RAS inh

1. 2018
Presented by
Sameh Saber Eid Mohamed
Department of Pharmacology and Biochemistry, Faculty of Pharmacy,
Delta University for Science and Technology

2. Drug repositioning
 drug repurposing, re-profiling, re-tasking or therapeutic
switching
 is the application of a known drug to treat different disease
or simply, existing drug for new indication
 Reasons: the current economic climate
 Already approved drug or safe drug candidates that failed
or succeeded in clinical trials

3. Success stories
 Examples:
 Duloxetine for stress urinary incontinence
 Ethacrynic acid for leukemia
 Aspirin for colorectal cancer
 Thalidomide for cancer, leprosy and multiple myeloma
 Tretinoin for leukemia
 Miltefosine (breast cancer) for leishmaniasis
 What is the most famous example of success worldwide ?

5. Our contributions (2018)
 Perindopril, Fosinopril and Losartan inhibited the progression
of diethylnitrosamine-induced hepatocellular carcinoma in
mice via the inactivation of nuclear transcription factor kappa-
B (toxicology letters) I.F. 3.92
 Renin-angiotensin system inhibition ameliorates CCl4-induced
liver fibrosis in mice through the inactivation of nuclear
transcription factor kappa-B (Canadian j. of physiology and
pharmacology) I.F. 2.3
 Lisinopril inhibits nuclear transcription factor kappa B and
augments sensitivity to silymarin in experimental liver fibrosis
(international immunopharmacology) I.F. 3.2

6. Our contributions (2018)
 Telmisartan, an AT1 receptor antagonist, attenuates murine
hepatocellular carcinoma via modulating ERK1/2, TAK1, NF-κB
and PPARγ signaling crosstalk (under review)
 Albendazole, mebendazole and Itraconazole as antitumor
agents (under review)
 Olmesartan ameliorates chemically-induced ulcerative colitis
in rats via modulating NFκB and Nrf-2/HO-1 signaling
crosstalk (accepted) Toxicology and Applied pharmacology I.F.
3.9

7. research activities 2019
 Epidermal growth factor (current research project)
 Ruxadustat (current research project)
 Itraconazole (in diabetic complications)
 Metformin (as antitumor agent)
 Sitagliptin (as antitumor agent: lung cancer, renal cell
carcinoma, HCC)
 Promising for therapeutic switching as anti-tumor agents:
o Diclofenac
o Cimetidine
o Chloroquine
o Clarithromycin
o 2-Methoxyestradiol
o Celecoxib

8. Members
 Dr. Sameh Saber (Pharmacology)
 Dr. Rania Khalil (Biochemistry)
 Dr. Amal Ghanim (Biochemistry)
 Dr. Ghada Sami (Pharmacology)
 Dr. Reham Goda (Microbiology)
 Dr. Wafaa Ezz (Microbiology)
 Dr. Ahmed Kira (pharmaceutics)
 Dr. Rasha Gamal (Pharmacology)

9. Introduction/liver fibrosis
 Liver fibrosis is a major cause of morbidity and mortality
worldwide due to chronic viral hepatitis and fatty liver
disease associated with obesity.
 Owing to the complex molecular pathogenesis, the
possibility for effective systemic treatment is relatively
limited.
 HSC activation characterizes the most critical event in
fibrosis
because these cells represent the primary source of ECM in
liver upon injury.

11. Introduction/Fibrosis
 In the healthy liver, quiescent HSCs have very low levels of
expression of components of the RAS and are unable to
secrete Ang-II.
 Following liver injury, activated HSCs express ACE and the
AT1 receptor, and acquire the ability to synthesize Ang-II,
which induces differentiation of HSCs into myofibroblasts
 Systemic infusion of Ang-II induces liver fibrosis.
Furthermore, studies using gene-deletion mice have
demonstrated that AT1-receptor-deficient mice are
protected from fibrosis.

12. Introduction/HCC
 HCC is a major global health problem
representing approximately 75% of primary liver
cancers, characterized by poor prognosis and is
considered the second most common cause of
cancer-related mortality
 Liver carcinogenesis is a complex process in
which different signaling pathways affecting cell
proliferation and angiogenesis are dysregulated.

