Regulating Liver Fibrosis with Tyrosine Kinase Inhibitors

Natalia Veronica (A0098827J)

Department of Pharmacy, Faculty Science
Final Year Project P2 presentation
Supervisor: Associate Professor Ho Han Kiat

What is liver fibrosis

1 of 28

Liver fibrosis:
Excessive accumulation of extracellular matrix in response to chronic
liver injury

Chronic injury:
Viral infection
(Hepatitis B and C)
Alcohol
Normal
liver

Fibrotic
liver

Cirrhotic
liver

Cancerous liver
(hepatocellular
carcinoma)

Rockey DC, Friedman SL. Hepatic Fibrosis and Cirrhosis. 2012:64-85.

Complications of liver fibrosis

2 of 28

Liver cirrhosis
Increases risk of
hepatocellular carcinoma
(HCC)
HCC accounts for 70 % to 85 % of the total
liver cancer burden worldwide

In men, liver cancer is the 5th

leading cause of cancer death.

In women, liver cancer is the 9th

leading cause of cancer death.

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: a cancer journal for clinicians. Mar-Apr 2011;61(2):69-90.
2. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.
International journal of cancer. Mar 1 2015;136(5):E359-386.

Regulation of extracellular matrix

(ECM) homeostasis
Matrix
synthesis

Normal
liver

Matrix
degradation
Matrix
homeostasis

Fibrogenesis

Matrix
synthesis

Fibrotic
liver

3 of 28

Fibrinolysis
Matrix
degradation

Matrix
homeostasis

Fibrogenesis

Factors contributing to fibrogenesis

Increase in matrix synthesis
Decrease in matrix
degradation

>

Fibrinolysis

Factor regulating fibrinolysis

Matrix metalloproteinase (MMPs):
matrix degradation
Tissue inhibitor of metalloproteinase (TIMPs):
inhibitors of MMPs
Rockey DC, Friedman SL. Hepatic Fibrosis and Cirrhosis. 2012:64-85.

Hepatic stellate cells (HSCs) in liver

fibrosis

4 of 28

Chronic liver injury

Injury
Recruitment of inflammatory cells
Inflammation
NK cells

T cells

Kupffer cells
Release of cytokines

Activation

Activation of HSCs
and fibrogenesis

Quiescent
hepatic stellate
cells (HSCs)

Activated HSCs

Increase ECM production

Collagen type I and III
Increase TIMPs production
Decrease MMPs
production

TIMPs
MMPs

ECM degradation
MMPs

MMPs activity
inhibited

ECM accumulation

Rockey DC, Friedman SL. Hepatic Fibrosis and Cirrhosis. 2012:64-85.

Current therapeutic options

5 of 28

Alcohol
abstinence
Antiviral therapy
(anti-hepatitis B
and C)
Risk of reversion
to fibrosis

Injury

Corticosteroids
*Incomplete
suppression
fibrogenesis

Inflammation

Target fibrogenic
signaling in HSCs

Activation of HSCs
and fibrogenesis

Chronic liver injury

Recruitment of
inflammatory cells

NK cells

T cells

Kupffer cells
Release of cytokines

Activation

Quiescent HSCs

Activated HSCs

Liver transplantation is the only curative treatment for end stage liver fibrosis
*Limited organ donor and compatibility
Rockey DC, Friedman SL. Hepatic Fibrosis and Cirrhosis. 2012:64-85.

Fibrogenic signaling in HSCs

6 of 28

Neurochemical
Cannabinoids
Opioids
Serotonin
*cannabinoid
inhibitions leads to
depression

Adipokines

Reninangiotensin
system
*less
progression in
inflammation
but not in
fibrosis

Activated HSCs

Nuclear
receptors:
PPAR
FXR
PXR

Endothelin 1 and
nitric oxide

Tyrosine
kinases

Cohen-Naftaly M, Friedman SL. Current status of novel antifibrotic therapies in patients with
chronic liver disease. Ther Adv Gastroenterol. 2011;4(6):391417.

Tyrosine kinases involved in liver

fibrosis

7 of 28

Activated HSCs have increase

expression of tyrosine kinase
receptors:
Vascular endothelium growth
factor receptors (VEGFR)
Fibroblast growth factor
receptors (FGFR)
Platelet derived growth factor
receptors (PDGFR)

Qu K, Huang Z, Lin T, et al. New Insight into the Anti-liver Fibrosis Effect of Multitargeted Tyrosine
Kinase Inhibitors: From Molecular Target to Clinical Trials. Frontiers in pharmacology. 2015;6:300.

