Evaluation of the effect of GIT 27 & TAK 242 on drug-induced liver injury

Republic of Iraq
Ministry of Higher Education and Scientific Research
Al-Mustansiriyah University
College of Medicine
Evaluation of the effects of GIT 27 and TAK 242 on
methotrexate-induced liver injury
A Thesis
Submitted to the College of Medicine and the Committee of Postgraduate
Studies of the University of Al-Mustansiriya in Partial Fulfillment of the
Requirements for the Degree of Master of Science in Pharmacology
By
Alaa Fadhel Hassan
(B.Sc. Pharmacy-2012)
Department of Pharmacology
Supervised by
Professor Assistant Professor
Bassim I. Mohammad Bassim Shehab Ahmed
M.B.Ch.B, MSc, PhD M.B.Ch.B, FICMS
Pharmacology Histopathology
2018 A.D.
1439 A.H.

"ُ‫ء‬‫آ‬َ‫ش‬َ‫ن‬ ‫ن‬َ‫م‬ ٍ‫ت‬‫ا‬َ‫ج‬َ‫ر‬َ‫د‬ ُ‫ع‬َ‫ف‬‫ر‬َ‫ن‬
ٍ‫لم‬ِ‫ع‬ ‫ي‬ِ‫ذ‬ ِّ‫ل‬ُ‫ك‬ َ‫ق‬‫و‬َ‫ف‬َ‫و‬
‫يـــــــــــــــــم‬ِ‫ل‬َ‫ع‬"
‫سورة‬‫يوسف‬
‫آ‬‫ية‬67

Declaration
We certify that the thesis entitled: “Evaluation of the effects of GIT 27
and TAK 242 on methotrexate-induced liver injury", prepared by
Alaa Fadhel Hassan under our supervisions. The study carried on in
the Department of Pharmacology, College of Medicine and Iraqi Center
for Cancer and Medical Genetic Researches, Al-Mustansiriyah
University-Baghdad in partial fulfilment of the requirements for the
degree of Master of Science in pharmacology.
Signature Signature
Professor Assistant Professor
Dr.Bassim I. Mohammad Dr. Bassim Shehab Ahmed
Supervisor Supervisor
In review of the available recommendations, I forward this thesis for debate by
examining committee
Signature
Professor
Dr. Ali Ismail A. Mohammed
Head of Department of Pharmacology
Date: /2018

Certification
We, the members of the examining committee, certify that; after reading
the thesis entitled, “Evaluation of the effects of GIT 27 and TAK 242
on methotrexate-induced liver injury”, in its contents, it is adequate
for the award of the degree of Master of Science in Pharmacology to the
postgraduate student Alaa Fadhel Hassan at the University of Al-
Mustansiriyah, College of Medicine, Department of Pharmacology.
Signature Signature
Assist. Professor Lecturer
Dr. Laith M. Abbas Dr. Ali K. Chelab
Member Member
Date: / /2018 Date: / /2018
Signature Signature
Professor Assist. Professor
Dr. Bassim I. Mohammad Dr. Bassim S. Ahmed
Supervisor (member) Supervisor (member)
Date: / /2018 Date: / /2018
Signature
Assist.Professor
Dr. Haidar M. Jawad
Chairman
Date: / /2018
Approved by the council of the College of Medicine, the University of
Al- Mustansiriyah
Signature
Professor
Dr. Ali Ismail A. Mohammed
The Dean
Date: / /2018

I
Dedication
To the person who made it possible to continue,
Professor G. H. Majeed

II
Acknowledgment
Precious Thanks to Almighty and Merciful GOD, Lord of Creation for
giving me the power and strength to accomplish the present work.
I would like sincerely to thank the Ministry of Higher Education and
Scientific Research, Al-Mustansiriyah University, College of
Medicine, Postgraduate Studies Department and the Department of
Pharmacology and Therapeutics, for their kind support and facilities
to the postgraduate students.
I am especially grateful and beholden to my supervisors: Professor
Bassim I. Mohammad AL-Sheibani (University of Al-Qadisiyah/
College of Pharmacy) and Dr. Bassim S. Ahmed (Al-Mustansiriyah
University, College of Medicine) for their continuous advices,
guidance and assistance, throughout my study.
I am very much grateful and thankful to the staff of Al-Mustansiriyah
University, The Iraqi center of cancer research and medical genetics,
department of experimental therapy for their kindly help and support
during the study.
It would be a pleasure to express my thanks and gratitude to Dr. Asma
A. Swadi, Dr. Ahmed S. Mahmoud, Nawras L. Wahab, Huda J. Merza
and Raghad A. Sabri for their long-last Support and cooperation.

III
Abstract
Background:
Methotrexate-induced liver injury is a common problem which is described
either as increased levels of hepatic biomarkers that is seen in patients with
inflammatory bowel disease and patients with rheumatoid arthritis or as
idiosyncratic induced liver injury that is seen in patients with inflammatory
bowel disease, or as fibrosis and cirrhosis in rheumatoid arthritis patients'
and of psoriatic patients. The typical profile of methotrexate-induced liver
injury is associated with abnormal level of hepatic biomarkers with fatty
changes in liver tissues accompanied with inflammation and oxidative
stress which rarely progresses to acute liver failure. The participation of
immune system that results in the production of proinflammatory cytokines
and chemokines is the probable link between methotrexate-induced
toxicity and toll like receptors pathways, which are the common participant
receptors of the immune system and their activation is required for
cytokines production and inflammatory processes, Both GIT 27 and TAK
242 are antagonist of toll like receptors.
Aim of study:
To investigate whether treating the animals with TAK 242 or GIT 27,
could reverse the injuries induced by methotrexate, or the tested drugs have
a valuable hepatoprotective potential, especially considering that both
drugs are anti-inflammatory and immunomodulating agents.
Materials and method:
Thirty five adult albino male rats (aged 4-6 months) (Weight 125-225 g)
were randomly divided into 5 groups (7 rats in each).

IV
Control group: rats were kept on regular diet and distilled water
throughout the fourteen experimental days.
Vehicle pre-treated group: rats were administered i.p. dimethyl sulfoxide
for 7 days followed by 7 days of oral methotrexate 0.2mg/kg/day.
Methotrexate group: rats were left untreated for 7 days followed by 7
days of oral methotrexate 0.2mg/kg/day.
TAK 242 pre-treated group: animals were administered i.p. TAK 242
5mg/kg/day for 7 days followed by 7 days of oral methotrexate 0.2mg/kg.
GIT 27 pre-treated group: rats were administered 4 i.p. challenge doses
of GIT 27 25mg/kg/day at 168, 120, 72 and 24 hours before starting
treatment with oral methotrexate 0.2mg/kg/day for 7 days.
At the end of experiment, the animals were anesthetized with i.m.
ketamine/xylazine and sacrificed. Heart blood was drawn and collected for
chemical analysis and the liver was preserved in formaline for
histopathological study.
Results:
Animals were treated with methotrexate show significant increase in
chemical markers in comparison with animals in control group, p value
was <0.05 with increase in serum values of: ALT, AST, ALPL, Bb, IL-6,
TNF-α, LPO and MDA and decrease in values of TSP and GSH as well as
sever histopathologic liver change with steatosis of grade (8) according to
NAFLD Activity Scores. Animals were pretreated with TAK-242 show
significant improvement in serum level of ALT, AST, ALPL, IL-6, TNF-
α, MDA and GSH; beside an improved histopthalogical profile of moderate
steatosis (grade 3). Animals were pretreated with GIT-27 show significant
changes in serum level of AST, ALPL, Bb, IL-6, TNF-α, MDA and GSH
with change in histopthalogical profile of moderate steatosis (grade 4).

V
Conclusion:
The present study suggests a hepato-protective effect of both TAK-242 and
GIT-27 against liver injury induced by methotrexate dependent on their
immunomodulation effect via antagonism of the inflammatory receptors
TLR4 and TLR2/6.

VI
List of contents
Subject Page
Dedication I
Acknowledgments II
Abstract III
List of Contents VI
List of Tables X
List of Figures XII
List of Abbreviations XIV
Chapter one: Introduction Page 1
1.1 Drug induced liver injury 2
1.1.1 Idiosyncratic versus Intrinsic drug induced
liver injury
2
1.1.2 Factors predispose to drug induced liver
injury
3
1.1.3 Patterns of drug induced liver injury 4
1.1.4 Histological patterns of drug induced liver
injury
6
1.1.4.1 Acute hepatitis 6
1.1.4.2 Acute liver failure (fulminant hepatitis) 6
1.1.4.3 Chronic hepatitis 7
1.1.4.4 Acute cholestatic injury 7
1.1.4.5 Chronic cholestasis and ductopenia 7
1.1.4.6 Granulomatous hepatitis 8
1.1.4.7 Steatosis and steatohepatitis 8
1.1.4.8 Vascular changes 9
1.1.4.9 Drug related neoplasm 9
1.1.4.10 Other patterns 9
1.1.5 Mechanisms of Drug induced liver injury 10
1.2 Toll like receptors in Pathophysiology of liver
injury
11

VII
1.2.1 Toll like receptors 11
1.2.2 Toll like receptors mechanism of action 12
1.2.2.1 MyD88-dependent pathway 13
1.2.2.2 MyD88 independent pathway 14
1.2.3 TLR2 14
1.2.4 TLR4 15
1.2.4.1 Co-receptors 16
1.2.4.2 Adaptor molecules 16
1.2.4.3 Transcription factors 16
1.2.4.4 Downstream signalling factors regulated by
TLR4
16
1.2.5 TLR6 17
1.2.6 Role in Pathophysiology of liver injury 17
1.2.6.1 Drug induced liver injury 17
1.2.7 TLR Therapeutic targeting 18
1.2.7.1 TLR2 18
1.2.7.2 TLR4 18
1.2.7.3 TLR7 19
1.2.7.4 TLR9 19
1.3 Methotrexate 20
1.3.1 Pharmacodynamics 21
1.3.2 Pharmacokinetics 22
1.3.3 Adverse effects and Toxicities 23
1.3.4 Methotrexate-induced Liver injury 25
1.3.4.1 Mechanism 25
1.3.4.2 Prevalence 26
1.3.4.3 Histological patterns 27
1.3.5 Prophylactic and protective approaches
against methotrexate-induced liver injury
28
1.3.5.1 Regular follow up and recommendations 28
1.3.5.2 Avoidance of risk factors (drugs
interactions)
28
1.3.5.3 Dosage regimen adjustment, switching and
withdrawal
30
1.3.5.4 Clinical trials with medications and
medicinal plants
30
1.3.5.5 Standard supplement and acute liver failure
antidote
31

VIII
1.3.5.6 Intervention by liver transplantation, plasma
exchange and bioartficial liver assist devices
31
1.4 TAK 242 31
1.4.1 TAK 242 Pharmacodynamics 32
1.4.2 TAK 242 Pharmacokinetic 33
1.4.3 TAK 242 in Clinical and Experimental trails 34
1.5 GIT 27 36
1.5.1 GIT 27 Pharmacodynamics 37
1.5.2 GIT 27 Pharmacokinetics 37
1.5.3 GIT 27 in Clinical and experimental trails 38
1.6 Aim of the study 41
Chapter two: Materials and Methods Page 42
2.1 Materials 43
2.2 Place and period of the Study 45
2.3 Experimental animals 46
2.3.1 Animal diet 46
2.4 Experimental design 46
2.4.1 Experimental model of MTX-induced liver
injury
47
2.5 Preparation of drugs 48
2.5.1 TAK 242 48
2.5.2 GIT 27 48
2.5.3 MTX 49
2.5.4 DMSO 49
2.6 Samples collection 49
2.6.1 Tissue samples collection 49
2.7 Chemical Markers 50
2.7.1 Markers of hepatic function 50
2.7.1.1 Total serum protein 50
2.7.1.2 Hepatocellular markers 50
2.7.1.3 Hepatobiliary bilirubin 51
2.7.2 Inflammatory markers 51
2.7.3 Biomarkers of oxidative stress 52
2.7.3.1 Lipid peroxide 52
2.7.3.2 Malondialdehyde 53
2.7.3.3 Reduced glutathione 53
2.8 Histopathological study 54

