Evaluation of hepatoprotective agents - Hemant Kanase

Evaluation of Hepatoprotective Agents
Dr. Hemant R Kanase
1

Flow of the seminar
- Introduction
- Ideal hepatoprotective agent
- Limitations of current treatment
- Preclinical test:
• In-vitro methods
• In-vivo methods
- Parameter of evaluation
- Clinical Assessment
2

Introduction
• Largest and the most important organ (weighing 1–1.5 kg and
representing 1.5–2.5% of the body mass.)
• Principle site for metabolism, secretory and excretory functions.
• Distinguishing fact: potential of hepatocyte to proliferate and
regenerate along with its dual blood supply
3

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Liver Toxicity
The main causes of liver damage:
Chemicals like carbon tetrachloride CCL4, phosphorous , aflatoxins, chlorinated hydrocarbon
etc
Drugs i.e. DILI ( drugs induced liver injury ) eg. Halothane, Paracetamol.
Alcohol
Autoimmune disorders : PBC, PSC, Wilson’s disease
Infections: Viral , bacterial, parasites

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 Caruaru tragedy – dialysis tragedy (1996)
• 126 pts on hemodialysis
• Intoxicatced Microcystis sp. Blue algae – Microcystin LR – Hepatoxic agent
 Case report:
• 76 yr female
• Presented with blister on foot, later died with sepsis, bleeding and hepatic
faillure
• Diagnosis : colitis
• Treatment : Mecaptopurine (thiopurine) with allopurinol
Drug interaction - hepatoxicity

On depletion of glutathione
Leads to accumulation of NAPQI
Causes Toxicity
8

An ideal hepatoprotective agent should be able to:
 control infection or counter the infective noxious and injurious elements, if these are the
etiological factors,
 protect the liver parenchyma, promote healing of damaged parenchyma to revert back to
a structurally normal or near normal state,
 help liver parenchymal cells to regain their functional capabilities,
 prevent complications both short term and long term leading to mortality and
morbidity,
 an ideal antifibrotic would be one that is liver specific, well tolerated and effective in
attenuating excessive collagen deposition without affecting normal extra cellular matrix
synthesis.
9

Currently available modalities of hepatoprotection:
 Removal of causative agent
• iron and copper overload, alcohol consumption, secondary biliary obstruction, drug
induced liver disease
 Inhibiting the inflammatory response of liver
• anti-inflammatory agents : Corticosteriods (lack of anti fibrotic activity)
Colchicine (Anti inflammatory + Anti Fibrotic )
 Inhibition of activation and proliferation of hepatic stellate cells
• Antioxidants: Vitamin E (-tocopherol), phosphatidylcholine, S-adenosyl-L-methionine
(SAMe)
• Prevent hepatic stellate cell proliferation and collagen formation: Interferon-,
pentoxifylline and amiloride:
• Antifibrotic activity: Vasodilators like PGE2 and nitric oxide donors
10

Limitations of currently available drugs
• Most of them don’t have proven efficacy conclusively.
• There is high risk of relapse
• Many of them produce side effects.
Treatment largely been supportive and symptomatic.
Shift in focus to alternative forms of therapy based on drugs derived from plants.
Thus there is a need for evaluation of new treatment modalities by using modern
scientific methodology
11

Models of hepatoxicity
Pre clinical test :
• In vivo
• In vitro
Clinical assessment
12

In vitro models of liver damage
1. Primary screening of the potential therapeutic agents
2. Primary hepatocyte cell culture
3. Stellate cell culture
4. Kupffer cell culture
5. Rat liver slice
6. Assays for measuring total cell population
7. Assays for measuring cell proliferation
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1. Primary screening of the potential therapeutic agents
 Different in vitro concentrations of the test drug are incubated with the culture and
effect of the drugs on the cell line is studied.
 Parameters : viability and proliferation of the cells in presence of the test drugs.
 Viability - cytotoxic effect of the drug.
 Effect on proliferation of cells - Antifibrotic effect, evaluated by the modulation of
monocyte macrophage system.
 The drugs can be incubated in various in vitro concentrations with macrophages
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2. Primary hepatocyte cell culture
Fresh hepatocyte preparations and primary cultured hepatocytes are used
The basic method:
 Isolation of hepatocytes by perfusion of liver with collagenase or utilization primary
cultured hepatocytes.
 Determination of the viability of the hepatocytes.
 Incubation of the cell culture with hepatotoxin and with or without the test drugs.
 Determination of the activity of the transaminases released into the medium by the
hepatocytes
Hepatotoxins: CCl4, paracetamol, d-galactosamine, tert-Butyl hydroperoxide and ethanol.
16