14.  Sorafenib (a multikinase inhibitor) is the only
systemic therapy currently showing a certain
degree of effectiveness for HCC
 It only allows the increase of patients’ survival
in a few months
 Therefore, a critical need exists to explore and
evaluate possible alternative strategies for
effective management of HCC
Introduction/HCC

15. Introduction/HCC
 Several studies pointed to the role of RAS
components in the development of different
types of malignancies such as breast
hyperplasia, prostate cancer, pulmonary
metastases of renal cell carcinoma, esophageal
and keratinocyte carcinoma.
 However, little is known about the role of RAS
in the pathogenesis of HCC.

16. TGF-β contribution to liver fibrosis and HCC
 TGF-β contribution to liver fibrosis through
a) Direct activation of HSCs and differentiation into myofibroblasts
b) Stimulating synthesis of ECM
c) Inhibition of ECM degradation by increasing synthesis of TIMPs
 TGF-β contribution to HCC development through
a) Stimulation of metastasis through activation of EMT by
upregulation of MMP-2 through ERK1/2, P38MAPK pathways
b) Stimulation of proliferation by increasing cyclin D1 expression
c) Stimulation of angiogenesis by increasing VEGF through ERK1/2
signaling which affects promotion of inflammation and release of
fibrosis enhancing molecules from activated endothelial cells

17. Aim
The present study was conducted
 To examine the effect of RAS inhibition using
the ACEIs, perindopril and fosinopril and the
ARB, losartan on DEN-induced HCC and
CCl4 induced liver fibrosis
 To elucidate some of the mechanisms
implicated in their potential hepato-
protective effects.

18. Methods (experimental design)
GP Fibrosis model
(CCl4 induced, 5 weeks)
No. HCC model
(DEN induced, 16 weeks)
No.
Gp 1 Vehicle (olive oil) 6 Vehicle (saline) 6
Gp 2
CCl4 (i.p. inj. of 40% sol in olive oil) at a
dose of 1 ul/gm mouse twice a week
25
DEN (i.p. inj. of 1% sol in saline) week 1 (30 mg
kg-1), 2 (50 mg kg-1), 3 (50 mg kg-1), 4 (70 mg kg-1), 5
(100 mg kg-1), 6-16 (50 mg kg-1) once a week
60
Gp 3 CCl4/Silymarin (30 mg kg-1) 15 DEN/Sorafenib (30 mg kg-1) 30
Gp 4 CCl4/Perindopril (1mg kg-1) 15 DEN/Perindopril (1mg kg-1) 30
Gp 5 CCl4/Fosinopril (2 mg kg-1) 15 DEN/Fosinopril (2 mg kg-1) 30
Gp 6 CCl4/Losartan (10 mg kg-1) 15 DEN/Losartan (10 mg kg-1) 30
Gp 7 CCl4/PE (1mg kg-1)+SI (30 mg kg-1) 15 DEN/PE (1mg kg-1)+SO (30 mg kg-1) 30
Gp 8 CCl4/FO (2mg kg-1)+SI(30 mg kg-1) 15 DEN/FO(2mg kg-1)+SO (30 mg kg-1) 30
Gp 9 CCl4/LO (10mg kg-1)+SI(30 mg kg-1) 15 DEN/LO (10mg kg-1)+SO (30 mg kg-1) 30
Drug
therapy
started
10
days
post
induction
of
fibrosis
model
and
45
days
post
induction
of
HCC
model

19. Results/histologic examination of selected RAS inhibitors
(fibrosis/monotherapy)
I. Liver specimens from the CCl4-treated mice showed extensive fibrous tissue
deposition, score (3-4)
II. The administration of PE, FO, or LO improved the histological picture of liver
showing reduction of fibrosis, score (1-2) which was comparable to SI
monotherapy
(normal; H&E x200), (CCl4, SI, PE, FO and LO; Masson trichrome x200)

20. I. Kaplan-Meier survival plots showed that CCl4-treated mice had a higher
mortality rate.
II. The administration of SI, PE, FO, or LO resulted in significant increase in over all
survival compared with CCl4 treated mice.
III. log rank analysis for comparison of survival probability revealed non-significant
differences in cumulative survival of mice treated with RAS inh. compared with
SI as a single agent.
Survival analysis (fibrosis/monotherapy)
Vs
CCl4
P=
SI 0.01
PE 0.02
FO 0.07
LO 0.04

