Presentation of experimental research

1. ‫الرحيم‬‫الرحمن‬‫هللا‬ ‫بسم‬
«ُ‫ع‬َ‫رف‬َ‫ن‬َ‫ن‬ ‫ن‬َ‫م‬ ٍ‫ات‬َ‫ج‬َ‫ر‬َ‫د‬َ‫وق‬َ‫ف‬َ‫و‬ ُ‫آء‬َ‫ش‬
ٍ‫م‬‫ل‬ِ‫ع‬ ‫ي‬ِ‫ذ‬ِِّ‫ل‬ُ‫ك‬‫ـم‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ـ‬‫ي‬ِ‫ل‬َ‫ع‬»
‫العظيم‬‫هللا‬ ‫صدق‬
‫سورة‬‫يوسف‬-‫اآلية‬76

2. EVALUATION OFTHE EFFECTS OF
GIT 27 ANDTAK 242 ON
METHOTREXATE-INDUCED LIVER
INJURY
By Alaa Fadhel Hassan
M.Sc. student
Department of Pharmacology

3. 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 cause of liver damage are ruled out”.
■ It is the most common reason of drug withdrawal (bromofenac &
troglitazone), denied approval (ximelagatran) & cessation of
development (fialuridine).
■ Primarily categorized as intrinsic (predictable, onset within days) &
idiosyncratic (non predictable, occur within 8 wk. –1 yr.).

4. Factors predispose 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
life style and heavy alcohol consumption.

5. Patterns of drug induced liver injury
Classification dependent on:
■ Hepatocellular biochemical laboratory assessment “the calculated R
value” hepatocellular, cholestatic or mixed injury.
■ Causality assessment scoring system “Ruossel Uclaf Causality
Assessment Method; Maria &Victorino systems”.
■ Histopathological finding identified by “Drug induced liver injury
network” acute &/chronic hepatitis & cholestasis, acute liver failure,
granulomatous hepatitis, steatosis, vascular injury, drug related
neoplasm & etc.

6. Methotrexate
■ It is a 4-amino,10-methyl folate analogue derived from aminopterin.
■ Methotrexate pharmacodynamics:
I. Block the cytoplasmic de novo synthesis of purines, pyrimidines &
polyamines (inhibit the enzymes: dihydrofolate reductase,
thymidylate synthase, amido phosphoribosyl transferase & methylene
tetrahydrofolate reductase).
II. Increase intracellular build-up of adenosine thus decrease the release
of proinflammatory cytokines (inhibit the enzyme 5-
aminoimidazole-4carboxamide ribonucleotide transformylase,
adenosine deaminase & the mammalian target of rapamycin).

7. Methotrexate pharmacokinetics
■ Absorption at small intestine.
■ Wide distribution to body tissue, its distributed to synovial fluid,
cerebrospinal fluid & appear in breast milk.
■ Its Vd is about IL/kg & 50% of plasma concentration bound to serum
albumin.
■ Mainly concentrated in hepatic &/ renal tissue, spleen & skin.
■ Its hepatic metabolism by enzymes aldehyde oxidase & tissue
metabolism by enzyme folypolyglutamate synthase.
■ Excretion: renally (major pathway) & biliary (minor).

8. Methotrexate-induced liver injury
■ Mechanism, mainly by 3 approaches:
long half-lived metabolites,
non-selective inhibition of folate pathway,
generation of free radicals.
■ Prevalence: methotrexate-induced liver injury is less common after high
dose methotrexate than low dose regimen, seems common in patients
with psoriasis than with rheumatoid arthritis, lowest incidence reported
with inflammatory bowel disease patients.
■ Histological pattern: typical changes are steatohepatitis, fibrosis &
cirrhosis.

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

10. Toll like receptors
■ These are type I integral transmembrane glycoprotein family, consist of
2 domain: extracellular domain & cytoplasmic domain.They recognizes
pathogen- &/ damage-associated molecular patterns.
■ A total of 13 toll like receptors exist in mammals with 10 detected in
human genome.The receptors (TLRs) targeted in this study includes:
TLR2 mainly detect gram +ve bacterial lipopeptides & peptidoglycan.
TLR4 detect lipopolysaccharide of gram –ve bacteria & viral proteins.
TLR6 detect microbial cell wall components (diacyl & triacyl
lipopeptides & lipoproteins).

