PLN-74809, a dual αVβ6/αVβ1 integrin inhibitor, inhibits fibrosis in precision-cut liver tissue from PSC and PBC patients and the Mdr2 knockout mouse

S Turner1, M Decaris1, S Ho1, C Chen1, G Lee1, V Rao1, M Marlow1, S Martin1, T Chen1, J Cha1, M Munoz1, T Hom1, K Leftheris1, Y Popov2 and J Schaub1

1Pliant Therapeutics, South San Francisco, CA, 2Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

RationaleIntegrins αVβ6 and αVβ1 are heterodimeric proteins that bind andactivate latent TGF-β. In primary biliary cholangitis (PBC) andprimary sclerosing cholangitis (PSC), αVβ6 and αVβ1 integrins arethought to play a role in the development and propagation offibrosis through TGF-β activation by cholangiocytes and stellatecells, respectively. We have identified an orally available,dual-selective, small-molecule inhibitor of αVβ6 and αVβ1,PLN-74809, and tested its ability to block fibrosis throughinhibition of integrin-mediated TGF-β activation.

MethodsIHC and ELISA-based assays were used to evaluate expression ofαVβ6 and αVβ1 in human tissue samples from PSC andPBC patients. Anti-fibrotic properties of PLN-74809 and a similarlypotent, dual-selective compound, PLN-75068, were evaluatedin vivo in two mouse models of biliary fibrosis (Mdr2-/- andDDC diet) and in precision-cut liver tissue slices (PCLivS) preparedfrom PSC and PBC patient liver explants by IHC,hydroxyproline (OHP) quantitation, and gene expression analysis.

Results1) Integrins αVβ6 and αVβ1 were significantly elevated infibrotic Mdr2-/- mice, and in PSC and PBC patient liver tissue2) PLN-75068 reduced relative hepatic collagen levels andserum ALP levels in DDC diet-injured mice3) PLN-74809 reduced hepatic TGF-β signaling, relative hepaticcollagen levels, and serum ALP levels in Mdr2-/- mice4) PLN-74809 reduced collagen gene expression in PCLivSprepared from PSC and PBC patient liver explants

Integrins αVβ6 and αVβ1 Activate Latent TGF-β, Resulting in Profibrotic Gene Expression

Conclusions

Dual inhibition of αVβ6 (on cholangiocytes) and αVβ1 (on fibroblasts) offers a targeted approach for blocking TGF-β signaling in biliary fibrosis

Levels of αVβ6 and αVβ1, two integrins that activate latent TGF-β through binding to LAP, were increased in biliary fibrosis in human and mouse samples

Dual inhibition of αVβ6/αVβ1 with PLN-74809 or PLN-75068 significantly reduced hepatic TGF-β signaling, hepatic collagen deposition, and serum ALP in either the Mdr2-/- or DDC mouse models of biliary fibrosis

Dual inhibition of αVβ6/αVβ1 with PLN-74809 reduced collagen gene expression in PCLivS prepared from PSC and PBC patient tissue explants PLN-74809 warrants further investigation as a direct anti-fibrotic in PSC and PBC clinical trials

References: Peng Z, et al. Hepatology 2016,6(1):217–232 Key abbreviations: ALP, alkaline phosphatase; DDC, 3.5-Diethoxycarbonyl-1.4-dihydrocollidine; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; LAP, latency-associated peptide; OCA, obeticholic acid; OHP, hydroxyproline; PBC, primary biliary cirrhosis; PCLivS, precision-cut liver slices; PSC, primary sclerosing cholangitis; QD, once daily

Disclosures: All authors, except YP, were employed by Pliant Therapeutics Inc. at the time of their contribution to the studies reported here

Control D

iet

Vehicl

e

7506

8 150

mg/kg

BID

7506

8 500

mg/kg

BID

OCA 30 m

g/kg Q

D0

100

200

SerumAlkaline Phosphatase

U/L

** *** p

= 0.

