PLN-74809, a dual αVβ6/αV 1 integrin inhibitor, inhibits fibrosis in ... · PLN-74809, a dual...
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PLN - 74809, a dual α V β 6 /α V β 1 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 Popov 2 and J Schaub 1 1 Pliant Therapeutics, South San Francisco, CA, 2 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA Rationale Integrins α V β 6 and α V β 1 are heterodimeric proteins that bind and activate latent TGF-β. In primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), α V β 6 and α V β 1 integrins are thought to play a role in the development and propagation of fibrosis through TGF-β activation by cholangiocytes and stellate cells, respectively. We have identified an orally available, dual-selective, small-molecule inhibitor of α V β 6 and α V β 1 , PLN-74809, and tested its ability to block fibrosis through inhibition of integrin-mediated TGF-β activation. Methods IHC and ELISA-based assays were used to evaluate expression of α V β 6 and α V β 1 in human tissue samples from PSC and PBC patients. Anti-fibrotic properties of PLN-74809 and a similarly potent, dual-selective compound, PLN-75068, were evaluated in vivo in two mouse models of biliary fibrosis (Mdr2 -/- and DDC diet) and in precision-cut liver tissue slices (PCLivS) prepared from PSC and PBC patient liver explants by IHC, hydroxyproline (OHP) quantitation, and gene expression analysis. Results 1) Integrins α V β 6 and α V β 1 were significantly elevated in fibrotic Mdr2 -/- mice, and in PSC and PBC patient liver tissue 2) PLN-75068 reduced relative hepatic collagen levels and serum ALP levels in DDC diet-injured mice 3) PLN-74809 reduced hepatic TGF-β signaling, relative hepatic collagen levels, and serum ALP levels in Mdr2 -/- mice 4) PLN-74809 reduced collagen gene expression in PCLivS prepared from PSC and PBC patient liver explants Integrins α V β 6 and α V β 1 Activate Latent TGF-β, Resulting in Profibrotic Gene Expression Conclusions Dual inhibition of α V β 6 (on cholangiocytes) and α V β 1 (on fibroblasts) offers a targeted approach for blocking TGF-β signaling in biliary fibrosis Levels of α V β 6 and α V β 1 , two integrins that activate latent TGF-β through binding to LAP, were increased in biliary fibrosis in human and mouse samples Dual inhibition of α V β 6 /α V β 1 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 α V β 6 /α V β 1 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 Diet Vehicle 75068 150 mg/kg BID 75068 500 mg/kg BID OCA 30 mg/kg QD 0 100 200 Serum Alkaline Phosphatase U/L ** ** * p = 0.06039 **p<0.01 TGF-β-Activating Integrins α V β 6 and α V β 1 Are Present at Elevated Levels in Liver Tissue with Biliary Fibrosis IHC staining showed α V β 6 expression and SMAD3 phosphorylation in PSC and PBC liver tissue ELISA-based assay showed elevated α V β 1 levels in PSC liver tissue α V β 6 levels were elevated in Mdr2 -/- mouse livers; Itgb6 -/- mice were protected from fibrosis α V β 1 levels were elevated in Mdr2 -/- mouse livers 1 Control PSC 0 5 10 15 Human Integrin α v β 1 pg/µg total protein ** Balb/c Mdr2 -/- 0.0 0.1 0.2 0.3 pg/µg total protein p = 0.0707 Integrin α v β 1 Balb/c Mdr2 -/- 0 2 4 6 pg/µg total protein *** Integrin α v β 6 Dual α V β 6 /α V β 1 Inhibition Reduced Hepatic Collagen Deposition in the DDC Biliary Fibrosis Model Week 2 Week 4 Week 6 Week 8 Tissue Collection Week 0 PLN-75068 oral BID DDC diet Picrosirius red β 6 Integrin 4 weeks of 0.03% DDC feeding induced collagen deposition and integrin α V β 6 expression in the liver Control Diet Vehicle 75068 150mg/kg BID 75068 500mg/kg BID OCA 30mg/kg QD 0.0 0.2 0.4 0.6 Relative Hepatic Collagen (OHP) ug OHP/mg liver * ** p = 0.06253 Dual α V β 6 /α V β 1 inhibition with PLN-75068 significantly and dose-dependently reduced collagen deposition and serum ALP in DDC-injured mice PSC Liver PBC Liver pSMAD3 β 6 Integrin Peng et al., 2016 Mdr2 -/- Mdr2 -/- ;Itgb6 -/- 2 ***p<0.001 *p<0.05; **p<0.01 Dual α V β 6 /α V β 1 Inhibition Reduced Hepatic Collagen Deposition in the Mdr2 -/- Biliary Fibrosis Model 3 Week 3 Week 6 Week 9 Week 12 Tissue Collection Week 0 PLN-74809 oral QD Mdr2 -/- 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 Picrosirius red PLN-74809 Dual α V β 6 /α V β 1 inhibition with PLN-74809 significantly and dose-dependently reduced SMAD3 phosphorylation and collagen deposition in Mdr2 -/- livers Balb/c Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0.0 0.1 0.2 0.3 pSMAD3 (Normalized to total protein) * *** Hepatic pSMAD3 PLN-74809 (QD) *** *p<0.05; **p<0.01; ***p<0.001 Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0 200 400 600 Serum Alkaline Phosphatase IU/L PLN-74809 (QD) ** Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0 1 2 3 4 Serum Total Bilirubin mg/dL PLN-74809 (QD) Dual α V β 6 /α V β 1 inhibition with PLN-74809 dose-dependently reduced serum markers of biliary injury Vehicle 100 mg/kg 300 mg/kg 1000 mg/kg 0 50 100 150 200 Relative Hepatic Collagen (OHP) µ g of OHP/100 mg liver PLN-74809 (QD) * **p<0.01 Dual α V β 6 /α V β 1 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 2 0.0 0.5 1.0 1.5 Viability Relative Viability PBC PSC DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 COL1A1 Relative expression PLN-74809 **** * p = 0.0897 DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 COL1A2 Relative expression PLN-74809 * DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 COL3A1 Relative expression ** PLN-74809 DMSO 100 nM 1 µ M 10 µ M Alk 5 Inh 0.0 0.5 1.0 TGFB1 Relative expression *** * PLN-74809 p = 0.0559 PCLivS were viable for 48 hours in culture Picrosirius red staining for collagen showed extensive fibrosis in PCLivS tissue PSC Liver PBC Liver *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001
Transcript of PLN-74809, a dual αVβ6/αV 1 integrin inhibitor, inhibits fibrosis in ... · PLN-74809, a dual...
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
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tal p
rote
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Balb/c
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Integrin αvβ1
Balb/c
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2
4
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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
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Mdr
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tgb6
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2
***p<0.001
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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
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0.0
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orm
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ed to
tota
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tein
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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
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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
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bilit
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PBCPSC
DM
SO
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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
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h
0.0
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Rel
ativ
e ex
pres
sion
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PLN-74809
DM
SO
100
nM
1 µM
10 µ
M
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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