21. I. Serum AFP level was not significantly different in CCl4-treated mice compared
with normal mice.
II. Treatment of mice with the RAS inh. and SI resulted in non-significant
differences in the level of AFP compared with CCl4-treated mice.
III. Serum level of ALT was significantly increased in CCl4-treated mice compared
with normal mice.
IV. There were significant decreases in serum ALT levels in the treatment groups
compared with CCl4 group.
Data are expressed as mean ± SEM (n=6), statistical analysis was performed using one-way ANOVA,
followed by Tukey’s post-test, +P <0.05 vs. normal, *P < 0.05 vs. CCl4.
Effect on ALT and AFP (fibrosis/monotherapy)
No significant
differences were found
among treatment
groups

22. I. Mice treated with CCl4 had significantly higher level of hepatic VEGF and
hydroxyproline compared with normal mice.
II. Drug treatment significantly diminished the CCl4 induced increase in VEGF level
and hydroxyproline content.
Data are expressed as mean ± SEM (n=6), statistical analysis was performed using one-way ANOVA, followed
by Tukey’s post-test, +P <0.05 vs. normal, *P < 0.05 vs. CCl4, @P < 0.05 vs. SI
Effect on VEGF and hydroxyproline (fibrosis/monotherapy)
No significant
differences were found
among treatment
groups
VEGF was sig.
increased in FO-
treated mice comp.
with SI

23. I. Serum levels of TNF-α, TGF-β, MMP-2 and TIMP-1 were significantly elevated
in CCl4-treated mice compared with normal mice.
II. Drug treatment showed significant decreases in TNF-α, TGF-β, and TIMP-1
levels compared with CCl4-treated mice.
III. On the other hand, no significant differences were observed in MMP-2 level in the
treatment groups compared with CCl4-treated mice.
Data are expressed as mean ± SEM (n=6), statistical analysis was performed using one-way ANOVA, followed
by Tukey’s post-test, +P <0.05 vs. normal, *P < 0.05 vs. CCl4.
Effect on TNF-α, TGF-β, MMP-2 and TIMP-1
(fibrosis/monotherapy)
No significant
differences in their
levels were found
among treatment
groups

24. I. MMP-2:TIMP-1 ratio was significantly decreased in CCl4-treated mice compared
with normal mice.
II. PE-treated mice showed significant increase in the ratio of MMP-2:TIMP-1
compared with CCl4-treated mice.
III. On the other hand, no significant differences were observed in the ratio of MMP-
2:TIMP-1 in SI, FO and LO treatment groups compared with CCl4-treated mice.
Data are expressed as mean ± SEM (n=6), statistical analysis was performed using one-way ANOVA, followed
by Tukey’s post-test, +P <0.05 vs. normal, *P < 0.05 vs. CCl4.
Effect on MMP-2/TIMP-1 (fibrosis/monotherapy)
No significant
differences in the ratio
were found among
treatment groups

25. I. Drug treatment significantly diminished the CCl4-induced increase in the protein
expression of α-SMA and NFкB p-p65/p65 ratio and increased the level of NFкBia
II. In this regard, comparable effects were observed between the different drugs used and SI.
Data are expressed as mean ± SEM (n=4), statistical analysis was performed using one-way ANOVA, followed by
Tukey’s post-test, +P <0.05 vs. normal, *P < 0.05 vs. CCl4.
Effect on α-SMA, NFкB p-p65/p65 ratio and NFкBia (fibrosis/monotherapy)
No significant
differences were found
among treatment
groups

26. Ikk
TNF-
TGF-Β
TIMP-1
VEGF
TNF-
TGF-Β
TIMP-1
VEGF
SI
SM
A
TNF-
TGF-Β
TIMP1
MMP-
2
VEGF
SM
A
TNF-
TGF-Β
TIMP1
MMP-
2
VEGF
Ikk