3. Stellate cell culture
 In chronic injury there is stellate cell activation
 There is secretion of matrix by activated stellate cells results in liver fibrosis and
ultimately cirrhosis.
 Stellate cells are isolated from rat liver by collagenase / pronase digestion
 The test drugs are incubated with the activated stellate cell culture by hepatotoxins
 Parameters studied :morphology of the cells, -SMA expression, cell count with
thymidine incorporation and inhibition in synthesis of collagen type I and III.
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4. Kupffer cell culture
 Isolation: Kupffer cells are isolated from the liver by perfusion of the liver with pronase
followed by differential centrifugation.
 The isolated cells are maintained in culture where their phagocytic properties are
retained.
 On exposure of the cells to phagocytic stimuli like zymosan particles, there is increased
oxygen consumption and superoxide production.
 The inhibitory effects of various agents or drugs on Kupffer cells are tested in the in
vitro cultures.
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5. Rat liver slice
 A liver slice consists of the various cell types of the organ where the cells maintain
normal architecture, acinar localization and cell-cell communications.
 The functional heterogeneity and biochemical capacity of the whole organ is retained.
 Histopathological assessment of the exposed tissue can be done.
 It may be possible to assess action of hepatotoxin on the liver even if only few
milligram of test drug is available.
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6. Assays for measuring total cell population
To determine the total cell population in the assay system
Dye exclusion test : dye is retained by dead cells and excluded by live cells
DiSC assay (Differential Staining Cytotoxicity) : live cells and dead cells are counted by two
different colours against an internal standard
MTT assay : colorimetric assay which depends upon the reductive capacity of live cells to
metabolize the MTT dye to a highly coloured formazan crystals.
• The UV absorbance of the solubilized crystals is measured spectrophotometrically and
directly correlates with the number of living cells in suspension.
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7. Assays for measuring cell proliferation
Clonogenic assay (Human tumour stem cell assay)
 Culturing of cells in petri dishes, capillaries or wells with or without drug.
 At the end of the culture period, the number of colonies is counted.
 The difference between the number with and without drug is taken as a
measure of drug effect.
 Thymidine incorporation assay:
Actively growing and multiplying cells Cells are incubated with radiolabelled
thymidine, they use radioactive thymidine for their DNA synthesis.
 The amount of incorporated thymidine is proportional to the proliferation.
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Advantages & disadvantages of in vitro models
 Advantages
• Ability to screen numerous samples at one time.
• Convenient
• Less variation in results.
• Good reproducibility.
Disadvantages
• Damage to hepatocytes may be mediated through mechanisms different from
those operating in vivo, and results may differ.
• The test drugs may not cross the GI barrier to reach the liver tissue in exactly the
same proportions as that added to the cell culture.
• Extrapolation of these findings to human beings is more difficult.
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In vivo models of hepatotoxicity
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In vivo models of hepatotoxicity
A. Non-invasive model
a. Chemically induced hepatotoxicity
b. Drug-induced hepatotoxicity
c. Radiation-induced hepatotoxicity
d. Metal-induced hepatotoxicity
e. Diet-induced hepatotoxicity
B. Invasive model
Bile duct ligation
C. Genetic model
Transgenic animal
model
Animals – rodents (wistar rats, spargue dawley, Swiss albino mice)
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Chemically induced hepatoxicity
Commonly used hepatotoxins
• Carbon tetrachloride (CCL4)
• Thioacetamide
• Dimethyl or diethyl nitrosamine
• Alfatoxin B1
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CCl4 induced
 Grain fumigant, dry cleaning fluid, fire extinguisher fluid
The formation and release of free radicals and lipid peroxides is mechanisms of
injury.
used for inducing both acute and chronic liver disease
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Principle:
CCl4 --- Cytochrome P-450/2E1 (CYPE2E1) in hepatocytes ---- CCl3 (Toxic metabolite)
The free radical (trichloromethylperoxyl radical) ------ attacks lipids on the membrane of
endoplasmic reticulum, induces an acute centrolobular necrosis.
Procedure :
• Wistar rats (120 – 150 gm)
• Before experiment, animals are deprived from food.
• Hepatotoxicity is produced in animals by different concentration of CCl4 by different
route (i.p., s.c., oral)and different time period .
• On the last day of the study the animals are anesthetized and killed for studying
biochemical parameter and Histopathological tests.
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Model of acute hepatitis:
Model of fibrosis:
• 1 mg/kg carbon tetrachloride twice a week orally dissolved in olive oil (1:1), over a
period of 8 weeks.
Dose mg/kg Duration Route of admin
1 mg/kg
0.5 ml/kg
0.2 ml/kg
0.125 ml/kg
1.0 ml/kg
2 ml/kg
2-5 days
3 days
2 weeks
7 days
Every 72 hrs for 10 days
Every 72 hrs for 10 days
Oral
i.p.
i.p.
i.p.
i.p.
s.c.
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Evaluation:
1. The following parameters are determined in the serum:
• Total bilirubin
• ALT, AST
• GGT
• 7S fragment of type IV collagen
• Procollagen III N-peptide
2. Histological analysis of the liver using a score from 0 to IV
Grade 0: Normal histology
Grade I: Tiny short septa of connective tissue without influencing the hepatic lobule
structure
Grade II: Large septa of connective tissue with penetration into parenchyma and tendency
to develop nodules
Grade III: Nodular liver architecture with lost hepatic lobule structures
Grade IV: Excessive connective tissue deposition subdividing the regenerating lobules
and development of scars 29

