Presentation on World Congress of Immunology & Microbiology (SciTech Immuno-Microbiology 2019) At 20/6/2019

1. THE EFFECT OF
PRETREATMENT WITH
TOLL-LIKE RECEPTOR 4
ANTAGONIST
RESATORVID ON
METHOTREXATE-
INDUCED LIVER INJURY
IN RATS:
HISTOPATHOLOGICAL
STUDY
Alaa Fadhel Hassan
BSc. Pharm. & MSc.
Pharmacology

2. The original
project
It was part of the
fulfilment of the
requirements for
the degree of
M.Sc. in
Pharmacology, and
It was my whole
life 1 year ago…

4. Drug-induced liver injury
• Point to any liver injury caused by xenobiotics or
chemicals including drugs or medicinal herbs;
described as “the diagnosis of exclusion, made
when all common causes of liver damage are
ruled out”.
• It is the most common reason for drug
withdrawal (bromofenac & troglitazone), denied
approval (ximelagatran) & cessation of
development (fialuridine).
• Primarily categorized as intrinsic (predictable) &
idiosyncratic (non predictable).

5. Factors predisposing to drug-induced
liver injury
• Clinical or host-related factors, non-
modifiable as: age, gender, genetic factors.
• Drug-related factors: reflect drug-drug
interactions and drug physical properties
defined as “The rule of two”.
• Environmental factors: associated with
unhealthy nutrition, sedentary lifestyle and
heavy alcohol consumption.

6. Classification of drug-induced liver
injury
• Hepatocellular biochemical laboratory
assessment “the calculated R value”
hepatocellular, cholestatic or mixed injury.
• Causality assessment scoring methods
“Ruossel Uclaf; Maria & Victorino systems”.
• Histopathological finding identified by “Drug-
induced liver injury network” hepatitis &
cholestasis, acute liver failure, granulomatous
hepatitis, steatosis, vascular injury & etc.

7. Methotrexate-induced liver
injury
• Mechanism, mainly by 3 approaches: long
half-lived metabolites, non-selective
inhibition of folate pathway& generation of
free radicals.
• Ending with: oxidative stress and imbalance
of cellular oxidant/antioxidant enzymes
leading to stimulation & production of pro-
inflammatory cytokines (CK) that involves
Toll-like receptor 4 induced inflammatory
pathways.

8. Methotrexate-induced liver
injury
• Prevalence: it is less common after high
dose methotrexate than low dose
regimen, seems common in patients
with psoriasis than with rheumatoid
arthritis, the lowest incidence reported
with inflammatory bowel disease
patients.
• Histological pattern: typical changes
associated with methotrexate-induced
liver injury are steatohepatitis, fibrosis &
cirrhosis.

9. Prophylactic and protective
approaches against drug-
induced liver injury
• Regular follow-up & recommendations.
• Avoidance of risk factors (ex. drugs
interactions).
• Dosage regimen adjustment, switching
& withdrawal.
• Clinical trials with medications &
medicinal plants.
• Standard supplement.
• Acute liver failure antidote.

10. Toll-like receptor 4 (TLR4)
• It is a member of pattern recognition receptor family
(type I integral transmembrane glycoprotein), that
detect lipopolysaccharide of gram negative bacteria,
viral proteins & endogenous ligands.
• Hepatocytes are the main site for TLR4 expression
despite their weak response to TLR4 ligands. Kupffer
cells, stellate cells & dendritic cells directly respond to
TLR producing pro-inflammatory CK while playing an
indefinite role in sinusoidal endothelial cells, also
expressed by lymphocyte.

11. Toll-like receptor 4 pathways

12. Resatorvid (TAK 242)
• Is a selective inhibitor of TLR4 signal transduction pathway
(covalently binding the nucleophilic cysteine 747 residue
located at toll/interleukin receptor domain that is
necessary for TLR4 homodimeraization phase).
• It prevents monocytes & macrophage proinflammatory CK
& nitrous oxide production, it is suggested to ameliorate
inflammatory process correlated with insulin resistance in
diabetes, cardiac disease, biliary obstruction as well as
sepsis.
• It’s highly lipophilic, pass through blood brain-barrier. Its
plasma level raises after 3 hr. & decrease after 24 hr.

13. Aim of the study
This research aims to evaluate the
histopathological changes after pre-
treatment with resatorvid against
methotrexate induced-liver injury in
an albino-wistar rat model.

14. Materials & Methods

15. Place, period & source of animals
• The experiment was performed at the Iraqi
centre of cancer research & medical genetics,
Baghdad, Iraq (Oct., 17/2017-lasted for 14 days),
after being approved by The ethical committee of
the Pharmacology Department, College of
Medicine, Mustansiriyah University.
• Involved a total of 28 male albino Wistar rats
(aged 4–6 months and weighed 125–225 g) taken
from Kut Technical Institute, University of Wasit,
Iraq.

