peter bunyard; inder bhamra; emily offer; kay eckersley; claire … · 2020. 12. 24. ·...

Log[RXC006], M

Pre-clinical data using candidate compound RXC006 demonstrates that porcupine is a novel and promising target

for the treatment of Idiopathic Pulmonary FibrosisPeter Bunyard; Inder Bhamra; Emily Offer; Kay Eckersley; Claire Chaplin; Simon Woodcock; Catherine Eagle; Clifford D. Jones; Caroline Phillips; Richard Armer

Redx Pharma, Block 33S, Mereside, Alderley Park, Cheshire, SK10 4TG, UK; e: [email protected]; t: +44(0)1625 469937; www.redxpharma.com

• Inhibition of Wnt signalling impacts on several mechanisms that underpin tissue remodelling in fibrotic diseases such as suppression of inflammation, reduction of apoptosis, prevention of epithelial mesenchymal transition and inhibition of fibroblast activation. Canonical and non-canonical mechanisms are involved5,6,7.

BACKGROUND RESULTS

RESULTS

• The Redx porcupine inhibitor RXC006 is a potent suppressor of both canonical and non-canonical signalling pathways.

• RXC006 can potently suppress the release of Wnt3a from L-Wnt3a cells and Wnt5a from HLFs.

• RXC006 is able to potently suppress pro-fibrotic gene expression and collagen deposition in murine models of kidney, liver and lung fibrosis over a range of different doses.

• Suppression of fibrosis in the models is achieved at in vivo concentrations that predict a safe and well tolerated dose can be achieved in patients.

• RXC006 has been nominated as a candidate for development in Idiopathic Pulmonary Fibrosis and IND-enabling studies have been initiated with first time in human studies expected to begin in 2020.

Conclusions: RXC006 was able to show strong therapeutic anti-fibrotic effects in the murine bleomycin lung fibrosis

models as shown by significant reductions in Ashcroft Scores, Wnt and pro-fibrotic pathway suppression and hydroxyproline.

References: 1. Nusse R, Varmus H. EMBO J. 2012 Jun 13;31(12):2670-84. 2. Castellone M, Laukkanen M. Front Biosci (Schol Ed). 2017 Jan 1;9:31-45. 3. Miao CG, Yang Y. Cell Signal. 2013 Oct;25(10):2069-78. 4. Herr P, Basler K. Dev Biol. 2012 Jan 15;361(2):392-402. 5. Moon J, Zhou H. Proc Natl Acad Sci U S A. 2017 Feb 14;114(7):1649-1654. 6. Chen CW, Beyer C. Ann Rheum Dis. 2016 Apr;76(4):773-778. 7. Thompson, BA. Cancer Res 2015;75(15 Suppl):Abstract nr 5071. 8. Madan B 2016 May;89(5):1062-74. doi: 10.1016/j.kint.

RXC006 displays potent in vitro Wnt pathway inhibition in mechanistic & phenotypic assays

ADME profile of RXC006 is suitable for further progression and clinical development RXC006 suppresses fibrosis therapeutically in murine lung bleomycin models • Wnt signalling is known to be important for tissue remodelling in several pathologies including cancer, auto-immunity and fibrosis1,2,3.

• Porcupine (PORCN) is a membrane-bound O-acyltransferase required for and dedicated to palmitoylation of all Wnt ligands, an essential step in the processing of Wnt ligands for secretion4.

Protocols: Mice between the ages of 6 to 8 weeks were given bleomycin* on day 1. Compounds where then administered by oral gavage in 0.5% CMC

+ 0.1% tween (QD/BID as shown below) from day 7 until day 21 or day 14 to 28 (male C57/bl6 n=10 per cohort).*In the 21 day model, 70l of bleomycin (1.5U/kg) administered via the oropharyngeal route. In the 28 day model, 50l of bleomycin administered via the intra-tracheal route (1.25mg/kg)

A) Donor L-Wnt 3a cells secreting Wnt3a were treated RXC006 for 24 hours. Inhibition of Wnt3a secretion was detected via the transfer of media harvested onto a β-catenin reporter cell line8. RXC006 was able to suppress -catenin reporter activity in a dose dependent manner.

B) Immuno-fluorescence imaging also showed that RXC006 suppresses β-catenin stabilization directly in the Wnt-3a expressing cell line.