Effect of CCl4 Morphological parameters
Normal liver Post - CCl4 liver
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SilymarinDistilled water CCl4
Liver histopathology post- CCl4 (Masson
Trichome Stain)
31

Modification:
Kawaura et al. (1993) produced liver cirrhosis with ascites in dogs by administration
of 2 ml CCl4 per kg body weight once a week for 4 weeks.
Wirth et al. (1997) studied the effects of a bradykinin B1 receptor antagonist in rats
with CCl4-induced liver cirrhosis.
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D – Galactosamine
Single dose or a few repeated doses of D-galactosamine cause acute hepatic necrosis in
rats.
• Prolonged administration leads to cirrhosis
Accumulation of uridine sugar nucleotide causing disruption of metabolism of
glycoproteins and glycolipids leading to increased cytosolic Ca+
Procedure
Wistar rats (100-180 gm) are used.
Single or divided doses of 100-400 mg/kg D-galactosamine are injected i.p. or i.v.
during one day.
Model of fibrosis: 500mg/kg of D-galactosamine is injected i.p. thrice weekly for 1-3
months
Evaluation:
Liver - HPE
Blood - Biochemical parameters
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Thioacetamide (TAA)induced hepatotoxicity.
 White crystalline solid, serves as source of sulphide ions in the synthesis of organic
and in organic compounds.
 TAA – not toxic, Thio acetamide S – oxide binds to hepatic macromolecules,
increases intracellular Ca+ concentration and causes cellular damage and necrosis.
 Widely used to induce fibrosis
 Procedure:
Wistar rats 100-150 gms.
I.P. dose of thioacetamide: 200 mg/kg - thrice weekly for 8 weeks
200 mg/kg - for 2 weeks
Evaluation:
Liver - HPE
Blood - Biochemical parameters
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Diethyl nitrosamine (DEN) induced
 Carcinogenic: used in the manufacture of some cosmetics, pesticides, rubber products
 DEN is hydroxylated to ethyldiazonium ion by CYP2E1 causes DNA damage and induces
hepatotoxicity
 This model is important in studying progression of fibrosis to HCC.
 Procedure
• Wistar Rats
• Free access to standard rat pellet food and tap water..
• Animals treated with diethyl nitrosamine having different concentration (50–200
mg/kg, i.p. for 4–12 weeks).
Evaluation:
Liver - HPE
Blood - Biochemical parameters 35

Alfatoxin B1 induced hepatotoxicity
 Mycotoxin – acute hepatotoxicity and liver carcinomas
 Metabolized by cytochrome P450 to highly reactive 8, 9-epoxide that binds to cellular
macromolecules, primarily in the periportal region of the liver.
 Liver injury manifests as periportal parenchymal cell necrosis, haemorrhage, and injury to
intrahepatic bile ducts.
 Used to establish models of cholestasis
 Procedure:
Wistar Rats
Disease is induced by administration of different concentration of alfatoxin (200 micro
gm/kg – 6 mg/kg PO) for 2days – 52 weeks.
 Evaluation:
Blood – Biochemical analysis
Liver - HPE 36

