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 Turner ,
1 M Decaris ,
1 S Ho ,
1 C Chen ,
1 G Lee ,
1 V Rao ,
1 M Marlow ,
1 S Martin ,
1 T Chen ,
1 J Cha ,
1 M Munoz ,
1 T Hom ,
1 K Leftheris ,
1 Y Popov2 and J Schaub 1
1Pliant Therapeutics, South San Francisco, CA, 2Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Rationale TGF-β-Activating Integrins αVβ6 and αVβ1 Are Present Dual αVβ6/αVβ1 Inhibition Reduced Hepatic Collagen Dual αVβ6/αVβ1 Inhibition Reduced
1 at Elevated Levels in Liver Tissue with Biliary Fibrosis 3 Deposition in the Mdr2-/- Biliary Fibrosis Model 4 Collagen Expression in PBC/PSC Patient Tissue
Integrins αVβ6 and αVβ1 are heterodimeric proteins that bind and
activate latent TGF-β. In primary biliary cholangitis (PBC) and IHC staining showed αVβ6 expression ELISA-based assay showed
primary sclerosing cholangitis (PSC), αVβ6 and αVβ1 integrins are and SMAD3 phosphorylation in elevated αVβ1 levels in Mdr2-/- PLN-74809 oral QD

thought to play a role in the development and propagation of PSC and PBC liver tissue PSC liver tissue
β6 Integrin pSMAD3 Week 0 Week 3 Week 6 Week 9 Week 12
fibrosis through TGF-β activation by cholangiocytes and stellate Tissue
Human Integrin α v β 1
cells, respectively. We have identified an orally available, Collection

PSC Liver
dual-selective, small-molecule inhibitor of αVβ6 and αVβ1, **

pg/µg total protein

15
PLN-74809, and tested its ability to block fibrosis through Picrosirius red staining showed a dose-dependent decrease in
hepatic collagen deposition with PLN-74809
inhibition of integrin-mediated TGF-β activation. 10

5 PLN-74809
Picrosirius red staining for collagen showed PCLivS were viable for

PBC Liver
Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg extensive fibrosis in PCLivS tissue 48 hours in culture
Methods 0

Picrosirius red
ol

C
PS
tr
IHC and ELISA-based assays were used to evaluate expression of

on
C
**p<0.01
αVβ6 and αVβ1 in human tissue samples from PSC and Viability

PSC Liver
PBC patients. Anti-fibrotic properties of PLN-74809 and a similarly αVβ6 levels were elevated in Mdr2-/- mouse livers; αVβ1 levels were elevated in
1.5
PSC PBC

potent, dual-selective compound, PLN-75068, were evaluated Itgb6-/- mice were protected from fibrosis Mdr2-/- mouse livers

Relative Viability
in vivo in two mouse models of biliary fibrosis (Mdr2-/- and Integrin α v β 6 Integrin α v β 1 Dual αVβ6/αVβ1 inhibition with PLN-74809 significantly and dose-dependently 1.0

Mdr2-/-
DDC diet) and in precision-cut liver tissue slices (PCLivS) prepared *** 0.3 p = 0.0707 reduced SMAD3 phosphorylation and collagen deposition in Mdr2-/- livers 0.5
pg/µg total protein

pg/µg total protein

from PSC and PBC patient liver explants by IHC,

PBC Liver
0.2 Hepatic pSMAD3 Relative Hepatic Collagen 0.0
hydroxyproline (OHP) quantitation, and gene expression analysis. 4

Mdr2-/-;Itgb6-/-
(OHP) Day 0 Day 2 Day 0 Day 2

(Normalized to total protein)

200

µg of OHP/100 mg liver
0.1
2 0.3 *
*** 150
*** *

pSMAD3
Results 0
Peng et al., 2016 0.0 0.2
100 PCLivS from PBC and PSC patient explants showed a reduction in collagen gene
- /-