IX
2.8.1 Steps of the paraffin method according to
Bancroft and Stevens (1987)
54
2.8.2 Assessment of liver histopathology 57
2.9 Statistical analysis 59
Chapter three: Results Page 60
3.1 Treatment effect on rats’ weight 61
3.2 Treatment effect on markers of hepatic function 61
3.2.1 MTX effect on markers of hepatic function 61
3.2.2 TAK 242, GIT 27 pre-treatment effect on
markers of hepatic function
62
3.3 Treatment effect on inflammatory and oxidative
stress markers
67
3.3.1 MTX effect on inflammatory and oxidative
stress markers
67
3.3.2 TAK 242, GIT 27 pre-treatment effect on
inflammatory and oxidative stress markers
68
3.4 Correlation coefficient among study markers 73
3.5 Treatment effects on liver histopathologicl
findings
75
Chapter four: Discussion Page 82
4.1 Treatment effect on rats’ weight 83
4.2 MTX effects on study markers 83
4.2.1 MTX effects on markers of hepatic function 83
4.2.2 MTX effect on inflammatory and oxidative
stress markers
85
4.2.3 MTX effect liver histopathological finding 87
4.3 Effect of pre-treatment with TAK 242 and GIT
27
88
4.3.1 Effect of pre-treatment with TAK 242 on
hepatic function, inflammatory and oxidative stress
markers
88
4.3.2 Effect of pre-treatment with GIT 27 on
hepatic function, inflammatory and oxidative stress
markers
90
Chapter five: Conclusions and
Recommendations
Page 92
5.1 Conclusions 93
5.2. Recommendations 94
References Page 95

X
List of Tables
Tables Page
Table (2-1): List of instruments with their providers
and origin
43
Table (2-2): List of chemicals and drugs with their
providers and origin
44
Table (2-3): List of chemical analysis kits with their
providers and origins
45
Table (2-4): Experimental design 47
Table (2-5) assessment of liver histopathology 58
Table (3-1): Weight changes among rats’ groups
after 1 week (day 7) and 2 weeks (day 14)
61
Table (3-2): Serum liver enzymes changes between
rats treated with MTX and control group for 2
weeks
62
Table (3-3): Serum liver enzymes changes among
rats treated with MTX and TAK 242 (pre-treatment
groups) for 14 days, (N=7) for each
63
Table (3-4): Serum liver enzymes changes among
rats treated with MTX and GIT 27 (pre-treatment
groups) for 14 days, (N=7) for each
64
Table (3-5): Serum inflammatory and oxidation
parameters between rats treated with MTX and
control group for 14 days, (N=7) for each
68
parameters changes among rats treated with MTX
and TAK 242 (pre-treatment groups) for 14 days,
(N=7) for each
69
parameters changes among rats treated with MTX
and GIT 27 (pre-treatment groups) for 14 days,
(N=7) for each
70
Table (3-8): Pearson correlation and significance
value (2-tailed) of the changes in the study
parameters among all rats groups for 14 days,
(N=7) for each
74
Table (3-9): The assessment of liver injury
according to NAFLD histopathological grading
scores among the treatment groups MTX, TAK 242
81

XI
and GIT 27 (pre-treatment groups) for 14 days,
(N=7) for each

XII
List of figures
Figures Page
Figure (1-1): Patterns of DILI depending on
calculated R value
5
Figure (1-2):TLR4 signaling pathways 15
Figure (1-3): Structure of TAK 242 32
Figure (1-4): Metabolism of TAK 242 33
Figure (1-5): Structure of GIT 27 36
Figure (2-1): Administration of oral methotrexate
0.2mg/kg/day via rats’ oral gavage to the animals
48
Figure (3-1): Error bar chart show mean serum
ALT differences among treatment groups
65
Figure (3-2): Error bar chart show mean serum AST
differences among treatment groups
65
ALPL differences among treatment groups
66
Figure (3-4): Error bar chart show mean TSP
66
Figure (3-5): Error bar chart show mean serum Bb
67
Figure (3-6): Error bar chart show mean serum IL-6
71
TNF-α differences among treatment groups
71
Figure (3-8): Error bar chart show mean serum LPO
72
MDA differences among treatment groups
72
GSH differences among treatment groups
73
Figure (3-11): Liver section of normal control rats
(no abnormality) showing normal lobular
rearrangement
76
Figure (3-12): Liver section of MTX treated rats
(moderate to severe steatosis) showing hepatocyte
degeneration, microvesicular (red arrow) and
macrovesicular (green arrow) fat vacuoles are
shown connecting and opening onto each other
forming fatty cystic chains (black arrow)
77

XIII
Figure (3-13): Liver section of MTX treated rats
(moderate to severe steatosis) showing hepatocyte
fatty degeneration (red arrow) with moderate
inflammatory cells infiltration (black arrow)
78
Figure (3-14): Liver section of TAK 242 pre-treated
rats (moderate to severe steatosis) showing
hepatocyte fatty degeneration. No inflammatory
cells shown
79
Figure (3-15): Liver section of GIT 27 pre-treated
rats (moderate to severe steatosis) showing
hepatocyte degeneration, microvesicular and
macrovesicular fatty cysts. No inflammatory cells
shown
80

XIV
List of Abbreviations
A2a, A2b and
A3
Adenosine receptor
AICART 5-aminoimidazole-4carboxamide ribonucleotide
transformylase
ALF Acute liver failure
ALL Acute lymphoblastic leukemia
ALPL/AP Alkaline phosphatase
AMP/TP Adenosine monophosphate/triphosphate
AMPK AMP- activated protein kinase
ANTs Adenine nucleotide translocases
AST Aspartate aminotransferase
BCG Bacilli Calmette-Guérin
Bcl-2 B cell lymphoma 2
BCRP Breast cancer resistance protein
BECs Biliary endothelial cells
CC Chemokine
CD 4/-14 Cluster of differentiation 4/-14
CDK2 Cyclin dependent kinase-2
CK Cytokine
CME Coronary micro-embolization
COX2 Cyclooxygenase 2
CpG-DNA Unmetlylated cytosine phosphate guanine containing
deoxyribonucleic acid
CSF Cerebrospinal fluid
CYP450 Cytochrome P450 oxidation isozymes
DAMPs Damage-associated molecular pattern molecules
DHFR Dihydrofolate reductase
DILI/N Drug induced liver injury/network
DISH drug induced steatohepatitis
DMARDs Disease modifying antirheumatic drugs
DMSO Dimethyl sulfoxide
ECD Extracellular domain
ECM Extracellular matrix
ELIZA Enzyme linked immunosorbent assay
ER Endoplasmic reticulum
ERK Extracellular signal-regulated kinases
FFA Free fatty acids

XV
FR Free radicals
GPRD Database of general practice research in UK
GPx Glutathione peroxidase
GSH Reduced glutathione
GST Glutathione S-transferase
HBsAg Hepatitis B s antigen
HBV/CV Hepatitis B/C
HCG Human chorionic gonadotropin hormone
HD-MTX High dose methotrexate
HLA Human leukocyte antigen
HRP Avidin-Horserdish peroxidase
HSCs Hepatic satellite cells
HSP Heat shock protein
I/R injury Ischemia/reperfusion injury
IBD Inflammatory bowel disease
ICCMGR Iraqi center of Cancer Research and Medical
Genetics
ICD Cytoplasmic domain
IFN Interferon
IgE Immunoglobulin E
IL Interleukin
INH Isoniazid
iNOS Inducible nitric oxide synthase
IP-3 Inositol triphosphate-3
IRAK IL-1 receptor associated kinase
IRF7 Interferon regulatory factor-7
IRS-1 Insulin receptor substrate-1
IκB/IKK Inhibitor of kappa-B/kinase
JNK Jun N terminal kinase
KCs Kupffer cells
LMW Low molecular weight
LPO Lipid peroxide
LPS Lipopolysaccharides
LTA Lipoteichoic acid
lyc Lycopene
MAPK Mitogen activated protein kinase
MCP-1 Monocyte chemotactic protein-1

XVI
MD-2 Lymphocyte antigen 96
MDA Malondialdehyde
MIF Macrophage inhibitory factor
MRP Multidrug resistance associated protein
MTHR Methylene tetrahydrofolate reductase
mTOR Mammalian target of rapamycin
MTX Methotrexate
MTX-PG Polyglutamate MTX form
MyD88 myeloid differential88
NAC N-acetyl cysteine
NADPH Nicotinamide adenine dinucleotide phosphate
NAFLD Nonalcoholic fatty liver disease
NAGL Neutrophil gelatinase associated lipocalin
NAS NAFLD assessment scoring components
NASH Nonalcoholic steatohepatitis
NEFA Non esterified fatty acid
NF-κB Nuclear factor-κB
NKs/Ts Natural killer cells/T-cells
NO Nitric oxide
NSAIDs Non-steroidal anti-inflammatory drugs
NZB/NZW Hybrid New Zealand murine model
OAT/P1 Organic anion transporters
OD Optical density
PAMPs Pathogen associated molecular patterns
pDCs Plasma dendritic cells
PDK1 Pyruvate dehydrogenase kinase-1
PGN Peptidoglycan
PI-3 Phosphoinositide-3
RA Rheumatoid arthritis
RFC Reduced folate carrier
ROS Reactive oxygen species
RUCAM Ruossel Uclaf Causality Assessment method
SECs Sinusoidal endothelial cells
SFA Saturated fatty acid
SNRNP Small nuclear ribonucleotide
SOD Superoxide dismutase
TAK-1 Transforming growth factor-β-activated kinase

XVII
TBK Serine/threonine binding kinase
TG Triglycerides
TGF Transforming growth factor
TGF-β Transforming growth factor beta
Th17 T helper cell 17
TICAM-1 TRIF/toll like receptor adaptor molecule-1
TIR Toll/IL-receptor domain
TIRAP/MAL TIR domain containing adaptor protein/MyD88
adaptor like
TLRs Toll like receptors
TMD/ICD Transmembrane domain
TNF-α Tumour necrosis factor-alpha
TRAF TNF receptor associated factor
TRAM TRIF related adaptor molecule
Tregs Regulatory T cells
TREM1 Triggering receptor expressed on myeloid cells-1
TRIF TIR domain containing adaptor protein inducing
interferon-β
TSP Total serum protein
UC Ulcerative colitis
VDAC1 Voltage dependent anion channels
vit. B9 Folic acid

Chapter One: Introduction
2
1.1 Drug induced liver injury
Drug induced liver injury (DILI) also termed hepatotoxicity) point
to any liver injury caused by xenobiotics or chemicals including drugs
or medicinal herbs; whether introduced in therapeutic doses or in
overdose (Pandit A. et al., 2012; Sharma N. et al, 2012). This term
described before 70 years, since then thousands of drugs were reported
to cause liver injury and thus it is the most common reason of drug
withdrawal after preclinical or clinical studies as example: bromofenac
and troglitazone, denied approval: ximelagatran and cessation of
development: fialuridine (Kleiner D., 2017; Alempijevic T. et al., 2017;
Chalasani N. et al., 2008; Licata A. et al., 2016).
(DILI) is the most frequent reason of admission to hospital, liver
transplantation, acute liver failure (ALF), and acute hepatitis. The
incidence of DILI estimated in American and European countries was
1-20/100000 cases while the database of general practice research in
UK (GPRD) estimated more than 100/100000 cases for the years 1994-
1999 (Kleiner D., 2017; Singh D. et al., 2016; Yu Y-c. et al., 2017).
DILI is excellently described by Licata A. et al. as: “the diagnosis of
exclusion, made when all common cause of liver damage are ruled out”
(Singh D. et al., 2016; Licata A. et al., 2017; Miele L. et al., 2017).
1.1.1 Idiosyncratic versus Intrinsic drug induced liver injury
DILI primarily categorized as intrinsic or idiosyncratic (Chalasani N. et
al., 2014; Licata A. et al., 2016). The intrinsic DILI; also known as
predictable or dose-dependent, are the major ones that affect human and
animal models, these are related to the dose of administered drug,
resulted from direct hepatocellular necrosis due to the drug or its
metabolites within time of days (Chalasani N. et al., 2014; Licata A. et
al., 2016; Ramachandram R. & Kakar S., 2008).

3
The idiosyncratic DILIs are associated with 10-15 % of ALF events, are
described as low incidence events and may occurs within 8 weeks to 1
year after drug administration (Licata A. et al., 2016; Roth A. & Lee
M., 2017). They have no predictable dose dependent basis neither
related to the drug actions, but associated with persistent hepatic
inflammation mainly due to hypersensitivity/or immunologic reactions
and metabolic abnormalities that are both related to genetic variation
(Chalasani N. et al., 2008; Kaplowitz N., 2004; Clare K. et al., 2017).
1.1.2 Factors predispose to drug induced liver injury
Factors increasing the risk of DILI are categorized into 3 groups:
clinical or host related, drug related, and environmental factors
(Kaplowitz N., 2004).
Host related factors may be described as non-modifiable as age, gender,
and genetic factors (Alempijevic T. et al., 2017). Age is associated with
body function development and which drugs are used at time as so
infant and children susceptible to DILI due to valproic acid, aspirin and
propylthiouracil while advanced age patients would suffer from
amoxicillin-clavulanate and isoniazid’s (INH)-DILI (Singh D. et al.,
2016; Chalasani N. et al., 2014). Gender was also found to reflect
variability, in females the most drugs associated with DILI are
methyldopa, diclofenac, and nitrofurantoin; even pregnancy was
associated with increased DILI caused by methyldopa, hydralazine,
propylthiouracil and high dose tetracycline. Concurrent disease as
diabetes suggested to increase susceptibility to DILI with methotrexate
and antibiotics (Chalasani N. et al., 2014). Genetic factors’ mostly
associated with differences among patient due to polymorphism in
genes encoding enzymes and transporters affecting drug
pharmacokinetics and human leukocyte antigen system (HLA)
(Kaplowitz N., 2004; Clare K. et al., 2017).