16. Experimental design
The animals were divided into random four groups (7
rats in each group) :
• Control group: kept on a regular diet and distilled
water (14 days).
• Vehicle pre-treated group: were administered
intraperitoneal (i.p.) DMSO (7 days) followed oral
methotrexate 0.2 mg/kg/d (7 days).
• Methotrexate treated group: left untreated (7 days)
followed by oral methotrexate 0.2 mg/kg/d (7 days).
• Resatorvid pre-treated group: were administered i.p.
resatorvid 5 mg/kg/d (7 days) followed by oral
methotrexate 0.2 mg/kg/d (7 days).

17. Chemicals & drugs
• Resatorvid (C15H17CIFNO4S): a white crystalline powder
[MedChemExpress, New Jersey, USA-DMSO solubility of
≥360 mg/mL], was dissolved in DMSO and diluted with
D/W to a final concentration of 17 mg/ml.
• Methotrexate (C20H22N8O5): 50 mg/ml injectable
solution[KOÇAK Farma, ÍSTANBUL, Turkey], diluted with
D/W to a final concentration of 0.333 mg/ml.
• Dimethyl sulfoxide (DMSO): purchased as 99.5% solution
[Central Drug House (P) Ltd., New Delhi, India]. It was
diluted with Distilled water (D/W) to 8% w/v solution
(the concentration used to dissolve resatorvid).

18. Assessment of liver histopathology
• 24 he after the end of treatment, rats were
anaesthetized, sacrificed, liver tissues were dissected out
and fixed in containers with 10% formalin (stored in
−28°C until processing).
• The traditional procedures of the paraffin-embedded
method were used to prepare liver tissues for
microscopic evaluation.
• Liver structure evaluation for the grade of the induced
histopathological changes done using non-alcoholic fatty
liver disease (NAFLD) scoring system.

19. NAFLD activity score (NAS)
components
Item Score Extent % Definition & comments
Steatosis 0
1
2
3
<5
5–33
>33–66
>66
Refers to the amount of surface area
involved by steatosis as evaluated on
low-to-medium power examination
40X–100X); minimal steatosis (<5%)
receives a score of 0 to avoid giving
excess weight to biopsies with very
little fatty change.
Lobular
inflammation
0
1
2
3
No foci
<2 foci/200x
2–4 foci/200x
>4 foci/200x
Acidophil bodies are not included in
this assessment nor is portal
inflammation.
Hepatocyte
ballooning
0
1
2
None
Few balloon cells
Many cells/ promin-
ent ballooning
The term “few” means rare but
definite ballooned hepatocytes.

20. Results

21. NAFLD activity score (NAS) components
of the treatment groups
Score component Groups*
Control
group**
Methotrexate
group**
Resatorvid pretreated
group**
Steatosis
Score 0 3 2
Extent <5% >66% 33–66%
Lobular inflammation
Score 0 2 0
Extent None 2–4 foci/200x No inflammation
Ballooning degeneration
Score 0 2 1
Extent None Many Few cells
Total scores*** 0 8 3
*n=28 rats for the treatment groups, **n=7 rats for each group,
***Total score graded as severe (>= 5), moderate (3-4) and mild (< 3).

22. Liver histopathological findings of the
control group
Liver section of normal control rats (no abnormality), H and
E ×100.

23. Liver histopathological findings of the
control group
Liver section of normal control rats (no abnormality),H and E
×100.

24. Liver histopathological findings of the
methotrexate-treated group
Liver section of methotrexate-treated rats (moderate to
severe steatosis) showing fatty degeneration of hepatic
cells & fatty cystic chains, H and E ×100.

25. Liver histopathological findings of the
methotrexate-treated group
Liver section of methotrexate-treated rats (moderate-to-
severe steatosis) showing fatty degeneration of hepatic cells,
fatty cyst chains with chronic inflammatory cells infiltration,
H and E ×100.

26. Liver histopathological findings of the
resatorvid pretreated group
Liver section of resatorvid pre-treated rats showing
moderate steatosis with hepatic cell fatty degeneration and
fewer number of fatty cysts chains. No inflammation
observed, H and E ×100.

27. Liver histopathological findings of the
resatorvid pretreated group
Liver section of resatorvid pre-treated rats showing moderate
steatosis with hepatic cell fatty degeneration and fewer
number of fatty cysts chains. No inflammation observed, H
and E ×100.

28. Conclusion
• Resatorvid hepatoprotective effect
against methotrexate-induced injury
was promising throughout resolving
the accompanying inflammation and
partial restoring histopathological fatty
alterations.
• The study support the role of
resatorvid inhibition of TLR 4 singling
pathways involved in the pathogenicity
of methotrexate-induced liver injury.

29. Recommendations
• Considering longer time pretreatment &/ co-
treatment courses with different
concentration of resatorvid to detect if there
is a better profile of protection against MTX-
induced liver injury.
• Other studies reported anti-inflammatory
and anti-cancer action of resatorvid in
preclinical trials, that’s would be very
interesting if an additive effect achieved in
both methotrexate-immune and anti-cancer
action besides inhibiting methotrexate-
induced liver injury.

30. Thank you