11. Toll like receptors mechanism of action,
TLR4 as ex.

12. TLR role in pathology of liver injury
■ Drug induced liver injury
■ Viral hepatitis, (HBV).
■ Hepatobiliary obstruction
■ Non-alcoholic fatty liver disease.
■ Alcoholic liver disease.
■ Fibrosis & cirrhosis.
■ Ischemic reperfusion injury &
allograft rejection.
■ Hepatic regeneration after partial
hepatectomy.
■ Hepatic autoimmune disease.
■ Hepatocarcinoma.

13. TAK 242 (Resatrovid)
■ It is a cyclohexene derivative.
■ It covalently bind the nucleophilic Cys747 located at
theTIR domain that is
necessary for the homodimeraization
phase ofTLR4.
■ It prevent monocytes & macrophage proinflammatory cytokine &
nitrous oxide production, thus it is suggested to ameliorate
inflammatory process correlated with insulin resistance in diabetes,
cardiac disease, biliary obstruction as well as sepsis.
■ Its highly lipophilic, can pass through blood brain barrier. Its plasma
level raise after 3 hr. & decrease after 24 hr.

14. GIT 27 (VGX-1027)
■ It is small isoxazoline compound, antagonize the
action of ligand stimulatedTLR 2/6 &TLR4 & affect
the genes involved in antigenic
presentation process.
■ Thus targets the secretory capacity of macrophages, inhibiting the
release of proinflammatory cytokines.
■ It’s been developed for treating inflammatory disorders such as type 1
diabetes mellitus, colitis, inflammatory bowel disease, pleurisy &
rheumatoid arthritis.
■ Its approximate half life is 90 min. with peak plasma concentration
achieved after 2 hr. & steady state concentration after 5 days.

15. AIM OFTHE STUDY
To investigate whether treating the animals withTAK 242 or GIT 27, would
reverses the changes induced by methotrexate or whether the tested drugs
have a valuable hepatoprotective potential especially considering that both
are anti-inflammatory immunomodulating agents.

16. MATERIALS AND METHODS

17. Placed & period of the study
■ The experiment was performed
at the Iraqi centre of cancer
research & medical genetics,
Baghdad, Iraq (Oct., 17/2017-last
for 14 days).
■ Included 35 male albino-wistar
rats (aged 4-6 months & weight
125-225 g) achieved from Kut
technical institute, Wasit, Iraq.
■ TAK 242 & GIT 27 were achieved
as powder & were dissolved in
dimethyl sulfoxide while
methotrexate achieved as
solution & diluted with D/W.

18. Experimental design
Animals were divided into random 5 groups:
■ Control group: kept on regular diet & D/W (14 days).
■ Vehicle pre-treated group: administered I.P. dimethyl sulfoxide (7 days)
then oral methotrexate (7 days).
■ Methotrexate group: left untreated (7 days) followed by oral
methotrexate (7 days).
■ TAK 242 pre-treated group: administered I.PTAK 242 (7 days) followed
by oral methotrexate (7 days).
■ GIT 27 pre-treated group: administered 4 I.P challenge doses of GIT 27
followed by oral methotrexate (7 days).

19. Samples & tissues collection
■ After 24 hr. of the end of the treatment, the rats were anesthetized with
ketamine/Xylazine & sacrificed.
■ Samples collected from heart blood by direct needle puncture, allowed
to be settled then centrifuged at 4000 rpm for 10 min., the collected
serum was stored for further analysis.
■ liver tissues dissected after cutting rat’s abdomen, they were fixed with
30 ml 10% formalin, then prepared for microscopic evaluation using the
traditional (paraffin-embedded method).