4
Drug related factors reflect polypharmacy with increased drug-drug
interactions increases DILI due to antibiotics and anticonvulsants. Also
associated with specific drug physical properties resulted from chemical
composition and dose, defined as “The rule of two” which state that the
higher lipophilic drug with high dose reflect the higher blood absorbed
amount and then higher rate of metabolites which leads to DILI
(Alempijevic T. et al., 2017; Singh D. et al., 2016; Chalasani N. et al.,
2014).
Environmental factors are associated with unhealthy nutrition, obesity,
sedentary life style and heavy alcohol consumption. The last is
associated with paracetamol, duloxetine and INH-DILI (Alemijevic T.
et al., 2017; Chalasani N. et al., 2014; Yu Y-c. et al., 2017).
1.1.3 Patterns of drug induced liver injury
DILI are classified depending on hepatocellular biochemical laboratory
values, or depending on causality assessment scoring system or
depending on Histopathological findings (Chalasani N. et al., 2008;
Chalasani N. et al., 2014; Yu Y-c. et al., 2017).
The former depend on calculated R value that represent the division of
serum alanine aminotransferase (ALT) /its upper limit of normal by
serum alkaline phosphate (AP) /its upper limit of normal that indicate
whether the injury is hepatocellular, cholestatic or mixed (Miele L. et
al., 2017; Kaplowitz N., 2004; Yu Y-c. et al., 2017) according to the
following figure:

5
Figure (1-1): Patterns of DILI depending on calculated R value, where
ALT=ALT/Upper normal limit and AP= AP/Upper normal limit (Licata A.
et al., 2017).
The causality assessment of “Ruossel Uclaf Causality Assessment
method (RUCAM) and Maria and Victorino systems depending on
scores calculated for each involved factor (like age, risk factors, timing
of starting and stopping drugs and other factors) ranged from -3 to +3 to
compute the likelihood of DILI, as highly probable (>8), probable (6 –
8), possible (3–5), unlikely (1 or 2), or excluded (<0) “(Chalasani N. et
al., 2008; Chalasani N. et al., 2014). Finally the drug induced liver
injury network (DILIN) identifies 18 histological patterns of damage,
among them the most common are hepatitis and cholestasis (acute or
chronic); as well as cholestatic hepatitis. Other patterns include
granulomatous, steatotic (microvascular or macrovascular),
steatohepatitic, necrosis (zonal or massive), vascular injury,
hepatocellular alteration, nodular regenerative hyperplasia, mixed
injury, minimal nonspecific change, and absolutely normal
(Alempijevic T. et al., 2017; Miele L. et al., 2017; Licata A. et al.,
2016).

6
1.1.4 Histological patterns of drug induced liver injury
1.1.4.1 Acute hepatitis: the term acute refers to less than 6 months
duration with around 10% of cases are attributed to DILI. Its
characteristic features are portal and parenchymal inflammation,
swollen hepatocytes, and necrosis (spotty, confluent, and centrizonal).
Commonly portal mononuclear infiltrate of lymphocytes, plasma cells,
kupffer cells (KCs) as well as acidophils. Cholestasis or hepatic
regeneration also present but grossly preserved liver architecture
(Sharma N. et al., 2012; Ramachandran R. & Kakar S., 2008).
Example of associated drugs: indomethacin, mefenamic acid,
phenytoin, valproic acid, ampicillin, sulphonamides, griseofulvin, INH,
refampin, ritonavir, ziduvodine, anakinra, azathioprine,
cyclophosphamide, tocilizumab, nefidipine, amiodarone, statins,
sulfonylureas, and allopurinol (Pandit A. et al., 2012; Kleiner D., 2017;
Ramachandran R. & Kakar S., 2008).
1.1.4.2 Acute liver failure (fulminant hepatitis): the term is
pathognomic of idiosyncratic DILI, occurs within 26 weeks of injury.
Clinical presentation appears like the acute hepatitis but with massive or
submassive parenchymal necrosis, it’s associated with severe
coagulopathy and hepatic coma (Ramachandran R. & Kakar S., 2008;
Privitera G. et al., 2014).
Examples of associated drugs: drugs that cause acute hepatitis could be
involved, clopidogrel, rivaroxaban, monoamine oxidase inhibitors,
sulphonamides, tetracycline, cocaine, 3,4-
methylenedioxymethylamphetamine (ecstasy), zalcitabine, ziduvodine
(Pandit A. et al., 2012; Licata A. et al., 2017; Miele L. et al., 2017).

7
1.1.4.3 Chronic hepatitis: the term chronic refers to local hepatic
inflammation with increase in serum aminotransferases for more than 6
months that developed in around 5-10% of DILI cases. Typically
characterized by enlarged liver with extensive hepatocellular loss,
mononuclear infiltrate, necrosis (whether piecemal or periportal,
parenchymal focal) resulting from tissue fibrosis. Jaundice may occur.
(Sharma N. et al., 2012; Kleiner D., 2017).
Examples of associated drugs: trazodone, phenytoin, phenelzine,
sulphonamides, lisinopril, tamoxifen, etanercept, infliximab and 5-
flurouracil (Pandit A. et al., 2012; Miele L. et al., 2017; Ramachandran
R. & Kakar S., 2008).
1.1.4.4 Acute cholestatic injury: clinically presented as pure
cholestasis or cholestatic hepatitis, both characterized by raised serum
alkaline phosphatase (ALPL) and γ-glutamyl transferase. But the former
is associated with hepatocellular or canalicular bile deposit while the
latter is associated with hepatocellular damage, inflammation and
increased number of biliary ductules characterized by
ploymorphonuclear leukocytes infiltrate and fibrosis (periportal)
(Ramachandran R. & Kakar S., 2008)
Examples of associated drugs: ezetimibe, chlorpromazine,
prochlorperazine, erythromycin, oral contraceptives, and warfarin
(Pandit A. et al., 2012; Singh D. et al., 2016; Ramachandran R. & Kakar
S., 2008).
1.1.4.5 Chronic cholestasis and ductopenia: it’s a condition in which
the symptoms of acute cholestasis persist for more than 3 months
showing pseudoxanthomatous change, with continuous decrease in
number of bile ducts due to injury and inflammation, and vanishing bile
duct syndrome may arise (Ramachandran R. & Kakar S., 2008).

8
Examples of associated drugs: ibuprofen, amoxicillin-clavulanic acid,
flucloxacillin, clindamycin, carbamazepine and amiodarone (Kleiner D.,
2017; Miele L. et al., 2017; Ramachandran R. & Kakar S., 2008).
1.1.4.6 Granulomatous hepatitis: the term granulomatous refers to
persistent inflammatory pattern characterized by the presence of fat
vacuoles enclosed by fibrin ring that are surrounded with epithelioid
hepatocytes in the portal tracts or liver parenchyma. It’s clinically
presented with fever (Kleiner D., 2017; Ramachandran R. & Kakar S.,
2008).
Examples of associated drugs: diazepam, phenytoin, chlorpropamide,
procarbazine, procainamide, methyldopa, diltiazim, allopurinol, Bacilli
Calmette-Guérin vaccine (BCG), and patients with hepatitis C (HCV)
who are treated with interferon (IFN) (Kleiner D., 2017; Ramachandran
R. & Kakar S., 2008).
1.1.4.7 Steatosis and steatohepatitis: it’s a reversible condition which
may progress to fibrosis, cirrhosis or even hepatocellular carcinoma.
The histopatholgic characteristic feature is the large fat vacuoles
resulted from triglycerides (TG) deposition in the liver (Sharam N. et
al., 2012; Cao L. et al., 2016; Fagone P. et al., 2015). Typically presents
as microvesicular, macrovesicular, and steatohepatitis.
The microvesicular term refers to the presence of small fat vacuoles
surrounding the nucleus at the centre of hepatocytes (liposomes) due to
mitochondrial injury. Example of associated drugs: cocaine,
tetracycline, ziduvodine, valproic acid, and vitamin A (Sharma N. et al.,
2012; Miele L. et al. 2017; Ramachandran R. & Kakar S., 2008).
While the macrovesicular term refers to “small and large fat droplets”
that occupies the whole hepatocyte which push the nucleus to the edge
of the cell causing distinct signet ring appearance.

9
These droplets can combine together resulting in the development of
irreversible fatty cysts. Examples of associated drugs: glucocorticoids,
indomethacin, ibuprofen, mefloquine, nitrofurantoin, methotrexate
(MTX), 5-flurouracil, cisplatin, tamoxifen, and oestrogen (Sharma N. et
al., 2012; Kleiner D., 2017; Ramachandran R. & Kakar S., 2008).
Steatohepatitis term refers to steatosis coexisting with hepatocellular
ballooning, fibrosis, and lobular inflammation. It’s also attributed to
oxidative stress and mitochondrial loss of function. Examples of
associated drugs: amiodarone, propranolol, valproic acid, haloperidol,
irinotecan, and tamoxifen (Pandit A. et al., 2012; Miele l. et al., 2017;
Ramachandran R. & Kakar S., 2008).
1.1.4.8 Vascular changes: like Veno-occlusive disease/sinusoidal
obstruction syndrome in which damage to venule endothelium causes
sinusoidal swelling and thrombosis in zone 3 leading to hepatocellular
necrosis, centrilobular fatty changes and failure seen after
chemotherapeutic agents (Kleiner D., 2017; Ramachandran R. & Kakar
S., 2008).
1.1.4.9 Drug related neoplasm: attributed to cellular mutations that
lead to uncontrolled cellular division; focal nodular hyperplasia,
hepatocellular carcinoma or adenoma some-times are linked with drugs
as: carbamazepine, clomiphene, danazol, and oral contraceptives
(Sharma N. et al., 2012; Ramachandran R. & Kakar S., 2008).
1.1.4.10 other patterns like hepatoportal sclerosis where there is portal
vein tightening and loss as well as dilation, and nodular regenerative
hyperplasia. Both seen with chemotherapy example: cyclophosphamide,
doxorubicin, prednisone, and oxaliplatin (Kleiner D., 2017;
Ramachandran R. & Kakar S., 2008). Other patterns also include
hepatic cytoplasm alteration and pigmentation patterns including
ground glass associated with endoplasmic reticulum (ER) multiplication

10
and show eosinophilic cytoplasm. Associated drugs: cyanamide,
diazepam, phenobarbital and mycophenolate mofetil. (Kleiner D., 2017;
Ramachandran R. & Kakar S., 2008)
1.1.5 Mechanisms of Drug induced liver injury
The first mechanism of DILI is mediated by direct toxicity resultant
from drug metabolism. In which the liver is exposed to high level of the
drugs, their electrophilic metabolites and the generated free radicals
(FRs). Also hepatotoxic drugs can induce cytochrome P (CYP450)
isozymes oxidation resulting in more and more FR. These FR bind to
macromolecules causing biological membrane damage, mitochondrial
vulnerability, DNA damage and preventing hepatobiliary flow (Sharma
N. et al., 2012; Kleiner D., 2017; Singh D. et al., 2016). These FR may
outweighs the intracellular defensive system involving superoxide
dismutase (SOD), catalases, peroxiredoxin, thioredoxin, glutathione
peroxidases and S-transferase, and tocopherols causing further
disturbances (Kaplowitz N., 2004; Simeonova R. et al., 2014). Free
radicals also causes stimulation of the adaptive response affecting the
permeability transition pore complex like voltage dependant anion
channels (VDAC1) and adenine nucleotide translocases (ANTs)
resulting in cellular programmed self-destruction “apoptosis” and
nuclear disassembly or swelling and lysosomal lysis “necrosis and
autophagy” (Brenner C. et al., 2013; Cao L. et al., 2016; Hikita H. et al.,
2015).
Second mechanism of DILI mediated by mitochondrial damage. Being
so important in cellular toxicity and viability; the mitochondrial
participation in DILI is somewhat unclear since most proapoptotic
signals begun with them so it is controversial whether their damage is
caused by or maintain hepatocellular toxicity. Drugs can either cause
direct mitochondrial damage throughout reactive oxygen species (ROS)