Drug induced hepatotoxicity
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Acetaminophen induced
 Paracetamol administration causes necrosis of the centrilobular hepatocytes
 Procedure:
• Wistar rats 150-200 gms.
• Single oral dose of 2gm/kg p.o. body weight
• Test compound is given for 6 days prior to PCM administration and on 7th day along with
PCM administration
• On last day animal are sacrificed.
• different doses (2-3 mg/kg, p.o. for 7 days–3 weeks)
 Evaluation:
Liver - HPE
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Anticancer drugs induced…
 Mechanism: Secondary to production of reactive oxygen species (ROS), intended to
induce tumor cell apoptosis.
Cisplatin induced hepatoxicity:
 Mechanism: Elevated CYP2E1 enhances production of ROS and oxidative stress which
induces hepatotoxicity
Procedure:
• Wistar rats (100-150 gms)
• Free access to food and water.
• Dosage and duration: 3.5–7 mg/kg i.p. for 1–5 days
 Evaluation:
Liver - HPE 39

Anti-TB drugs induced…
 INH , Rifampicin
 Principle Isoniazid is chemically isonicotinyl hydrazine which is used to treat tuberculosis.
N - acetyltransferase CYP2E1/Cyt chrome p450
INH --------------------- acetyl hydrazine -------------------------------- reactive acylating sp.
Rif (+) Rif (+)
 Reactive species covalently with liver cell macromolecules cause hepatocyte injury.
 Procedure:
• Wistar rats 150–200 g, free access to food and water.
• Rats are treated with isoniazid (INH) 50mg/ kg, co-administered with rifampicin (RIF)
100mg/kg i.p.of 10–28 days
 Evaluation:
Liver - HPE 40

Radiation induced hepatotoxicity:
Principle :
 Activation of the coagulation cascade leading to accumulation of fibrin and
formation of clots in the central veins and hepatic sinusoids.
 Cause hepatic dysfunction by death of centrilobular hepatocytes and atropy of the
inner hepatic plate.
Procedure:
• Animals allowed free access to a standard diet and clean drinking water.
• Animals are irradiated
• Single dose of 5 Gy of gamma-radiation for 2 days
• single dose level of 3 - 6 Gy for 7 days
• Single dose of gamma rays 6 Gy for 15 consecutive days
 Evaluation:
Liver - HPE 41

Metal induced hepatoxicity
 Toxic and carcinogenic metal compounds.
 It promotes the formation of reactive oxygen species (ROS) such as hydrogen peroxides.
These ROS enhance the production of lipid peroxides and the highly reactive hydroxyl
radical
Procedure:
• Animals are treated by mercury,
• Mercury chloride (HgCl2) (80 mg/l) as drinking water for 4 weeks.
• 5 mg/kg s/c inj of HgCl3 on the 7th day of experiment
 Evaluation:
Liver - HPE 42

Diet induced hepatoxicity
Alcohol induced..
 Acetaldehyde: is toxic and may cause membrane damage and cell necrosis via
generation of highly reactive oxygen species (ROS) (alpha-hydroxyethyl)
Procedure:
• Ethanol 2.0 ml/l00 g p.o. for 21 days
• Ethanol 7.9 g/kg p.o. daily for 45 days
• Ethanol 5 g/kg/day p.o. for 60 days
• Ethanol 3.76 gm/kg twice a day p.o. for 25 days
 Evaluation:
Liver - HPE
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Models of acute hepatitis:
1. CCl4 – 1 mg/kg for 5 days
2. D- galactosamine – 100 – 400 mg/kg single / divided dose on single day
3. Paracetamol induced – 2mg/kg for 6 days
Limitations:
• Lesions produced in above mentioned models resemble lesions produced in viral
hepatitis, i.e. necrosis, the underlying mechanism may be totally different.
• The viruses may not act in the same way as hepatotoxins.
There are models of acute hepatitis using virus:
• MHV A 59, MHV 3 (focal necrosis), woodchuck hepatitis virus (Hep B like hepatitis)
• Induce hepatitis by macrophage virus interaction
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Models of chronic hepatitis
Liver appearances in chronic hepatitis of viral and non-viral origin are similar.
Features: infiltration of mononuclear cells into the liver and signs of parenchymal
damage.
Various models:
i.p. injection of Corynebacterium parvum (0.7 mg/mouse i.v.).
i.p. injection of LPS from Escherichia coli (25 g per mouse)
weekly i.p. injection with syngenic liver homogenate fractions containing a self
antigen liver specific proteins (LSP) and Freund’s adjuvant (FCA) for 8 weeks
 Evaluation:
Liver - HPE
45