0603

9

**p<0.01

TGF-β-Activating Integrins αVβ6 and αVβ1 Are Present at Elevated Levels in Liver Tissue with Biliary Fibrosis

IHC staining showed αVβ6 expression and SMAD3 phosphorylation in

PSC and PBC liver tissue

ELISA-based assay showed elevated αVβ1 levels in

PSC liver tissue

αVβ6 levels were elevated in Mdr2-/- mouse livers; Itgb6-/- mice were protected from fibrosis

αVβ1 levels were elevated in Mdr2-/- mouse livers

1

Control

PSC0

5

10

15

Human Integrin αvβ1

pg/µ

g to

tal p

rote

in **

Balb/c

Mdr2-/-

0.0

0.1

0.2

0.3

pg/µ

g to

tal p

rote

in

p = 0.0707

Integrin αvβ1

Balb/c

Mdr2-/-

0

2

4

6

pg/µ

g to

tal p

rote

in ***

Integrin αvβ6

Dual αVβ6/αVβ1 Inhibition Reduced Hepatic Collagen Deposition in the DDC Biliary Fibrosis Model

Week 2 Week 4 Week 6 Week 8Tissue

Collection

Week 0

PLN-75068 oral BIDDDC diet

Picrosirius red β6 Integrin

4 weeks of 0.03% DDC feeding induced collagen deposition and integrin αVβ6 expression

in the liver

Control D

iet

Vehicl

e

7506

8 150

mg/kg BID

7506

8 500

mg/kg BID

OCA 30mg/kg

QD

0.0

0.2

0.4

0.6

Relative Hepatic Collagen(OHP)

ug O

HP/m

g liv

er * **p = 0.06253

Dual αVβ6/αVβ1 inhibition with PLN-75068 significantly and dose-dependently reduced collagen deposition and serum ALP in DDC-injured mice

PSC

Live

rPB

C Li

ver

pSMAD3β6 Integrin

Peng et al., 2016

Mdr

2-/-

Mdr

2-/- ;I

tgb6

-/-

2

***p<0.001

*p<0.05; **p<0.01

Dual αVβ6/αVβ1 Inhibition Reduced Hepatic Collagen Deposition in the Mdr2-/- Biliary Fibrosis Model3

Week 3 Week 6 Week 9 Week 12Tissue

Collection

Week 0

PLN-74809 oral QDMdr2-/-

Picrosirius red staining showed a dose-dependent decrease in hepatic collagen deposition with PLN-74809

Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg

Picr

osiri

us re

d

PLN-74809

Dual αVβ6/αVβ1 inhibition with PLN-74809 significantly and dose-dependently reduced SMAD3 phosphorylation and collagen deposition in Mdr2-/- livers

Balb/c

Vehicl

e

100 m

g/kg

300 m

g/kg

1000

mg/kg

0.0

0.1

0.2

0.3

pSM

AD

3(N

orm

aliz

ed to

tota

l pro

tein

)

****

Hepatic pSMAD3

PLN-74809 (QD)

***

*p<0.05; **p<0.01; ***p<0.001

Vehicl

e

100 m

g/kg

300 m

g/kg

1000

mg/kg

0

200

400

600

SerumAlkaline Phosphatase

IU/L

PLN-74809 (QD)

**

Vehicl

e

100 m

g/kg

300 m

g/kg

1000

mg/kg

0

1

2

3

4

SerumTotal Bilirubin

mg/

dL

PLN-74809 (QD)

Dual αVβ6/αVβ1 inhibition with PLN-74809 dose-dependently reduced serum markers of biliary injury

Vehicl

e

100 m

g/kg

300 m

g/kg

1000

mg/kg

0

50

100

150

200

Relative Hepatic Collagen(OHP)

µg

of O

HP/1

00 m

g liv

er

PLN-74809 (QD)

*

**p<0.01

Dual αVβ6/αVβ1 Inhibition Reduced Collagen Expression in PBC/PSC Patient Tissue

PCLivS from PBC and PSC patient explants showed a reduction in collagen gene expression with PLN-74809 treatment

4

Day 0 Day 2 Day 0 Day 20.0

0.5

1.0

1.5

Viability

Rela

tive

Via

bilit

y

PBCPSC

DM

SO

100

nM

1 µM

10 µ

M

Alk

5 In

h

0.0

0.5

1.0

COL1A1

Rel

ativ

e ex

pres

sion

PLN-74809

****

*p = 0.0897

DM

SO

100

nM

1 µM

10 µ

M

Alk

5 In

h

0.0

0.5

1.0

COL1A2

Rel

ativ

e ex

pres

sion

PLN-74809

*

DM

SO

100

nM

1 µM

10 µ

M

Alk

5 In

h

0.0

0.5

1.0

COL3A1

Rel

ativ

e ex

pres

sion

**

PLN-74809

DM

SO

100

nM

1 µM

10 µ

M

Alk

5 In

h

0.0

0.5

1.0

TGFB1

Rel

ativ

e ex

pres

sion

****

PLN-74809

p = 0.0559

PCLivS were viable for48 hours in culture

Picrosirius red staining for collagen showed extensive fibrosis in PCLivS tissue

PSC

Live

rPB

C Li

ver

*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001