11
generation, or by competing with medium chain fatty acid for β-
oxidation, or binding to mitochondrial DNA causing their destruction
(Alempijevic T. et al., 2017; HO S., 2015). This in turn result increasing
hepatocellular susceptibility to hypoxia or nutrient loss even mild level
(Hikita H. et al., 2015; Brenner C. et al., 2013).
Third important drug effect is throughout immune system. In which
drugs may act like hatpen (especially those with low molecular weight)
through binding with intracellular protein or CYP450 enzymes that are
recognized as foreign body initiating immune response,
immunoglobulin E (IgE) release and T-cell activation (Licata A. et al.,
2016; HO S., 2015). Drugs may act by danger signal hypothesis,
suggesting that mild drug induced cellular destruction will induce
danger signal beside the released antigen that will stimulate damage-
associated molecular pattern molecules (DAMPs) release and expand
the resultant inflammation and toxic effect. In both situation
hepatocellular damage stimulate the release of cytokine (CK) and
chemokine (CC) by KCs, natural killer cells (NKs) and NKTs. (Sharma
N. et al., 2012; Licata A. et al., 2016; Kaplowitz N., 2004).
1.2 Toll like receptors in Pathophysiology of liver injury
Toll like receptors are the target receptors which are blocked by the
drugs used in this study (TAK 242 and GIT 27)
1.2.1 Toll like receptors: These are type I integral transmembrane glycoprotein
family of very conserved structure (Matsunaga N. et al., 2011; Zhang E. &
Lu M., 2015), consist of two domains: an extracellular domain (ECD) of leucine
rich repeat motifs for the detection of ligands and cytoplasmic domain (ICD) with
an IL-1 receptor comparable region called Toll/IL-receptor (TIR) domain that is
responsible for the consequence inflammatory signal. It is the ECD which confer

12
specific ligand detection and is differ among TLRs family members. A total of
13 toll like receptors (TLRs) exist in mammals with 10 TLRs detected in human
genome (Kiziltas S., 2016; Guo J. & Fridman S., 2010) depending on their similar
morphology with Toll, which is a gene product participate in embryonic polarity
development- as well as adult fly -antimicrobial response of the species
Drosophila melanogaster (Matsunaga N. et al., 2011; Guo J. & Fridman S.,
2010).
TLRs function as a family of pattern recognition receptors (PRRs), that
recognizes pathogen associated molecular patterns (PAMPs) derived
from pathogen (Kiziltas S., 2016; Zhang E. & Lu M., 2015); like gram
negative bacterial lipopolysaccharides (LPS), gram positive bacterial
lipoteichoic acid (LTA) and peptidoglycan (PGN), mycobacterial
lipopeptides, yeast zymosan, viral and bacterial ribonucleic acid (RNA),
and unmetlylated cytosine phosphate guanine containing- (CpG)
deoxyribonucleic acid (DNA) (Min H. et al., 2014; Takashima K. et al.,
2009). And DAMPs as: damaged organelles structures, extracellular
matrix, cytosolic and nuclear proteins, Heat shock protein-60 (HSP-60)
and HSP-70, hyaluronic acid fragments and free fatty acids (FFA)
(Zhang Y. et al., 2015; Kiziltas S., 2016; Broering R. et al., 2011). They
causes activation of the innate and inflammatory immune responses,
epithelial regeneration and sterile inflammation control (Hadi N. &
Jabber H., 2016; Guo J. & Fridman S., 2010).
1.2.2 Toll like receptors mechanism of action
Upon binding to their ligands, TLRs undergo conformational changes,
dimerization as well as interaction with adaptor molecules passing
series of intracellular signal transduction pathways; resulting in the
secretion of pro-inflammatory mediators including nitric oxide (NO),
CK like TNF-α, IL-6 and IL-1β, and CC (Takashima K. et al., 2009;
Matsunaga N. et al., 2011; Zhang E. & Lu M., 2015).

13
Four adaptor molecules are included in these complicated pathways
starting with myeloid differential88 (MyD88), TIR domain containing
adaptor protein/MyD88 adaptor like (TIRAP/MAL), TIR domain
containing adaptor protein inducing interferon-β (TRIF), and TRIF
related adaptor molecule (TRAM). These pathways also includes one or
more TIR containing adaptor molecule such as IL-1 receptor associated
kinase-1 (IRAK-1), IRAK-4, TNF receptor associated factor-6
(TRAF6), serine/threonine binding kinase (TBK-1), mitogen activated
protein kinase (MAPK), and inhibitor of kappa-B (IκB) kinase (IKK)
(Takashima K. et al., 2009; Kiziltas S., 2016).
There are two intracellular signalling pathway: MyD88-dependent and
independent signal transduction pathway.
1.2.2.1 MyD88-dependent pathway: it is utilized by all TLRs but not
TLR3 (Takashima K. et al., 2009; Broering R. et al., 2011). This
pathway activate the IRAKs, TRAF6, transforming growth factor
(TGF)-β-activated kinase (TAK-1) and the IKK complex (Zhang E. &
Lu M., 2015). It causes the nuclear translocation of NF-κB and adaptor
protein-1 (AP1) (Li M. et al., 2006; Kiziltas S., 2016) and end with the
secretion of pro-inflammatory cytokine IL-6, IL-10, IL-12 and TNF-α
(Broering R. et al., 2011; Guo J. & Fridman S., 2010). MyD88 also
activate the extracellular signal-regulated kinases (MAPK/ERK), Jun
(N) terminal kinase (JNK) and phosphoinositide-3 (PI3) kinase which
stimulate the AP1 pathway, and activate the interferon regulatory
factor-7 (IRF7) causing release of type-I IFN or co-stimulatory
molecules associate with antimicrobial response by endosomal TLRs 3,
7, 8 and 9 (Zhang N. et al., 2015; Hussey S. et al., 2013; Broering R. et
al., 2011).

14
1.2.2.2 MyD88 independent pathway: the main pathway of TLR3 and
4, involve TRIF signalling pathway activation which results in inositol
triphosphate (IP3) phosphorylation and induction of IFN-β gene
expression as well as activation of TRAF6 (Takashima K. et al., 2009;
Broering R. et al., 2011). Surprisingly the same outcome obtained from
plasmatoid dendritic cells (pDCs) stimulated by TLR 7 and 9
throughout activation of MyD88/IRF7 dependent pathway (Gárate I. et
al., 2014; Zhang E. & Lu M., 2015).
TLR4 further utilizes TIRAP to activate MyD88 and TRAM to bridge
the TRIF activation, which means that TLR4 uniquely utilizes both the
MYD88 dependent and independent pathways (Zhao Y. et al., 2015;
Takashima K. et al., 2009; Broering R. et al., 2011). As stated by
Kiziltas S. et al. (2016) “ The final outcome of TLRs activation differ
dependent on the nature of PAMPs, the concomitant activated TLR, the
level of cytokine and the cell stimulated. Moreover, chronically
activated signalling pathway is likely to induce transcription of
oncogenic factor which add further level of complexity to the
intracellular signalling for these receptors”.
1.2.3 TLR2: cell membrane expressed receptor, mainly detect gram
positive bacterial lipopeptides, and PGN (Takashima K. et al., 2009;
Bhattacharyya S. et al., 2016). It uniquely forms heterodimers of
TLR1/2 and TLR2/6 complex which are able to distinguish between
triacylated and diacylated bacterial and synthetic lipopeptides
(Bhattacharyya S. et al., 2016; Guo J. & Fridman S., 2010). This
receptor shares TLR4 MyD88/TIRAP dependent pathway (Kiziltas S.,
2016; Zhang E. & Lu M., 2015).
It is expressed by hepatocyte under inflammation, triggers hepatic KCs,
HSCs, and sinusoidal endothelial cells (SECs) expression of

15
costimulatory molecules such as T-cells, production of IFN-γ and TNF-
α by KCs, biliary endothelial cells (BECs) and SECs respectively. It
also mediate the feedback of intrahepatic BECs by regulating IRAK-M
expression. Furthermore TLR2 self-expression induced by TLR4 in
HSCs (Kiziltas S., 2016; Guo J. & Fridman S., 2010).
1.2.4 TLR4: surface expressed receptor, the first expressed homolog
which detect LPS of gram negative bacteria mainly, viral proteins,
endogenous ligands as low molecular weight (LMW) hyaluronic acid,
heparin sulphate, saturated fatty acids (SFA), fibrinogen, fibronectin,
HSP-60 and-70, high mobility group bax-1 and degraded matrix (Li M.
et al., 2006; Guo J. & Fridman S., 2010).
TLR4 signalling pathway [Figure (1-2)] involve co-receptors, adaptor
molecules, signal transcription factor.
Figure (1-2):TLR4 signalling pathways (Matsunaga N. et al., 2011).

16
1.2.4.1 Co-receptors: including CD14 and lymphocyte antigen 96
(MD-2). CD-14 is protein linked to glycophosphatidyl inositol
responsible for LPS translocation within MD-2 causing TLR4
activation. It is expressed on innate immune cells as macrophage and
monocytes. MD-2 is glycoprotein associated with TLR4 ECD on cell
membrane, mainly at myeloid and endothelial cells. It is essential for
both TLR4 cellular expression as well as activating signalling pathways
(Takashima K. et al., 2009; Li M. et al., 2006; Guo J. & Fridman S.,
2010).
1.2.4.2 Adaptor molecules: the four adaptor proteins including
MyD88, TIRAP/MAL, TRIF/toll like receptor adaptor molecule-1
(TICAM-1), and TRAM being utilized in TLR4 signalling cascade (Li
M. et al., 2006).
1.2.4.3 Transcription factors: the main three factors involved
including NF-κB, AP1, and IRF. NF-κB is an endoplasmic expressed
pleotropic protein complex, responsible for regulation of genes of pro-
inflammatory CK , chemokine, and adhesion molecules, cell cycle and
survival regulating proteins as cyclin D1 and B cell lymphoma 2 (Bcl-
2). AP1 composed of dimers of the Jun and Fos protein families which
is responsible for regulation of cellular replication and survival. Finally
the IRFs protein regulating IFNs, are responsible for signal activation
via TLR4/TRIF dependent pathway (Guo J. & Fridman S., 2010).
1.2.4.4 Downstream signalling factors regulated by TLR4 : including
prototypic inflammatory mediators IL-6, monocyte chemoattractant protein1
(MCP1), proinflammatory enzyme iNOS and cyclooxygenase-2 (COX2) (Zhao
Y. et al., 2015; Zhang Y. et al., 2015; Gárate I. et al., 2014), reactive oxygen
species, adhesion molecules, type I IFN as well as cellular cycle regulating
protein and apoptotic proteins (Kong J. et al., 2016; Lin X. et al., 2015; Guo J. &
Fridman S., 2010).

17
TLR4 being expressed in low level hepatically unless an injury exist; it
is expressed by- and activate hepatocytes, KCs, SECs, HSCs, adaptive
immune cell helper cell (CD4), T-cells, and T regulatory cells (Tregs).
It further mediate cholangiocytes response (Guo J. & Fridman S., 2010).
1.2.5 TLR6: this receptor expressed on cell membrane, detect microbial
cell wall component as well as distinguish subtle differences between
triacyl and diacyl lipopeptides, lipoproteins via heterodimerization with
TLR2 (Bhattacharyya S. et al., 2016; Zhang E. & Lu M., 2015), it
stimulate KCs promoting T-cells replication, releasing IFN-γ, SECs
induce allogeneic T-cell activation and producing TNF-α (Kiziltas S.,
2016; Guo J. & Fridman S., 2010).
1.2.6 Role of TLRs in Pathophysiology of liver injury
Since the liver is main organ that captures gut derived endotoxin
exposed by portal circulation (Oya S. et al., 2013), so TLRs would have
significant role in hepatic injuries attributed to frequent activation of the
hepatic innate immune system, which contribute in the induction of
inflammation in acute injuries. Whilst pathogenic dependent
suppression of TLRs mediate chronic hepatic injuries/disorders like
hepatitis, fibrosis, alcoholic and non-alcoholic liver injuries,
ischemia/reperfusion, and carcinoma (Kiziltas S., 2016).
1.2.6.1 Drug induced liver injury: In paracetamol human
hepatotoxicity, endogenous chemical injury derive extracellular matrix
(ECM), the ligand which activate TLR4 to release TNF-α, induce iNOS,
peroxynitrite, glutathione depletion, amplified immune response,
sequestering leukocytes, increase serum hyaluronic acid, steatosis,
necrosis, and congestion (Guo J. & Fridman S., 2010). In another
experimental trial, the selective blockade of TLR4 was successful in
ameliorating the hepatic oxidative stress resultant from paracetamol