Models of fibrosis
 Persistant exposure to inducing agent may lead to fibrosis and serves as a good model
for anti-fibrotic drugs.
 Models for such injury should meet the following criteria:
• It must be reproducible.
• Mortality rate of animals should be low.
• If recovery is to be studied the injury should be reversible.
• Stages from the onset of acute injury to the development of fibrosis must be distinct
 The models of chronic liver injury can be induced by
• 1 mg/kg carbon tetrachloride twice a week orally 8 weeks
• 500mg/kg of D-galactosamine is injected i.p. thrice weekly for 1-3 months
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Using surgical/ invasive techniques:
Bile duct ligation (BLD) induced fibrosis in rats.
 Useful experimental model to study fibrosis.
 BDL stimulates the proliferation of biliary epithelial cells, resulting in proliferating
bile ducts, cholestasis, portal inflammation, and fibrosis, causing secondary biliary
cirrhosis
 Procedure:
 Male Sprague Dawley rats (250 g) are anesthetized.
• midline abdominal incision
• isolation of the common bile duct above the duodenum
• two ligatures were placed to the proximal and on distal part of the bile duct.
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• Animals are given normal diet and water ad libitum throughout the experiment.
• Test compound is administered twice daily for 6 weeks.
• After 6 weeks animals are sacrificed
• Blood: Hepatic enzymes, bile acids, 7S fragment of type IV collagen, and
procollagen III N-peptide.
• The liver: histological studies and for hydroxyproline determinations
 At 4 – 6 weeks: features resemble biliary cirrhosis in humans
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Models of Cholestasisis
A. Endotoxin induced cholestasis:
 Mechanism: inhibits the uptake and secretion of bile acids and organic
anions.
 7.5 micro gm /kg dose is administered i.p.
B. Ethinyl estradiol induced cholestasis:
5 mg/kg body weight orally.
The cholestatic effect is due to the endogenous estrogen metabolite
estradiol-17-D-glucoronide (leads to impairment of transport mechanism
in basolateral and canalicular hepatocyte membrane.)
Other drugs:
 Cyclosporine A, Rifamycin, rifampicin glibenclamide, Alpha napthyl
isothiocynate inhibit bile salt transport and induce cholestasis.
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Model of obstructive cholestasis:
 Physical obstruction to the biliary system
 Total: Impaired excretion and retention of bile within the hepatocytes
increased ductular proliferation and periportal necrosis
 Partial: Incomlete obstruction to the bile duct causing mild ductular proliferation and
portal fibrosis
Good model for chronic fibrosing cholangitis.
 Selective obstruction: Selected bile ducts draining selected lobes.
For studying reserve secretory capacity.
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Models of steatosis:
Lipid content of hepatocyte regulated by: Uptake, synthesis, oxidation and export
Hepatic steatosis : input of fat exceeds the capacity for fatty acid oxidation i.e
output
For studying human nonalcoholic fatty liver disease
1. Increased hepatic `input’ of fats
 Genetically modified mice with
increased hepatic lipogenesis
 Environmental causes of increased fatty
acid input
2. Decreased hepatic `output’ of fats
 Genetically modified mice with
decreased hepatic elimination of fats
 Environmental causes of decreased
hepatic elimination of fats
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1. Targeted over expression
of genes that promote
lipogenesis
Eg.
A-BZIP/F mice
Adipose specific C/EBPKO
aP2-Diptheria Toxin mice
• Enlarged, fatty livers at a very
young age
• Deficient in the adipocyte
hormone leptin
2. Naturally occurring
mutations that promote
lipogenesis
Eg.
• ob/ob mice - naturally occurring
mutation that prevents the
synthesis of leptin
• db/db mice - mutation of leptin
receptors
• fa/fa mice - exhibit mutation of
the gene for leptin receptor.
• Resistance to leptin
I. Increased hepatic `input’ of fats
 Genetically modified mice with increased hepatic lipogenesis
52