20. RESULTS

21. MTX effect on biomarkers of hepatic
function
Parameters Groups P value
Control MTX
ALT 41.55±15.49 56.31±10.87 0.022
AST 25.94±3.05 32.50±3.46 0.000
ALPL 13.22±3.80 19.61±4.49 0.009
TSP 2419.39±340.80 2009.44±313.96 0.030
Bb 1.23±0.18 1.80±0.85 0.047

22. TAK 242, GIT 27 pre-treatment effect on
biomarkers of hepatic function
Parameters Groups P value Groups P value
MTX TAK 242
+ MTX
MTX GIT 27
+ MTX
ALT 56.31±
10.87
37.50±
10.11
0.005 56.31±
10.87
44.19±
6.29
0.057
AST 32.50±
3.46
28.94±
3.17
0.042 32.50±
3.46
28.98±
2.76
0.044
ALPL 19.61±
4.49
13.82±
2.79
0.016 19.61±
4.49
14.60±
3.56
0.035
TSP 2009.44±
313.96
2439.57±
294. 28
0.024 2009.44±
313.96
2357.62±
414.52
0.063
Bb 1.80±
0.85
1.19±
0.64
0.035 1.80±
0.85
1.23±
0.11
0.086

23. MTX effect on inflammatory and oxidative stress
biomarkers
Parameters Groups P value
Control MTX
IL-6 50.21±13.04 76.60±14.92 0.006
TNF-α 137.77±38.39 196.18±65.56 0.012
LPO 181.24±9.04 199.76±7.89 0.002
MDA 50.59±6.28 65.35±16.24 0.035
GSH 0.25±0.05 0.19±0.07 0.039

24. TAK 242, GIT 27 pre-treatment effect on
inflammatory and oxidative stress
biomarkers
Parameters Groups P value Groups P value
MTX TAK 242 +
MTX
MTX GIT 27 +
MTX
IL-6 76.60±
14.92
55.95±
22.19
0.029 76.60±
14.92
54.53±
18.96
0.020
TNF-α 196.18±
65.56
150.39±
23.76
0.045 196.18±
65.56
149.12±
34.52
0.040
LPO 199.76±
7.89
189.76±
7.31
0.075 199.76±
7.89
188.89±
14.14
0.054
MDA 65.35±
16.24
43.68±
14.81
0.003 65.35±
16.24
45.51±
9.59
0.006
GSH 0.19±
0.07
0.26±
0.02
0.008 0.19±
0.07
0.25±
0.03
0.031

25. Treatment effects on liver histopathologicl
findings
NAFLD Activity Score (NAS) Components
Score
components
Groups
Control MTX TAK 242 GIT 27
Steatosis Score 0 3 2 2
Extent <5% >66% 33-66% 33-66%
Lobular
inflammation
Score 0 2 0 1
Extent None 2-4 foci
/200x
No
inflamma-
tion
<2 foci
/200x
Balloning
degeneration
Score 0 2 1 1
Extent None Many Few cells Few
cells
Total scores 0 8 3 4

26. Treatment effects on liver histopathologicl
findings – control group & methotrexate
group.
liver section of control group rat.
Stained with H & E 100X.
liver section of methotrexate group rat.
Stained with H & E 100X.

27. Treatment effects on liver histopathologicl
findings –TAK 242 & GIT 27 pretreated
groups
liver section ofTAK 242 pre-treated rat.
Stained with H & E 100X.
liver section of GIT 27pre-treated rat.
Stained with H & E 100X.

28. Conclusions
The results shown in this study conclude the followings:
■ Supporting the role ofTLR 4 andTLR2/6 singling pathways in the
pathogenicity of MTX-induced liver injury.TAK 242 (TLR4 blocker) and
GIT 27 (TLR2,TLR4 &TLR6 non-selective inhibitor) inhibition ofTLR
signaling pathways.
■ Pretreatment with daily doses ofTAK 242 was able to counteract the
biochemical markers associated with MTX-induced liver injury (serum
transaminases, Bb,TSP,TNF-α, IL-6 , MDA and GSH).
■ 4 x doses pretreatment with GIT 27 was able to counteract the serum
levels of (AST, ALPL,TNF-α, IL-6 , MDA and GSH) only, with decreased
histolopathological alteration of MTX-induced liver injury to a moderate
grade.

29. Recommendations
■ Considering longer time pretreatment courses with different
concentration of bothTAK 242 and GIT 27 to detect whether there is
better profile of protection against MTX-induced liver injury.
■ Considering co-treatment courses with different concentration of both
TAK 242 and GIT 27 to detect whether there is better profile of
protection against MTX-induced liver injury
■ Other studies reported anti-inflammatory and anti-cancer action of
bothTAK 242 and GIT 27 in preclinical trials, that’s would be very
interesting if additive effect achieved in both MTX-immune and anti-
cancer action besides inhibiting of MTX-induced liver injury.

30. THANKYOU