18
induced liver injury in mice model (Salama M. et al., 2015). Wei C-B.
et al., reported that TLR4/MyD88-NF-κB signalling pathway is
involved in the T helper cell 17 (Th17)/Treg cellular imbalance that
result from high dose triptolide-induced liver injury. TLR4
pharmacologic blockade was shown to counteract the resultant
hepatocellular inflammation throughout the restoration of the balance of
T cells that paly key role in both innate and adaptive immune response
(Wei C-B. et al., 2017).
1.2.7 TLR Therapeutic targeting
TLRs, being receptors for variety of ligands and effectors of both innate
and adaptive immune response in the liver; with multiple steps
signalling pathway can be very attractive as therapeutic targets that
could be modified by synthetic agonist, antagonist or naturalized
antibodies (Kiziltas S., 2016).
1.2.7.1 TLR2: Pam2/3CSK4 TLR2 ligands covalently linked to CD8+
or B-cell epitopes associated peptides were found to enhance
therapeutic response in tumour models, by stimulating TLR2 induced T-
cell activation (Zhang E. & Lu M., 2015)
1.2.7.2 TLR4: various antagonist starting with the peptide P13, an
inhibitor of TIR domain signalling pathway that was found to
ameliorate inflammatory response and improve surviving in a TLR4-
mediated hepatic injury of murine model (Guo J. & Fridman S., 2010),
the Lipid A mimetics E5564 and CRX526 which bind to TLR4-MD2
complex showing valuable inhibition of CK production in LPS treated
animal models as well as septic shock patients in phase III clinical trial
(Broering R. et al., 2011; Guo J. & Fridman S., 2010), And finally
TLR4/MD2/IgG-Fc fusion protein inhibitor of NF-κB and JNK
activation provides interesting biologic therapy for liver fibrosis,

19
alcoholic and non-alcoholic steatohepatitis (NASH) by decreasing IL-6
and MCP-1 production (Guo J. & Fridman S., 2010).
While a TLR4-synergizer Fc/fusion protein antagonist of the triggering
receptor expressed on myeloid cells-1 (TREM1) and TLR4 ligand α-1
acid glycoprotein where found to inhibit hepatic macrophage LPS-
induced activation: by TERM-1 blockade, and boosting immune
response against infection respectively. The theoretically interesting
scenarios in treating viral hepatitis became real when monophosphoryl
lipid A derivatives were formed in 2 adult hepatitis B virus (HBV)
vaccine administered intravenously (I.V.) (Zhang E. & Lu M., 2015;
Guo J. & Fridman S., 2010).
1.2.7.3 TLR7: selective agonist I.V. isatoribine given once daily to
patient with HCV infection was found to cause viral load decrement
with a mild to moderate adverse effect profile. Another lGS-9620 ligand
was tested on HBV animal model resulted in decreased HBV viral load
and hepatitis B s antigen (HBsAg) serum level as well as dose
dependent increment of IFN-α (Broering R. et al., 2011; Zhang E. & Lu
M., 2015).
1.2.7.4 TLR9: the selective TLR9 agonist 1018 ISS
(immunomodulatory sequences) that contains repeated CpG motifs was
found to modulate TLR9 signalling pathway involved in HBV infection
and been tested in phase III clinical trials, while the intracellular
signalling inhibitors ST2825 and RO0884 designed to block IRAK1 and
-4/MyD88 singling pathway caused inhibition of the NF-κB, IL-1β, and
TNF-α activation as well as decreased hepatic IL-6 secretion (Broering
R. et al., 2011; Zhang E. & Lu M., 2015).

20
1.3 Methotrexate
MTX is a 4-amino,10-methyl folate analog, derived from aminopterin
(Olayinka E. et al., 2016; Lun B.and Rodway G., 2017); first used in
oncology since 1940.
High dose methotrexate (HD-MTX) used to treat acute lymphoblastic
leukemia (ALL), osteosarcoma, head and neck cancer, non-Hodgkin
and Hodgkin lymphomas, trophoblastic neoplasms (Levéque D. et al.,
2017; Campbell J. et al., 2016). Also it’s the first line agent used orally
in treating rheumatoid arthritis (RA) naïve patients, eczema and
psoriasis. As cited by Carrascosa J. et el., “Rodanovich et al. suggest
that it is further beneficial in decreasing cardiovascular risk in these
patients” (Bianchi G. et al., 2016; Carrascosa J. et al., 2016).
Parenteral MTX administered in inflammatory bowel disease, ulcerative
colitis (UC) and in steroid dependent Crohn’s disease (Coskun M. et al.,
2015; Gabbani T. et al., 2016). Considering combination, it is used to
decrease the dose and increase the effect of narrowband-UV-
phototherapy, to improve pharmacokinetics and inhibit formation of
adalimumab and infliximab antibodies (Carrascosa J. et al., 2016;
Quetglas E. et al., 2015; Howard S. et al., 2016), and in tubal ectopic
pregnancy in combination with mifepristone (Wan S. et al., 2016; Yang
C. et al., 2017).
Off label MTX indicated in bladder, and breast cancers,
medulloblastoma, and prophylaxis in acute graft vs. host disease after
allogeneic hematopoietic stem cell transplantation (Levéque D. et al.,
2017; Yee J. & Orchard D., 2016).

21
1.3.1 Pharmacodynamics
Both MTX and its polyglutamate metabolite (with 1000x activity), are
known blocker of the cytoplasmic de novo synthetic pathways of
purines, pyrimidines as well as polyamines (Pandit A. et al., 2012;
Levéque D. et al., 2017; Howard S. et al., 2016); depending on their
inhibition of the enzymes dihydrofolate reductase (DHFR), thymidylate
synthase, amido phosphoribosyl transferase involved in the machinery
of the dividing cells. Also indirectly inhibit methylene tetrahydrofolate
reductase (MTHR) involved in homocysteine conversion to methionine.
These inhibitory effects firstly mediate anti-cancer action (Pandit A. et
al., 2012; Akbay T. et al., 2010; Inoue K. & Yuasa H., 2014), secondly
provoke MTX toxicity by affecting proliferative tissue such as intestinal
mucosa, dermal epithelium, and bone marrow (Lun B. & Rodway G.,
2017), thirdly it is attributed to the inhibition of the division of nourish
cells and the release of circulatory Human chorionic gonadotropin
hormone (β-HCG) promoting the necrosis and absorption of embryonic
tissue in ectopic pregnancy (Coskun M. et al., 2015; Wan S. et al.,
2016), and fourthly, it prevent the proliferation of leukocytes which is
another participant mechanism in the anti-inflammatory effect of MTX
(Gabbani T. et al., 2016).
The main anti-inflammatory effect is accomplished by inhibiting the
enzyme 5-aminoimidazole-4carboxamide ribonucleotide transformylase
(AICART) causing the accumulation of its substrate which is an analog
of adenosine monophosphate (AMP) that inhibit adenosine deaminase
and the mammalian target of rapamycin (mTOR) (Restrepo L. et al.,
2016). The inhibition of adenosine deaminase causing intracellular
buildup of adenosine, its consequent release in the circulation and
stimulation of the macrophages and monocytes adenosine receptors

22
(A2a, A2b and A3), thus decreasing the release of proinflammatory
cytokines interleukin-2 (IL-2), IL-4, tumor necrosis factor-α (TNF-α),
and the transcription of IL-1 receptor inhibitor at messenger RNA
(mRNA) basal level while affecting the endothelial cell receptors
preventing the release of IL-6, IL-8, E-selectin as well as the release of
leukotrienes (Restrepo L. et al., 2016; Inoue K. & Yuasa H., 2014).
MTX apoptotic effect is generated by upregulation of p21 and p53
protein expression as well as through production of free radicals
(Gabbani T. et al., 2016; Gutierrez J. & Hwang K., 2016; Khafaga A. &
El-Sayed Y., 2018).
1.3.2 Pharmacokinetics
MTX rapidly absorbed at small intestine, widely distributed to body
tissue despite being hydrophilic anionic compound at physiologic pH so
require facilitated transport by reduced folate carrier (RFC) to cross
biologic membranes. MTX is mainly concentrated in the tissues of the
liver, kidney, spleen, and skin. It’s also distributed to the synovial fluid,
cerebrospinal fluid (CSF) approximately 0.5-11% of its plasma
concentration (Levéque D. et al., 2017; Inoue K. & Yuasa H., 2014;
David A. et al., 2016), and around 8% appear in breast milk (Lun B. &
Rodway G., 2017). MTX volume of distribution is around 1L/kg, with
around 50% is bound to serum albumin (Levéque D. et al., 2017; Inoue
K. & Yuasa H., 2014).
MTX is exposed to hepatic metabolism by the enzyme aldehyde oxidase
to the less active form 7-hydroxy methotrexate, as well as 2,4-diamino-
N10-methylpteroic acid (Coskun M. et al., 2015; Patel H. et al., 2017)
and tissue metabolism by the enzyme folypolyglutamate synthase to the
biological active polyglutamate form (MTX-PG) that trapped
intracellularly allowing elongated MTX intracellular level, this

23
metabolite converted back to MTX by the enzyme c-glutamyl
hydrolase. MTX is not a substrate or inducer or inhibitor to the CYP450
enzymes (Levéque D. et al., 2017; Gabbani T. et al., 2016).
Both MTX and its metabolites excreted renally (major pathway around
90% after parenteral administration) and biliary with partial amount
exposed to enterohepatic circulation (minor around 1-2%); transporters
that control its hepatic and renal clarence are: RFC, organic anion
transporters (OATP1B1 and 3), multidrug resistance associated protein
(MRP2, MRP3, MRP4) and breast cancer resistance protein (BCRP2) in
the liver while OAT1, OAT3, MRP2, MRP4, BCRP and RFC in the
kidney (Levéque D. et al., 2017; Ogungbenro K. et al., 2014). Its half-
life (t1/2) ranged 8-15 hr. approximately 10.2 hr. for methotrexate and 9
hr. for its hepatic metabolites and clearance rate is 50-135 ml/min/m2
after parenteral administration (Levéque D. et al., 2017; Inoue K. &
Yuasa H., 2014; David A. et al., 2016).
1.3.3 Adverse effects and Toxicities
MTX toxicity incidences differ between low dose and high dose regimen (Lun B.
& Rodway G., 2017; Heidari-Soreshjani S. et al., 2017). Low dose MTX regimen
used in the inflammatory disorders is defined as two or three log orders lower
than the regimen used in the treatment of malignancy “5–25 mg/week versus
5000 mg/week” (Cronstein B., 2005).
Neurotoxicity reported after intravenous or intrathecal HD-MTX
regimen is attributed to the inhibition of transmethylation reaction that
disturb central protein, lipid, and myelin, high CSF level of cysteine and
S-adenosyl homocysteine (Gaies E. et al., 2012). This precipitate
cortical blindness and seizures; emesis also reported after HD-MTX
(Howard S. et al., 2016).

24
Pulmonary toxicity seems to be rare after HD-MTX likewise infrequent
after low dose regimen-but still serious and unpredictable. It is
attributed to idiosyncratic hypersensitivity reactions involving
stimulation of T-cells, type 2 alveolar cells, pulmonary fibroblasts and
eosinophils mobilization. Commonly appeared as bronchitis and
pneumonitis (Gabbani T. et al., 2016; Gaies E. et al., 2012).
Gastrointestinal toxicity caused by low dose typically present as
increase transaminases level; risk of pancreatitis reported within first
week of treatment (Lun B. & Rodway G., 2017; Coskun M. et al.,
2015).
Renal toxicity is major complication after HD-MTX due to
accumulation and crystal formation of MTX and its hepatic metabolites
(Levéque D. et al., 2017; Howard S. et al., 2016; Heidari-Soreshjani S.
et al., 2017).
Hematologic toxicity manifest as anemia, thrombocytopenia, leucopenia
and neutropenia that predispose to opportunistic infection. Also
manifest as malignant melanoma, nonmelanoma skin cancer, and
Epstein-Barr V. associated lymphoma with immunocompromised
patient that’s why regular complete blood picture and platelets count is
recommended (Gabbani T. et al., 2016; Gaies E. et al., 2012).
Cutaneous toxicity is another complain of HD-MTX appears as
stomatitis, urticarial, photosensitivity, and palmoplanter
erythrodysthesia “hand-foot syndrome” (Gaies E. et al., 2012).
Finally MTX is cytotoxic, teratogenic, embryotoxic and mutagenic that
impair male fertility and associated with female miscarriage, congenital
deformities affecting CNS, and causing craniofacial and lower
extremities defects especially within 1st
trimester. Thus, it is