Preliminary studies
8 of 28

In-vitro liver fibrotic model: activation of LX-2 cells with

2 ng/mL transforming growth factor-1(TGF-1) for 24
hours
Fibrogenesis
-Fibrotic markers: -smooth
muscle actin (-SMA) and
collagen type I

Fibrinolysis
-TIMPs and MMPs

Optimal increased
expression of -SMA and
collagen type I
Tyrosine kinase inhibitors
reduced expression of SMA and collagen type I

Hypothesis
9 of 28

Tyrosine
kinase
inhibitors

Increase
MMPs
Decrease
TIMPs

Promote
ECM
degradation

Tyrosine kinase inhibitors can increase expression of MMPs and reduce

expression of TIMPs, thereby promoting degradation of ECM and
amelioration of liver fibrosis

Objectives
10 of 28

To optimise in-vitro liver fibrotic model using LX-2 cells stimulated

with TGF-1

To determine using this optimised model, tyrosine kinase inhibitors

efficacy in reversing liver fibrosis specifically by reducing TIMPs
expression and increasing MMPs expression
Tyrosine
kinase
inhibitors

Optimised
model

TIMPs
MMPs

Xu, L., Hui, A. Y., Albanis, E., Arthur, M. J., O'Byrne, S. M., Blaner, W. S., . . . Eng, F. J. (2005). Human hepatic stellate cell lines,
LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut, 54(1), 142-151. doi: 10.1136/gut.2004.042127

Methodology-in vitro fibrotic model

optimisation

11 of 28

Methodology-tyrosine kinase
inhibitors efficacy

12 of 28

Methodology-gene and protein

analysis

13 of 28

MMP2, MMP13, TIMP1 and TIMP2 expression analysis

Analysis of protein
expression with western blot

Analysis of gene expression

with quantitative RT-PCR

Tyrosine kinase inhibitors

14 of 28

AZD4547
BGJ398
PD173074

Sorafenib

BIBF1120

Concentration dependent effects of

TGF-1
Gene expression following different concentrations of TGF-1

Increased TIMP1 gene expression

Increased TIMP2 gene expression

Increased MMP2 gene expression

No significant changes in MMP13

gene expression

15 of 28

Time dependent effects of TGF-1

16 of 28

Gene expression following 2 ng/mL TGF-1

Highest TIMP1 gene expression at 8-hour

MMP2 gene expression plateau at 24-hour

TIMP2 gene expression plateau at 24-hour

No observable trend for MMP13 gene

expression

Optimised conditions
17 of 28

At 2 ng/mL TGF- 1 for 24 hour:

-SMA

collagen
type I

TIMP1

TIMP2

MMP2

No change
MMP13

Extensive fibrogenesis (matrix synthesis) and minimal

fibrinolysis (matrix degradation)

Increased
fibrogenesis

Matrix
accumulatio
n
Matrix
homeostasis

Reduced
fibrinolysis

Tyrosine kinase inhibitors

decreased TIMP1 gene expression
AZD4547

BGJ398

PD173074

Sorafenib

18 of 28

BIBF1120

FGFRs inhibitors;
AZD4547, BGJ398
and PD173074
reduced TIMP1 gene
expression more than
multikinase inhibitors;
BIBF1120 and
sorafenib

Tyrosine kinase inhibitors

decreased TIMP2 gene expression
AZD4547

BGJ398

PD173074

Sorafenib

19 of 28

BIBF1120

BGJ398 showed
greatest TIMP2 gene
suppression
FGFRs inhibition can
be an attractive
antifibrotic approach

Tyrosine kinase inhibitors decreased TIMP1

and TIMP2 protein expression
AZD4547

Decreased TIMP1 and TIMP2 protein expression

BGJ398

Decreased TIMP1 and TIMP2 protein expression

20 of 28

Tyrosine kinase inhibitors decreased TIMP1

and TIMP2 protein expression
BIBF1120

Decreased TIMP1 and TIMP2 protein expression

PD173074

Decreased TIMP1 and TIMP2 protein expression

21 of 28

Tyrosine kinase inhibitors decreased TIMP1

and TIMP2 protein expression
Sorafenib

Decreased TIMP1 and TIMP2 protein expression

22 of 28

Tyrosine kinase inhibitors increased MMP2

gene expression
AZD4547

BIBF1120

PD173074

Sorafenib

23 of 28

BGJ398

AZD4547 showed
greatest increase in
MMP2 gene
expression

Tyrosine kinase inhibitors increased MMP13

gene expression
AZD4547

BIBF1120

PD173074

Sorafenib

25 of 29

BGJ398

AZD4547 showed
greatest increase in
MMP13 gene
expression

Tyrosine kinase inhibitors treatment

24 of 28

Tyrosine
kinase
inhibitors

TGF-1 activated LX-2

cells
TIMPs

MMPs

BGJ398 showed more prominent reduction in

TIMPs
AZD4547 showed more prominent increase in
MMPs

TIMPs
MMPs

ECM degradation
MMPs

MMPs activity
inhibited

ECM accumulation

Future studies
26 of 28

Perform more biological

replicates to confirm the
findings

Determine changes in
protein expression of
MMPs

Investigating
downstream signalling
pathways involved in
tyrosine kinases
signalling such as Erk
and Akt

Conclusion
27 of 28

Tyrosine
kinase
inhibitors

TGF-1 activated LX-2

cells

Tyrosine kinase inhibitors:

Alter the balance between extracellular matrix degradation and synthesis
Increase MMPs and decrease TIMPs expression increase net
protease activity promote extracellular degradation
FGFRs inhibition as an attractive anti-fibrotic target

Acknowledgement
28 of 28

Associate Professor Ho Han Kiat

Ms. Carrie Soh