27. I. Liver specimens from the DEN-treated mice showed solid sheets of malignant
enlarged hepatocytes with moderate to marked nuclear atypia, nuclear
hyperchromasia, increased nucleocytoplasmic ratio, pleomorphism and appearance of
tumor giant cells (grade 3).
II. The administration of PE, FO, or LO improved the histological picture of liver
showing moderate regression of malignancy to (grade 1) that was comparable to SO
monotherapy. (H&E x200)
Results/histologic examination of selected RAS
inhibitors (HCC/monotherapy)

28. I. Kaplan-Meier survival plots showed that DEN-treated mice had a higher mortality
rate.
II. The administration of SO, PE, FO, or LO resulted in higher survival probability
compared with DEN-treated mice.
III. log-rank analysis for comparison of survival probability revealed non significant
differences in cumulative survival of mice treated with the RAS inhibitors compared
with SO-treated mice.
Survival analysis (HCC/monotherapy)
Vs
DEN
P=
SO 0.029
PE 0.08
FO 0.04
LO 0.07

29. I. Serum levels of ALT and AFP were significantly increased in DEN-treated mice
compared with normal mice.
II. There were significant decreases in serum ALT and AFP levels in the treatment
groups compared with DEN treated mice.
Data are expressed as mean ± SEM (n=6), statistical analysis was performed using one-way ANOVA, followed
by Tukey’s post-test, +P <0.05 vs. normal, *P < 0.05 vs. DEN, @P < 0.05 for indicated pairs
Effect on ALT and AFP (HCC/monotherapy)
= No significant
differences were found
among treatment
groups compared with
SO
= No significant
differences were found
among treatment
groups compared with
SO

30. I. Levels of TGF-β, TNF-α, MMP-2 and VEGF were significantly elevated in DEN-treated
mice compared with normal mice.
II. Significant decreases in their levels were found in the treatment groups compared with DEN-
treated mice.
Data are expressed as mean ± SEM (n=6), statistical analysis was performed using one-way ANOVA, followed by Tukey’s
post-test, +P <0.05 vs. normal, *P < 0.05 vs. DEN, @P < 0.05 for indicated pairs
Effect on TGF-β, TNF-α, MMP-2 and VEGF (HCC/monotherapy)
= No significant
differences were found
among treatment
groups compared with
SO
= No significant
differences were found
among treatment
groups compared with
SO
= No significant
differences were found
among treatment
groups compared with
SO
Significant decrease in
TNF- level in PE-
treated mice compared
with SO

31. I. Mice treated with DEN had significantly higher level of cyclin D1 mRNA relative
expression, higher ratio of NFкB p-p65/p65, and lower NFкBia level presented as fold
change vs Normal control.
II. Drug treatment significantly diminished the DEN-induced effect on their levels.
Data are expressed as mean ± SEM (n=4), statistical analysis was performed using one-way ANOVA, followed by
Tukey’s post-test, +P <0.05 vs. normal, *P < 0.05 vs. DEN.
Effect on cyclin D1, NFкB p-p65/p65 ratio and NFкBia
(HCC/monotherapy)

32. Ikk
Cyclin
D1
TNF-
TGF-Β
MMP-
2
VEGF
Cyclin
D1
TNF-
TGF-Β
MMP-
2
VEGF
SO

33. Conclusion
 The present study, demonstrated the first evidence that NFкB
pathway is involved in the observed anti-fibrotic and anti-
tumor effect of RAS inhibitors
 as indicated by lower hepatic levels of the NFĸB p-p65/p65
ratio and higher levels of NFĸBia representing inactivation of
NFкB signaling pathway.
 The inhibition of transactivation of NFкB p65 by the selected
RAS inhibitors might be responsible for decreased expression
level of cyclin D1 mRNA, decreased levels of TNF- and TGF-
β, consequently, levels of VEGF and TIMP-1 were down
regulated. Additionally, inhibition of myofibroblast activation
and lower hepatic collagen content were established

34. Conclusion
 the tissue affinity of the ACEI has no impact
on its antifibrotic or anti-tumor effect in the
liver.
 interfering the RAS either through the
inhibition of ACE or the blockade of AT1
receptors has the same therapeutic benefit.

35. Finally,
The present study encourage clinical trials
to examine the prospective use of ACEIs
or ARBs in managing patients with
hepatic fibrosis and its consequences of
cirrhosis and HCC.
Conclusion