- /-
expression with PLN-74809 treatment
c

c
b/

b/

2
1) Integrins αVβ6 and αVβ1 were significantly elevated in
dr
al

dr
0.1

al
***p<0.001 50
M
B

M
B
COL1A1 TGFB1
fibrotic Mdr2-/- mice, and in PSC and PBC patient liver tissue 0.0 0
Dual αVβ6/αVβ1 Inhibition Reduced Hepatic Collagen

Relative expression
Relative expression
2) PLN-75068 reduced relative hepatic collagen levels and 2

kg

kg
c

le

kg

kg

kg
le
kg
1.0 p = 0.0897 * 1.0 p = 0.0559

b/

ic

ic
g/

g/

g/

g/

g/
g/
al
Deposition in the DDC Biliary Fibrosis Model

h
*

m
B

Ve

Ve
serum ALP levels in DDC diet-injured mice

00

00
0

30

10

30
10
***

10

10
0.5 0.5
PLN-74809 (QD) PLN-74809 (QD)
3) PLN-74809 reduced hepatic TGF-β signaling, relative hepatic DDC diet PLN-75068 oral BID
*p<0.05; **p<0.01; ***p<0.001
****

collagen levels, and serum ALP levels in Mdr2-/- mice Dual αVβ6/αVβ1 inhibition with PLN-74809 dose-dependently 0.0 0.0
Week 0 Week 2 Week 4 Week 6 Week 8

Alk 5 Inh
100 nM
DMSO

1 µM

10 µM
Alk 5 Inh
100 nM
DMSO

1 µM

10 µM
4) PLN-74809 reduced collagen gene expression in PCLivS Tissue reduced serum markers of biliary injury
Collection
prepared from PSC and PBC patient liver explants Picrosirius red β6 Integrin Serum Serum PLN-74809 PLN-74809
Alkaline Phosphatase Total Bilirubin
COL1A2 COL3A1
600 4
4 weeks of 0.03% DDC feeding

Relative expression
Relative expression
Integrins αVβ6 and αVβ1 Activate Latent TGF-β, induced collagen deposition 400
3 1.0 1.0

mg/dL
**
Resulting in Profibrotic Gene Expression **

IU/L
and integrin αVβ6 expression 2 *
in the liver 200
1
0.5 0.5

0 0 0.0 0.0

Alk 5 Inh
100 nM
DMSO
Alk 5 Inh
100 nM
DMSO

1 µM

10 µM
1 µM

10 µM
kg

kg

kg
le
Dual αVβ6/αVβ1 inhibition with PLN-75068 significantly and dose-dependently

kg

kg

kg
le
ic

ic
g/

g/

g/

g/

g/

g/
h

h
m

m
Ve

Ve
reduced collagen deposition and serum ALP in DDC-injured mice

00

00
10

30

10

30
PLN-74809

10
PLN-74809

10
PLN-74809 (QD) **p<0.01 PLN-74809 (QD)
*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001
Relative Hepatic Collagen Serum
(OHP) Alkaline Phosphatase
253

Conclusions
0 6
0.6 0.
p=
**
39

*
ug OHP/mg liver

60

200
 Levels of αVβ6 and αVβ1, two integrins that activate latent TGF-β through binding to LAP, were increased in biliary fibrosis in human
0.0

0.4
*
p=

and mouse samples

U/L

100
** **
0.2

Dual inhibition of αVβ6 (on

 Dual inhibition of αVβ6/αVβ1 with PLN-74809 or PLN-75068 significantly reduced hepatic TGF-β signaling, hepatic collagen deposition,
cholangiocytes) and αVβ1 (on
0.0 0
and serum ALP in either the Mdr2-/- or DDC mouse models of biliary fibrosis
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ID

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fibroblasts) offers a targeted

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 Dual inhibition of αVβ6/αVβ1 with PLN-74809 reduced collagen gene expression in PCLivS prepared from PSC and PBC patient tissue explants
D

kg
h

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kg

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approach for blocking TGF-β

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30

30
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 PLN-74809 warrants further investigation as a direct anti-fibrotic in PSC and PBC clinical trials
15

50

15

50
A

signaling in biliary fibrosis

C
8

C
8

8
06

06

06

06

*p<0.05; **p<0.01
75

75

75

75

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