25
contraindicated in pregnancy, lactation as well as contraceptive methods
are recommended for males (Carrascosa J. et al., 2016; Gutierrez J. &
Hwang K., 2016; Mazaud C. & Fardet L., 2017).
1.3.4 Methotrexate-induced Liver injury
1.3.4.1 Mechanism: methotrexate supposed to induce liver injury
mainly by 3 mechanisms: long half-life metabolites (Akbay T. et al.,
2010; Miele L. et al., 2017), non-selective inhibition of folate pathway
(Gaies E. et al., 2012; Abo-Haded H. et al., 2017), and generation of
free radicals (Mahmoud A. et al., 2017). As the main organ for drug
metabolism, the liver is exposed to high level of the 7-hydroxy
methotrexate as well as high level of the MTX-PG stored in hepatocyte
build up intracellularly; this is the first risk associated with methotrexate
whether administered in low dose or HD-MTX regimens (Sharma N. et
al., 2012; Miele L. et al., 2017; Khafaga A. and El-Sayed Y., 2018).
Methotrexate inhibition of cytoplasmic de novo synthetic pathways of
purines, pyrimidines as well as polyamines by inhibiting folate
conversion pathways is non selective effect, thus it will not affect
cancer cells only but would rather affect normal cell including hepatic
cells. This will diminish hepatic folate reservoir, restrain folate entrance
to the mitochondria, affect nucleic acid synthesis and thus render
mitochondrial dysfunction and generate ROS (Khokhar A. et al., 2017;
Abo-Haded H. et al., 2017; Mahmoud A. et al., 2017).
Furthermore in association with ROS generation, methotrexate
increasing plasma homocysteine which will cause increased superoxide
and proxy nitrite, and decrease of the level of reduced nicotinamide
adenine dinucleotide phosphate (NADPH) dependent dehydrogenase,
increasing the oxidized glutathione and decrease cellular reduced

26
glutathione (GSH) (Pandit A. et al., 2012; Khafaga A. & El-Sayed Y.,
2018; Mahmoud A. et al., 2017). Both mechanisms will obstruct hepatic
cholesterol and TG metabolism resulting in fatty infiltration, these
mechanisms increase cellular sensitization to FR leading to stimulation
of immune system starting with hepatic satellite cells (HSCs) leading to
fibrosis, leukocyte accumulation, neutrophils secretion of pro
inflammatory enzymes and cytokines like inducible nitric oxide
synthase (iNOS), Nuclear factor-κB (NF-κB) and TNF-α which in turn
causes more production of FR causes sinusoidal congestion, dilation ,
hepatic fatty vacuolation focal necrosis and portal inflammation which
is the typical pattern of drug induced steatohepatitis (DISH) produced
by FR (Miele L. et al., 2017; Khokhar A. et al., 2017; Cure E. et al.,
2015). This participation of immune system that results in the
production of proinflammatory CK and CC is the link between
methotrexate induced toxicity and toll like receptors pathways, which
are the common participant receptors of the immune system that
activation is required for CK production.
1.3.4.2 Prevalence: Methotrexate-induced liver injury is preceded by
low doses as well as HD-MTX, despite its less common after HD-MTX
than low does regimens (Howard S. et al., 2016), also seems common in
patient with psoriasis than with RA, lowest incidence reported with
inflammatory bowel disease (IBD) patients (Akshay S. et al., 2017).
Howard S. et al., (2016) reported that transient toxicity incident after
60% of courses manifest as reversible hepatitis and 25% as
hyperbilirubinemia. Transient and asymptomatic increase in liver
enzymes reported during 1st month of administrating MTX in HD-
MTX, low dose, and even very low doses regimen. Up to 14-25% of
patients with IBD presented with increased level of transaminases after

27
MTX with 1% incidence described as idiosyncratic induced liver injury
(Coskun M. et al., 2015; Gabbani T. et al., 2016; Mazaud C. & Fardet
L., 2017). Around 49% of RA patients reported abnormal transaminases
level with 17% of 2-3x upper than normal limit with 17% reveal
different stages of fibrosis and cirrhosis after 4 years of MTX treatment
mentioned by Gabbani T. et al., (2016), and 25% of psoriatic patients
had fibrosis (Khokhar A. et al., 2017). A review study analyzed the data
of the Organ Procurement and Transplantation Network (1987-2011)
found that incident of end stage liver disease requiring transplantation
was 0.07% (Akshay S. et al., 2017).
1.3.4.3 Histological patterns: the typical changes of MTX induced
liver injury are steatohepatitic (Miele L. et al., 2017; Cure E. et al.,
2015), fibrotic and cirrhotic patterns that involves hepatocellular
hypertrophy, anisonucleosis and fatty degeneration, sinusoidal dilation
and portal inflammation, and focal to massive necrosis (Khafaga A. &
El-Sayed Y., 2018; Khokhar A. et al., 2017); they are categorized into 4
groups according to Roengik classification depending on the grade of
the present fatty degeneration as cited from Gaies E. et al., (2012)
“Grade I: mild fatty infiltration, nuclear variability, with or without
portal inflammation; Grade II: moderate to severeee fatty infiltration,
nuclear variability, and portal tract expansion, inflammation and
necrosis. Grade IIIA: mild fibrosis; Grade IIIB: moderate to severeee
fibrosis and Grade IV: cirrhosis”.

28
1.3.5 Prophylactic and protective approaches against methotrexate-
induced liver injury
1.3.5.1 Regular follow up and recommendations: according to the
U.S. and European guidelines as well as the American College of
Rheumatology as cited by Mazaud C. & Fardet L., (2017) and Mecoli
C. et al., (2016). Monitoring of the liver and renal function tests as well
as complete blood count is recommended after starting or changing the
dose and regularly for patients treated with low dose MTX while liver
biopsy is recommended for psoriasis patients receiving cumulative dose
of 1500 mg (Gabbani T. et al., 2016), arthritic patients with risk factors
and high aspartate aminotransferase (AST) before treatment begins and
those with persistent high transaminase or hypoalbuminemia during
treatment course, those with abnormal liver stiffness estimated by
FibroScan (also known as transient elastography - it’s the most recent
noninvasive method considered to diagnose and follow up fibrosis and
cirrhosis patient on MTX treatment) (Gabbani T. et al., 2016; Akshay S.
et al., 2017).
1.3.5.2 Avoidance of risk factors (drugs interactions): hepatotoxic
medication that increase risk for high transaminases level such as Non-
steroidal anti-inflammatory drugs (NSAIDs), Disease modifying
antirheumatic drugs (DMARDs), and retinoids. It is better to estimate
MTX level when it is used with phenylbutazone, phenytoin and
sulfonamides because they displace MTX bound to albumin, salicylate
and penicillin since they reduce MTX tubular secretion while renal
transport affected by probencid, also MTX is better avoided in HBV
infected patients because it is associated with viral activation that would
complicate liver toxic effect (Lun B. & Rodway G., 2017; Carrascosa J.
et al., 2016; Quetglas E. et al., 2015; Mecoli C. et al., 2016).

29
1.3.5.3 Dosage regimen adjustment, switching and withdrawal:
despite MTX discontinuation is reported in about 30% of patients due to
liver injury, this is not recommended. The same for rechallenge or
switching to another class for fear of more severe injury induced. Still
dosage adjustment and reduction recommended in psoriatic patients at
risk and patient older than 65 years to start with 5-7.5 mg/wk (Olayinka
E. et al., 2016; Carrascosa J. et al., 2016).
1.3.5.4 Clinical trials with medications and medicinal plants:
experimental and clinical studies proceeding to seek which antioxidant
or anti-inflammatory agent can reverse MTX-induced liver injury.
Considering drugs associated with lower DILI, the most suggested are
those with antioxidant and immunomodulatory effect like melatonin,
amifostine, ascorbic acid and sitagliptin as examples (Abo-Haded H. et
al., 2017). Starting with the neuro-hormone melatonin, an electron and
hydrogen donor that stabilize mitochondrial electron transport chain,
prevents mitochondrial GSH deprivation and adenosine triphosphate
(ATP) production. It shows beneficial effect in nonalcoholic fatty liver
disease (NAFLD) while sitagliptin used in experimental trial found to
modulate NF-κB signals in addition to its effect on lipid metabolism
and oxidative stress (Yucel Y. et al., 2017; Khokhar A. et al., 2017).
Starting with medicinal plants’ they are hypothesized to act as anti-
hepatotoxic and directly counteract DILI, hepatotropic that facilitate
liver healing and regeneration, or hepatoprotective that prevent DILI.
As example chamomile, curcumin, gallic acid, gingko, glycyrrhizin,
lycopene, resveratrol, and silymarin (Oya S. et al., 2013; Ghiliyal P. &
Bhatt A., 2012).
Gallic acid and lycopene (lyc) are reported to alleviate high
transaminases and superoxide dismutase level, decreased GSH level,

30
because of their antioxidant and free radical scavenging effect, anti-
inflammatory and anti-apoptotic action. lyc also decrease hepatocellular
damage, sinusoidal dilation and liver congestion (Olayinka E. et al.,
2016; Yucel Y. et al., 2017).
Silymarin action is dependent on cytoprotective effect resulted from
antioxidant effect of flavonoid and interaction with biologic membrane
molecules preventing lipid peroxidation, and cellular regenerative
promoting effect. It induces polymerase I ribosomal RNA (rRNA)
transcription urging its rate of synthesis, inducing antioxidant enzyme
expression thus protecting against free radical caused damage.
Furthermore it decreases leukotriene production by KCs by inhibiting 5-
lipoxygenase pathway (Oya S. et al., 2013).
Resveratrol and quercetin effects mostly depend on deactivation of
CYP450 enzymes. Resveratrol action depends on its phenolic group and
substitution of its hydroxyl group affecting isozymes involved in
metabolic generation of FR as CYP2E1 and CYP1A2, also itself being
metabolized by CYP1B1 result in an tyrosine kinase inhibitor and
anticancer metabolite piceatannol (Akbay T. et al., 2010; David A. et
al., 2016).
Curcumin reported to inhibit cyclooxygenase-2 and decrease NF-κB
production by its conjugated metabolites while ginkgo modulates
antioxidant enzymes and increase the hepatic elimination of other drugs.
Spirulina would act as antioxidant, FR scavenger as well as another
CYP450 inhibitor added to the list of hepatoprotective substance (Oya
S. et al., 2013; Kafaga A. & El-Sayed Y., 2018).

31
1.3.5.5 Standard supplement and acute liver failure antidote: folate
supplement as folic acid (vit. B9) 5-15 mg/wk. or folinic acid 15mg/wk.
administered before MTX treatment suggested to alleviate MTX induce
high transaminases level, reduce the incidence of gastrointestinal
adverse effect. However folic acid was found to inhibit aldehyde
oxidase thus preventing MTX rapid metabolism in fast metabolizers
besides improving its adverse effects (Carrascosa J. et al., 2016; Coskun
M. et al., 2015; Akshay S. et al., 2017). While ursodeoxycholic acid and
glucocorticoid were found beneficial in treating cholestatic DILI, the
only FDA approved antidote since 2004 for ALF is N-acetyl cysteine
(NAC) 50-250 mg/kg for 3days of early stage ALF and merely in adults
(Olayinka E. et al., 2016; Mangano K. et al, 2008).
1.3.5.6 Intervention by liver transplantation, plasma exchange and
bioartficial liver assist devices: are the last and best management for
confirmed irreversible ALF (Yucel Y. et al., 2017).
1.4 TAK 242
TAK 242 also known as resatrovid. A cyclohexene derivative with
chemical structure of Ethyl-(6R)-6-[N-(2-chloro-4-
flurophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate, designed as a
novel antiseptic agent (Hussey S. et al., 2013). Figure (1-3).
It is a selective inhibitor of TLR4 signal transduction pathway, that
interferes with IC TIR adaptor molecules interaction (Zhang Y. et al.,
2015; Gárate I. et al., 2014) thus preventing monocytes and MQ
proinflammatory CK and NO production both in vitro and in vivo
(Zhang N. et al., 2015).

32
It is suggested to be effective in ameliorating inflammatory process
correlated with insulin resistance in diabetes, cardiac diseases, biliary
obstruction as well as sepsis (Oya S. et al., 2013; Wang X-t. et al.,
2017).
Figure (1-3): Structure of TAK 242 (Hussey S. et al., 2013)
1.4.1 TAK 242 Pharmacodynamics
Molecular studies performed by Matsunaga N. et al. (2011) and
Takashima K. et al., (2009) proved that TAK 242 is selective inhibitor
of TLR4, that means it does not inhibit its protein expression (Zhao Y.
et al., 2015; Zhang Y. et al., 2015). TAK 242 with α-β unsaturated
carbonyl group would act as Michael acceptor, via its cyclohexene ring
covalently bind nucleophilic Cys747 located at helix αC/αC
at TIR
domain that is necessary for the homodimeraization phase which in
consequence inhibits MyD88 and TRIF pathway, association with
adaptor molecules TIRAP/Mal and TRAM at time and concentration
dependent manner (Takashima K. et al., 2009; Matsungan N. et al.,
2011).
They prove the selectivity of TAK 242 and TLR4 to each other by
studies utilizing:
I. Different TLR4 ligands induced activation, stating that TAK 242
action is independent on the type of stimulus neither on TNF-α
production.