β-catenin reporter assay A) β-catenin staining in L-WNT3a cells(representative images)

untreated RXC006 300 nM

B)

RXC006 is a potent inhibitor of canonical Wnt secretion and signalling

Wnt5a staining in HLFs (representative image)

TGF-β 10 ng/mL TGF-β 10ng/mL+ RXC006 300nM

Wnt5a staining in HLFs (IC50)

IC50 0.14 nM

A)

TGF-β 10ng/mL

TGF-β 10 ng/mL+ RXC006300 nM

Wnt5a supernatant concentration

Wnt5a

Coomassieloading Control

B)

RXC006 is a potent inhibitor of non-canonical Wnt5a release in human lung fibroblasts

Human lung fibroblasts (HLFs) were incubated with TGFβ 10 ng/mL RXC006A) Wnt5a expression in HLFs was analysed by immunofluorescence and RXC006 was shown to inhibit the release of Wnt5a is a dose dependent manner B) Wnt5a expression in concentrated supernatant was analysed by Western blot (L-Wnt5a cells) and RXC006 was shown to suppress Wnt5a release

-SMA staining in HLFs

L-Wnt3a CM

L-Wnt3a CM+ 300 nM RXC006

-SMA staining in HLFs (representative image)

L-Wnt3a CM L-Wnt3a CM+ 300 nM RXC006

-S

MA

(gre

en)

/ D

AP

I (b

lue)

Wnt3a expressing L-Wnt3a cells were incubated RXC006 300nM for 48 hours and the supernatant (described as conditioned medium (CM)) removed.

HLFs were then incubated with 50% conditioned medium for 48 hours.

-SMA expression was measured byImmuno-fluorescence and RXC006 was shown to suppress -SMA

RXC006 reduces Wnt3a induced α-SMA expression in human lung fibroblasts (HLFs)

IC50 0.4 nM

WNT974 doses with demonstrated target engagement are well tolerated in patients with no reported bone or GI effects.

Predicted RXC006 exposure in patients that will deliver anti-fibrotic efficacy based on matching the exposure that was efficacious in the lung bleomycin model dosed at 25 mg/kg.

30 mg WNT974

Human RXC006target exposure

Human exposure of WNT974* and predicted efficacious human exposure for RXC006

IC50 was determined from cellular phenotypic assays (Capan-2 & Wnt5a release from fibroblasts) Human exposure scaled from in vitro and in vivo pre-clinical studies.*Wnt974 is a Porcupine inhibitor (Novartis) currently in phase 1/2 clinical trials for oncology.

10 mg WNT974

NanoString gene expression analysis of lung tissue from 21-day model: gene suppression with RXC006 treatment versus vehicle

Log2 (fold change)

Wnt signalling Pro-fibrotic

Log2 (fold change)

Inflammation

Log2 (fold change)

Wnt/TCF pathway

Log2 (fold change)

Results: 28-day model: 14-28 day dosing

Results: 21 day model: 7-21 day dosing Representative Histology: 21 day model

Image representative of group mean Ashcroft scores. Scale Bar indicates 1.2mm. Small regions of dense collagenous connective tissue (fibrosis; black arrows demarcate) and lymphocyte infiltrates/aggregates (*) are present. A bronchiole (Br) and blood vessels (BV) are Present. A bronchiole (Br) and blood (BV) are indicated.

Vehicle RXC006 25 mg/kg QD

Vehicle

RXC006 25 mg/kg QD

Pirfenidone 100 mg/kg BID

No bleomycin

Ashcroft Score

Ashcroft Score Hydroxyproline

Vehicle

RXC006 25 mg/kg QD

Nintedanib 60 mg/kg QD

No bleomycin

Lung weights after inflation with PFA

Sirius Red

X50 magnification

1-way ANOVA with Fischer’s LSD


• RXC006 is highly potent in reporter gene and phenotypic cellular assays

• Selectivity demonstrated in secondary pharmacology screens

• Micromolar solubility in physiologically relevant media

• Orally bioavailable with scalable exposure in preclinical species

Rodent PK profile (PO)

Gen

es

RXC006 demonstrates therapeutic efficacy in a CCl4 liver fibrosis model Protocol: C57/Bl6 male mice (n=10 per cohort) aged 10-12 weeks were administered carbon tetrachloride (CCl4) in mineral oil i.p. 2x per week for 3

weeks, sham mice received 0.9% saline in mineral oil. Starting at day 15 compounds were administered BID/QD (as shown below) by oral gavage in 0.5% CMC + 0.1% tween. Animals were sacrificed on day 32.

