Acknowledgments


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

(AIM:REDX)

Nicolas Guisot

Research Fellow

[email protected]

Documents

ROCK2 selective inhibitors for the treatment of fibrosis · ROCK2 is a nodal point in cell signalling pathways associated with fibrotic diseases ROCK2 selective inhibitors for the