33
II. Substituting cysteine and arginine residue at TLR4 tranmembrane
domain (TMD/ICD) to examine TAK 242 binding to mutant model,
proving the exact binding site at Cys747.
III. Utilizing enantiomer of TAK 242 to inhibit TLR4/TNF-α which
found less potent in about 350 times, proving that TAK 242 itself being
recognized by TLR4 (Takashima K. et al., 2009; Matsungan N. et al.,
2011; Li M. et al., 2006).
1.4.2 TAK 242 Pharmacokinetic
It is a low molecular weight molecule 360.1 and associated with high
lipophilicity. It can pass through blood brain barriers, and its
distribution rate depend on specific organs. Its plasma level raises after
3hr. and decreases after 24hr. (Zhang N. et al., 2015; Zhao Y. et al.,
2015; Yousif N. et al., 2009). Experimentally metabolized to two
moieties: Phenyl ring moiety converted either to 2-chlorofluoroaniline
and M-1 which is acylated to M-2 and conjugate with glucuronic acid,
or M-3 and M-4 which conjugate with sulphate. The cyclohexene ring
conjugate with both glutathione and mercapturic acid (Yousif N. et al.,
2009). Figure (1-4).
Figure (1-4): Metabolism of TAK 242 (Yousif N. et al., 2009).

34
1.4.3 TAK 242 in Clinical and Experimental trails
I. Despite being effective in treating experimental porcine and murine
models of sepsis (Oya S. et al., 2013), undergoing two clinical trials of
septic shock patient, a phase III study published by Clinical Trials.gov
website (2013) for sepsis induced cardiovascular and respiratory failure,
It fail to demonstrate anti-inflammatory action with no concern of safety
or efficacy (Oya S. et al., 2013). Another phase III study published by
the former site (2011) of checking safety and efficacy in adults with
severee sepsis is still ongoing (Gárate I. et al., 2014).
II. In endotoxic shock model it improve surviving profile but fail to or
not significantly decrease mortality rate in human patient besides its
action on proinflammatory CK in both (Gárate I. et al., 2014; Zhang N.
et al., 2015). Still TAK 242 combination with antibiotics like
ceftazidime or imipenem improve murine survival, decrease level of
proinflammatory CK, and thrombocytopenia after Escherichia coli
induced sepsis and murine cecal ligation and puncture (Takashima K. et
al., 2009; Sha T. et al., 2011). Also protect against organ dysfunction
including liver, kidney and lung injury attributed to its effect on
proinflammatory CK level in combination with imipenem. Despite both
of the studies report that alone, TAK 242 lack effect on bacterial growth
and blood count. Another study in BCG primed mice revealed that
bacterial blood count is not increased after TAK-24 treatment
suggesting that it’s not associated with susceptibility to subsequent
infection as well (Takashima et al., 2009; Sha T. et al., 2011). TAK 242
administered as early as possible before bacterial replication resulted in
more efficacious protection in sepsis model (Sha T. et al., 2011; Li M.
et al., 2006).
III. Considering its action on CNS, TAK 242 was found to decrease
both heat and mechanical induced pain threshold, ameliorate

35
neuropathic pain in murine model in dose dependent manner (Zhao Y.
et al., 2015). It decrease neuroinflammation in rat brain frontal cortex
after stress, by decreasing proinflammatory enzyme COX2 and iNOS,
TNF-α, and IL-1β activation (Zhang Y. et al., 2015; Gárate I. et al.,
2014). It protect nerves against CNS acute injury as in ischemic
reperfusion (I/R) and traumatic injury (Oya S. et al., 2013).
IV. It also inhibits cardiac dysfunction, inflammation, fibrosis and hypertrophy
that correlate with hypertension or induced experimentally by aldosterone salt
(Zhang Y. et al., 2015), it is reported to improve cardiac function and reduce
apoptosis microinfarction area after coronary micro-embolization (CME)
experimental model (Wang X-t. et al., 2017).
V. In chronic low grade inflammatory status correlate with diabetes;
insulin resistance- and obesity TAK 242 administration improve CK
level resulted from high level LPS and non esterified fatty acid
(NEFA)-activated TLR4 (Hussey S. et al., 2013). In in vitro muscle cell
experiment, TAK 242 inhibits both inflammation and insulin resistance,
mRNA expression of inflammatory genes, block phosphorylation of
human insulin receptor substrate-1 (IRS-1) at Ser312
by JNK and IKKβ -
the mechanism which impairs insulin signalling and also restore glucose
transport action of insulin (Zhang N. et al., 2015; Hussey S. et al.,
2013). Whilst in in vivo acute lipid infusion study in rats it improve
insulin muscle action, restore insulin suppression of hepatic glucose
level despite revealing no effect on body weight and feeding rate of the
animals (Zhang N. et al., 2015).

36
1.5 GIT 27
A small isoxazoline compound (4,5-dihydro-3-phenyl-5-isoxazole
acetic acid) which is also known as VGX-1027, possess very interesting
immunomodulatory effect throughout antagonizing the action of ligand
stimulated toll like receptors, with preferable low toxicity and high
efficacy (Hadi N. & Jabber H., 2016; Stosic-Grujicic S. et al., 2007;
Min H. et al., 2014) [figure (1-5)]; this drug has been developed for
treating miscellaneous inflammatory disorders such as type 1 diabetes
mellitus, colitis, inflammatory bowel disease, pleurisy, and rheumatoid
arthritis (Fagone P. et al., 2014; Mangano K. et al., 2008).
It has been tested on severeal in vivo and ex-vivo studies as stated by
Mangano K. et al., (2008) ; showing protective effect against LPS
induced uveitis, multiple low doses streptozotocin induced diabetes,
carrageenan induced pleurisy in mice as well as cellular type II collagen
induced arthritis, also protect against rat model ischemic-induced renal
injury (Hadi N. & Jabber H., 2016; Fagone P. et al., 2014) while further
chemical modification of the molecule, attachment of NO donating
group; even lead to antitumor effect in both human and animal model
cancer cell (Maksimovic-Ivanic D. et al., 2008).
Figure (1-5): structure of GIT 27 (Stosic-Grujicic S. et al., 2007).

37
1.5.1 GIT 27 Pharmacodynamics
GIT 27 antagonize toll like receptors-2/6 and 4 signalling pathways thus
inhibiting the release of proinflammatory CK as TNF-α, IL-1β,
macrophage inhibitory factor (MIF), and the soluble phototropic
immunomodulatory NO furthermore inhibits the LPS activated NF-
κB/p38-MAPK signalling pathways at the molecular level (Hadi N. &
Jabber H., 2016; Saurus P. et al., 2015; Saurus P. et al., 2016).
Fagone P. et al., (2014) found that GIT 27 also affect genes involved in
antigenic presentation process as well. At cellular level it primarily
targets secretory capacity of macrophages, DCs improving systemic
lupus erythematosus (Stosic-Grujicic S. et al., 2007; Fagone P. et al.,
2014), immune-stimulated β islet pancreatic cells destruction as well as
podocytes, renal differentiated cells that function mainly in glomerular
integrity; loss due to apoptosis in autoimmune diabetes (Saurus P. et al.,
2015; Saurus P. et al., 2016). Interestingly Stosic-Grujicic S. et al.
(2007) stated that “GIT 27 affect macrophage function in an IFN-γ
independent manner so that it preserves the IL-12 - IFN-γ axis which
could be an indication of less immunosuppression effect to provoke
immunocompromised state with the consequential reduced immunity to
opportunistic pathogens” .
Generally, these pharmacological scenarios are responsible for
improvement of immune response to exogenous antigen or stimuli
(Fagone P. et al., 2014).
1.5.2 GIT 27 Pharmacokinetics
Stosic-Grujicic S. et al., (2007) carried a modest analysis of the
pharmacokinetic profile for GIT 27 that discovered some interesting
properties in murine experimental model such that: approximate “t1/2”
of 90 min with peak plasma concentration “T max” achieved after 2 hr.
, also their chromatographic peaks analysis suggests no extensive

38
biotransformation of the drug in vivo. Also they reported rapid and
intermediate lasting pharmacological effect after in-vitro administration
(30 min and 5 hr. respectively) while drug accumulation stated after 24
hr. and steady state concentration achieved after 5 days (Yousif N. et
al., 2009).
GIT 27 was found to be effective after both oral and intraperitoneal
routes with no toxic effect in both acute and subacute toxicological
studies (Stosic-Grujicic S. et al., 2007; Mangano K. et al., 2008; Fagone
P. et al., 2014). Saurus P. et al., (2015) negate renal toxicity on their
experiment carried on both cultured human podocytes and murine
models. Approximate 90% of dug excreted renally unchanged (Yousif
N. et al., 2009).
Stosic-Grujicic S. et al., (2007) and Min H. et al., (2014) reported state
of well general appearance, lack of/or slight body weight reduction with
no change in feeding and drinking behaviour of murine model after
treatment with GIT 27.
1.5.3 GIT 27 in Clinical and experimental trails
GIT 27 is been evaluated in ongoing two phase I clinical studies
published by Clinical Trials.gov website started at (2009): for safety
pharmacokinetics and multiple dose administration in healthy subjects
(Maksimovic-Ivanic D., et al., 2008). As non-cellular specific toll like
receptor antagonist, GIT 27 affects multiple inflammatory disorders
(Min H. et al., 2014).
Starting with systemic lupus erythematosus, in their study carried on
strains of hybrid New Zealand (NZB/NZW) murine model; Fagone P. et
al., (2014) described very complex anti-inflammatory pattern beginning
from genetic expression modulation passing by TLR pathway inhibition
and ends with pro-inflammatory/anti-inflammatory CKs and CCs
regulation. At the genetic level GIT 27 up- and downregulate a total of

39
774 gene associated with LPS induced inflammatory pathways.
Interestingly this down regulation of the induced genes result in
enriched lupus immune-inflammatory pathways beginning with the
“systemic erythematosus” and “antigen processing and presentation”
pathways. The drug modifies genes responsible for presenting antigen
to CD4+
T cell which are human leukocyte antigen class II molecules as
well as class I human leukocyte antigen peptides subunit regulator
coding gene, systemic erythematosus antibody immune-stimulant
antigens, immunoglobulin like receptors that participate in natural killer
cell and T cells activating signal (Fagone P. et al., 2014).
Second the “spliceosome” pathway genes like the small nuclear
ribonucleotide (SNRNP) core protein family, intron binding spleosomal
proteins involved in pre-mRNA linking, members of the cyclophilin
peptidylylpropyl isomerases enzymes required in protein overlapping,
immunosuppressant drug action and virion infection, and modify the
molecular chaperons associated with DNA binding and dimerization
processes. Going through CCs, CKs and their receptor reaching to the
noticeable inhibition of IL-10, GIT 27 markedly improve the disease
due to the direct action at the genetic expression level (Fagone P. et al.,
2014). The next disease underwent the GIT 27 activities is uveitis, the
net effect observed was lower level of TNF-α, NO and decreased
number of the infiltrated cells in the aqueous humour, maintenance of
intact epithelium of the iris and milder signs of uveitis only during the
acute phase of the inflammatory response in rat model (Managano K. et
al., 2008).
Another trials shows renal protective effect against ischemic induced
renal injury and obesity metabolic disorder associated kidney disease
where the typical inhibitory action resulted in lower TNF-α, IL-1β and
plasma neutrophil gelatinase associated lipocalin (NAGL); a novel

40
biomarker for acute/chronic kidney diseases-level (Hadi N. & Jabber
H., 2016). The later murine model experiment further reported
reduction in the level of IL-2, genetic expression of IL-1, AMP-
activated protein kinase (AMPK), and type IV collagen, proteinuria,
urinary albumin excretion, renal oxidative stress and also renal fat
content through the improvement of renal tissue lipid metabolism and
glucose tolerance (as an additional indirect effect) (Min H. et al., 2014).
Considering autoimmune diabetes and diabetic nephropathy, GIT 27
inhibits the decrement of the anti-apoptotic proteins, pyruvate
dehydrogenase kinase-1 (PDK1) and cyclin dependent kinase-2 (CDK2)
expression in renal podocytes thus prevented podocytes apoptosis
induced by LPS and obesity induced kidney injury. Thus finally
improve diabetic nephropathy in both animal models and human
cultured podocytes (Saurus P. et al., 2015; Saurus P. et al., 2016; Min
H. et al., 2014). Its anti-diabetogenic effect preceded throughout
protecting β cells from apoptosis and autoimmune destruction,
inhibiting pancreatic mRNA gene expression of TNF-α, IL-1β, as well
as iNOS which results in lower level of NO, improving pancreatic
insulitis and dose depended reduction of blood glucose level. These in
vitro, in vivo, and ex- vivo effects last even after drug cessation and
suggests leading prophylactic and therapeutic role in managing human
autoimmune diabetes (Stosic-Grujicic S. et al., 2007).
Min H. et al., (2014) incidentally reported improvement of lipid content
in the liver tissue by GIT 27 within their metabolic effect of high fat
diet (HFD) induced obesity in murine model.