HydroxyprolineLiver weight / body weight index

Results: 32-day CCl4 model: 15-32 day dosing

Conclusions: RXC006 significantly reduced the increased liver weight and hydroxyproline content caused by CCl4 suggesting porcupine suppression was able to ameliorate tissue remodelling caused by hyperplasia and fibrosis.

no CCl 4

Vehic

le

RXC00

6 1

mg/k

g

RXC00

6 5

mg/k

g

Pirfe

nidone

100

mg/k

g

0

1

2

3

4

5

Hyd

roxyp

rolin

e (

mg

/liv

er) *

no CCl 4

Vehic

le

RXC00

6 1

mg/k

g

RXC00

6 5

mg/k

g

Pirfe

nidone

100

mg/k

g

4

6

8

10

12

Liv

er

weig

ht/

bo

dy w

eig

ht*

100

******

***

Untr

eate

d

Veh

icle

RXC00

6 1

mg/k

g QD

RXC00

6 5

mg/k

g QD

Pirfe

nidone

100m

gs/kg

BID

0

5

10

15

GLS

Cq

*

***

Glutaminase

Vehicle

RXC006 5 mg/kg QD

Pirfenidone 100 mg/kg BID

0.9% saline

RXC006 1 mg/kg QD


RXC006 demonstrates efficacy when dosed therapeutically in the UUO model Protocol: C57/Bl6 male mice (n=10 per cohort) aged 8-12 weeks were subjected to the a unilateral ureteral obstruction (UUO) procedure. At day 6,

RXC006 5mgs/kg QD or vehicle was administered by oral gavage in 0.5% CMC + 0.1% tween for 6 days.

Results: 12-day UUO model: 6-12 day dosing

Vehicle RXC006 5mgs/kg QD

Representative Histology

Conclusions: RXC006 showed strong therapeutic anti-fibrotic effects in the UUO model of kidney fibrosis via the

suppression of Axin-2, collagen deposition and pro-fibrotic mediators such as anti-CTGF.

Vehicle RXC006 5 mg/kg0

200

400

600

800

1000

IL-1

b/R

PS

18

(Rela

tive e

xp

ressio

n) **


50

100

150

200

250

TN

Fa/R

PS

18

(Rela

tive e

xp

ressio

n)

*


50

100

150

Axin

-2/R

PS

18

(Rela

tive e

xp

ressio

n) ****


100

200

300

Co

l1A

1/R

PS

18

(Rela

tive e

xp

ressio

n)

***


50

100

150

200

CT

GF

/RP

S18

(Rela

tive e

xp

ressio

n)

****


20

40

60

80

100

120

FN

/RP

S18

(Rela

tive e

xp

ressio

n)

**

Axin-2 FibronectinCollagen 1

TNFα CTGFIL-1ßVehicle RXC006 5 mg/kg

0

5

10

15S

iriu

s R

ed

po

sit

ive (

%)

***

Sirius Red

Vehicle RXC006 5 mg/kg Vehicle RXC006 5 mg/kg Vehicle RXC006 5 mg/kg

Vehicle RXC006 5 mg/kg Vehicle RXC006 5 mg/kg Vehicle RXC006 5 mg/kg

RXC006 is efficacious prophylactically in a Rat 10 day bleomycin model of lung injury

Conclusions: Prophylactic administration of RXC006 dose dependently suppressed tissue damage, immune cell infiltration, fibroblast recruitment and hyperplasia as measured by the Ashcroft score. The data indicated that RXC006 was engaging porcupine in vivo and that porcupine inhibition was able to suppress pro-fibrotic responses in the lung.

Vehicle

RXC006 10 mg/kg QD

RXC006 1 mg/kg QD

Untreated

RXC006 30 mg/kg QD

Representative HistologyAshcroft Score

Protocol: Male Sprague Dawley rats (n=8 per cohort) aged 10 weeks (300-400g) were administered 1.5U/kg of bleomycin (intra-tracheal route in

400 mls) on day 0. From day 1 until day 10, RXC006 was dosed by oral gavage in 0.5% CMC + 0.1% tween. Animals were sacrificed at day 10.

30 mg/kg 10 mg/kg 1 mg/kgVehicle Untreated

RXC0061-way ANOVA with Fischer’s LSD

Results: 10-day rat bleomycin model: 1-10 day dosing

Log[RXC006], M

% P

OS

cells

Time (h)

Co

nce

ntr

atio

n (

µM

) Mouse 25mg/kg

Rat 5mg/kg

Mouse 5mg/kg

Rat 20mg/kg

RXC006

Wnt pathway reporter assay IC50 (nM) 0.4

HPAF2 proliferation GI50 (nM) 0.8

% free PPB (hu/m) 10/3

logD 2.9

Thermodynamic solubility FeSSIF pH 5.0, mg/L 153

Mouse hepatocyte clearance T1/2 (h) 0.7

Human hepatocyte clearance T1/2 (h) 3.9

Re

lati

ve e

xpre

ssio

n

peter bunyard; inder bhamra; emily offer; kay eckersley; claire … · 2020. 12. 24. ·...

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