41
1.6 Aim of the study
To investigate whether treating the animals with TAK 242 or GIT 27,
could reverse the injuries induced by methotrexate or the tested drugs
have a valuable hepatoprotective potential, especially considering that
both drugs are anti-inflammatory immunomodulating agents.

Chapter Two
Materials and
Methods

Chapter Two: Materials & Methods
43
2.1 Materials
Instruments, drugs, chemicals and analytical Kits used in the present
study listed with their suppliers and origins below:
Table (2-1): List of instruments with their providers and origin
Instruments Providers Origins
Autoclave Nüve Turkey
Centrifuge Andreas Hettich GmbH and Co. KG
ZONESUN
Germany
Germany
China
Digital balance Furi China
ELISA reader BioTek USA
Glass wares
(Flask, Graduated cylinders and etc.)
Simax
NALGENE
Volac
China
China
UK
Incubator JRAD
CYAN
Syria
Belgium
Light microscope Motic Germany
Oral gavage Kent Scientific Corporation. USA
Oven CYAN Belgium
Sensitive balance OHAUS Switzerland
Spectrophotometer Biochrom Ltd. UK
Surgical set HHH ROSTFRICE Germany
Vortex mixer CYAN
K GEMMY
Belgium
Taiwan
Water bath CYAN Belgium
Water distillation unit Running Waters, Inc. USA

44
Table (2-2): List of chemicals and drugs with their providers and
origin
Chemicals and Drugs Providers Origins
Dimethyl sulfoxide 99.5% Central drug house (P) Ltd India
Eosin Central drug house (P) Ltd India
Ethanol 70% APCO Jordan
Ethanol 99.9% DiamonD France
Formalin 37% Solvochem UK
Glacial acetic acid 99.5% Scharlab Spain
GIT 27 MedChemExpress USA
Hematoxylin Central drug house (P) Ltd India
Ketamine 10% Alfasan woerden Holland
Methotrexate KOÇAK pharma Turkey
Normal saline 0.9% Shahid Ghazi pharmaceutical co. Iran
Paraffin Wax Sakura Finetek UK
TAK 242 MedChemExpress USA
Xylazine 20% Kepro Holland
Xylene Sakura Finetek Germany

45
Table (2-3): List of biochemical analysis kits with their providers
and origins
Kit Providers Origins
Rat ALT ELISA kit Elabscience USA
Rat AST ELISA kit Elabscience USA
Rat ALPL ELISA kit Elabscience USA
Rat IL-6 ELISA kit Elabscience USA
Rat TNF-α ELISA kit Elabscience USA
Rat LPO ELISA kit Elabscience USA
MDA ELISA kit Elabscience USA
GSH assay kit Elabscience USA
The total protein assay kit Elabscience USA
Bb ELISA kit Elabscience USA
2.2 Place and period of the Study
The experiment was performed at the Department of Pharmacology,
College of Medicine, Al-Mustansiriyah University and The Iraqi Center
of Cancer Research and Medical Genetics (ICCMGR), Al-
Mustansiriyah University, Baghdad, Iraq. It started on the 17th
of
October, 2017 and lasted for 9 months, following the approval by the
ethical committee of the pharmacology department, College of
medicine, Al-Mustansiriyah University.

46
2.3 Experimental Animals
A total of 35 male albino-wistar rats (aged 4-6 months) (weight 125-225
g) were taken from Kut technical Institute, University of Wasit. They
were maintained under nonspecific pathogen free conditions in wire-
meshed cages (7 rats in each cage) with Ad libitum access to water and
food. Under a constant temperature 24 ± 3ο
C with 12:12 hr. light-dark
cycle (normal laboratory conditions) (Hadi N. & Jabber H., 2016;
Olayinka et al., 2016; Yucel Y. et al., 2017).
Animal handling and housing were proceeded in accordance with the
International Guidelines for the care and use of laboratory animals of
the National Research Council (Hadi N. & Jabber H., 2016; Olayinka E.
et al., 2016).
2.3.1 Animal diet: Rats were kept on the regular rat diet that consist of
wheat, barley, corn, soybean and vegetable protein with 1kg/ton of both
multivitamins and antioxidants.
2.4 Experimental design
The animals were divided into random 5 groups (7 rats in each group)
as follow (Olayinka E. et al., 2016; Zhao Y. et al., 2015):
Control group: Rats were kept on regular diet and distilled water
throughout the 14 experimental days.
Vehicle pre-treated group: Rats were administered i.p. dimethyl
sulfoxide (DMSO) for 7 days followed by 7 days of oral methotrexate
0.2mg/kg/day.
Methotrexate group: Rats were left untreated for 7 days followed by 7
days of oral methotrexate (MTX) 0.2mg/kg/day via rat oral gavage
(Olayinka E. et al., 2016).

47
TAK 242 pre-treated group: Animals were administered i.p. TAK 242
5mg/kg/day for 7 days followed by 7 days of oral methotrexate
0.2mg/kg/day via rat oral gavage (Olayinka E. et al., 2016; Zhao Y. et
al., 2015).
GIT 27 pre-treated group: Rats were administered 4 i.p. challenge
doses of GIT 27 25mg/kg at 168, 120, 72 and 24 hours before starting
treatment with oral methotrexate 0.2mg/kg/day for 7 days via rat oral
gavage (Hadi N. & Jabber H., 2016; Olyinka E. et al., 2016).
Table (2-4): Experimental design
Groups
Treatment
Day 1-7 Day 8-14
I:Control - -
II: DMSO DMSO i.p. MTX 0.2mg/kg oral
III: MTX - MTX 0.2mg/kg oral
IV: MTX + TAK 242 pre-treatment TAK 242 5mg/kg i.p. MTX 0.2mg/kg oral
V: MTX + GIT 27 pre-treatment GIT 27 25mg/kg
(Day 1,3,5 and 7) i.p.
MTX 0.2mg/kg oral
2.4.1 Experimental model of MTX-induced liver injury:
It was achieved via the administration of 0.2mg/kg/day MTX orally via
rat oral gavage for 7 days for all the groups except control group
dependent on previous literature (Olayinka E. et al., 2016). Figure (2-1).

48
Figure (2-1): Administration of oral methotrexate 0.2mg/kg/day via
rats’ oral gavage to the animals (as a model of MTX-induced liver
injury).
2.5 Preparation of drugs
2.5.1 TAK 242: Its chemical formula (C15H17CIFNO4S) with DMSO
solubility of ≥ 360 mg/mL [according to the manufacturer]. TAK 242,
white crystalline powder, dissolved in DMSO and diluted in D/W to
final concentration of 17mg/ml 1 hr. before it was administered i.p.
according to rat weight (Zhao Y. et al., 2015; Yousif N. et al., 2009).
2.5.2 GIT 27: Its chemical formula (C11H11NO3), supplied as off-white
crystal with ≥65 mg/ml solubility in DMSO [according to the
manufacturer]. It has been dissolved in DMSO.D/W to final
concentration of 7mg/ml 1 hr. before being administered i.p. according
to rat weight (Hadi N. Jabber H., 2016; Yousif N. et al., 2009).

49
2.5.3 MTX: Its chemical formula (C20H22N8O5) purchased as 50mg/5ml
solution, and was diluted with D/W to a final concentration of
0.333mg/ml to be administered orally via rat oral gavage according to
rat weight.
2.5.4 DMSO: It was purchased as 99.5% solution, diluted with D/W to
same as the volume used to dissolve both of the drugs [TAK 242 + GIT
27] and administered i.p. according to their protocols and rat weight
(Olyinka E. et al., 2016; Zhao Y. et al., 2015; Yousif N. et al., 2009).
2.6 Samples collection
After 24 hr. of the end of the treatment, the rats were anesthetized with
Ketamine 91mg/kg-Xylazine 9mg/kg I.M. (Hawk C., Leary S.and
Morris T., 2005; IQ 3Rs Leadership group, 2016).
Blood Sample collection done after sacrificing the animals (Akbay T.
et al., 2010), heart blood was obtained using direct needle puncture
(Matsungana N. et al., 2011). Samples were allowed to be settled in 10
ml sterile labeled gel tubes, then centrifuged at 4000 rpm for 10 min at
25ο
C (Olayinka E. et al., 2016).
The collected serum was stored in 2ml Eppendorf tubes at -20ο
C until
used for further analysis (Hadi N. & Jabber H., 2016).
2.6.1 Tissue samples collection: A cut was done to rats’ abdomen
using sharp scissor, the liver was dissected out immediately. Liver
tissue samples were fixed in containers with 30 ml of 10% formaline for
preservation of tissue structure from autolysis then stored until they
were processed (Zhao Y. et al., 2015).

50
2.7 Biochemical Markers
2.7.1 Markers of hepatic function
2.7.1.1 Total serum protein: Total serum protein (TSP) was measured
according to the assay kit steps that is depending on Cu+ reduction in
alkaline medium by proteins, then forming purple complex after
combination with the provided BCA reagent. That complex has peak
absorption at 562 nm and the obtained optical density (OD) values are
proportional to the present protein concentration; so total proteins was
calculated according to this formula (Olayinka E. et al., 2016):
Total serum protein concentration (μg/mL)
=
𝐎𝐃 𝐒𝐚𝐦𝐩𝐥𝐞−𝐎𝐃 𝐁𝐥𝐚𝐧𝐤
𝐎𝐃 𝐒𝐭𝐚𝐧𝐝𝐚𝐫𝐝−𝐎𝐃 𝐁𝐥𝐚𝐧𝐤
× Concentration of standard (563 𝜇𝑔/𝑚𝐿) ×
Dilution factor of sample before tested.
2.7.1.2 Hepatocellular markers: The estimation of serum activities of
ALT, AST and ALPL were done via enzyme linked immunosorbent
assay (ELISA) kit according to the manufacturers’ procedures, which
are shared among the kits as well as the principles.
These ELISA kit uses Sandwich-ELISA as their technique, in which
micro ELISA plates were covered with antibodies specific to rat ALT,
AST and ALPL respectively. Standards and/or samples added to these
plates’ wells to be combined with these antibodies followed by the
addition of biotinylated detection antibodies specific for rat ALT, AST
and ALPL and Avidin-Horseradish Peroxidase (HRP) conjugate to each
micro plate well.
These wells should be incubated at 37ο
C, washing should be done to
remove extra components, substrate reagents then added so that only
wells that implicates rat ALT, AST and ALPL biotinylated detection
antibody and Avidin-HRP conjugate would be blue coloured.

51
Addition of Substrate reagent would put an end to these enzyme-
substrate interactions yielding a yellow colour, finally the outcomes
estimated by measuring OD with spectrophotometer at a wavelength of
450 nm. These OD values are proportional to the present concentration
of rat ALT, AST and ALPL. Rat samples of unknown concentrations
could be calculated by comparing the OD of the samples with the
standard curve (Akbay T. et al., 2010, Olayinka E. et al., 2016; Yousif
N. et al., 2009).
2.7.1.3 Hepatobiliary bilirubin: Serum level of bilirubin (Bb) was
measured by ELISA kit; this kit utilized the technique of Competitive-
ELISA, in which the micro ELISA plate supplied in this kit has been
covered with an antigen specific to Bb. Samples and/standards’
bilirubin competes with the solid phase constant amount of Bb for the
Biotinylated Detection Ab specific to Bb throughout the reaction, then
Excess conjugate and unbound sample or standard are washed out
followed by addition of Avidin conjugated to Horseradish Peroxidase
each microplate well. A period of incubation at 37ο
C. The Substrate
Reagent is added to each well would end of the enzyme-substrate
reaction established by the addition of Stop Solution and the color
change can be measured spectrophotometrically by ELISA reader at a
wavelength of 450 nm. The Bb unknown concentration in samples then
calculated by comparing the OD of the samples with the standard curve
(Olayinka E. et al., 2016).
2.7.2 Inflammatory markers:
TNF-α and IL-6 serum levels were estimated again via ELISA kit which
uses Sandwich-ELISA as their technique, in which micro ELISA plates
were covered with antibodies specific to rat TNF-α and IL-6
respectively. Standards and/samples added to these plates’ wells to be

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