Environmental causes of increased fatty acid input
(i) Diet-Induced Obesity
High sucrose/ fructose diet (70 % sucrose, 10% fructose)
High fat diet (60% fat) given for 8 weeks
(ii) Diets that induce hepatic lipogenesis without Obesity
Arginine-deficient diet (abnormal orotic acid metabolism)
53

II. Decreased hepatic `output’ of fats
A. Genetic causes of decreased hepatic elimination of fats
(i) Mice with targeted deletion of genes that regulate fatty acid oxidation
PPARK-/- mice
AOX KO (Acyl CoA oxidase)
Aromatase KO (female)
(ii) Mice with naturally occurring mutations that inhibit fatty acid oxidation
Juvenile visceral steatosis (carnitine deficiency)
B. Environmental causes of decreased hepatic elimination of fats
• Methionine and or choline deficient diet (0.05%)
54

Problems faced with animal studies
1. Most of the animal models are based on chemical injury whilst the most common form
of clinical disease is viral in etiology.
2. Liver damage caused by toxins may be different from effect of natural hepatotoxins.
3. Chronic liver disease is hard to mimic in laboratory.
4. The drug used is usually administered along with or before injection of toxin, and
hence focus is on preventing damage due to toxin. However, patients present after
liver injury has set in.
5. Costly
6. Inconsistency in serum indicators used in studies.
55

Parameters of evaluation
In a particular model system of liver injury induced by any agent,
 the extent of functional and structural damage to the liver
 the regenerative capacity of the liver following such damage
This can be assessed by
 gross and histopathological examination of the liver using various stains,
 biochemical methods which measure various enzymes or substances in serum
liberated after injury to cells,
 evaluation of the clearance of substances from the blood.
56

Gross and Histopathological Examination
 Gross examination : Done by by isolating the organ from the body.
• Gives an idea about changes in margin, consistency and colour of the liver e.g. in
cirrhosis the liver is shrunken, nodular where as in obstructive jaundice it is enlarged.
 Histopathological examination: liver is sectioned,fixed and,stained
 The different stains:
• Haematoxylin-Eosin stain - nucleus and cytoplasm
• Masson trichome or Sirius red stain - collagen fibers
• Reticulin stain - reticulin fibers of the liver
57

Advantages of HPE
Information on nature of injury.
Eg. hepatocytes (degeneration, necrosis, and regeneration), inflammatory reaction,
structure of bile ducts and vascular distortion can be studied.
The structural changes can be graded using scores: knodell score, NASH activity score
The most widely used is the histological activity index (HAI) also known as the Knodell
score.
• Periportal +/- Bridging necrosis
• Intralobular degeneration and focal necrosis
• Portal inflamation
• Fibrosis
• Total score of 22.
58

Limitations:
• Histological grades sometimes poorly correlate with clinical and biochemical
assessment.
• May be due to sampling error, and a tendency for histological features to change
more slowly in response to therapy than biochemical ones
59

Biochemical parameters
 Assessment of liver function
Aspartate Serum Transferase (AST), Alanine Amino Transferase (ALT), Alkaline Phosphatase
(ALP)Lactate dehydrogenase (LDH), Total Bilirubin (TB) Total protein (TP),Triglycerides (TG)
Gammaglutamyl transferase (GGT) levels
• Endogenous substances that are liberated in the serum after injury to the liver
• Tests are always done in ‘batteries’ to minimize the false negative results. Therefore, the
predictive accuracy is very high, 95 % or even better.
• The conventional tests are of prognostic help.
60

Conventional liver function tests
Pathological feature Endogenous substance Comments
Cellular injury + cholestasis Bilirubin Confirmatory evidence for the
presence of liver disease
Hepatocellular damage AST, ALT Rough estimate of necrosis
Prothrombin time
Coagulation proteins Reflect status of synthetic
capacityAlbumin, prealbumin
Pseudocholinesterase
Inflammatory reaction Acute phase protein -  globulin,
CRP, MDG, SOD, GSH
Non-specific for chronic liver
disease
Cholestasis Alkaline phosphatase Non-specific marker
5ʹ nucleotidase Specific marker
 glutamyl transpeptidase Marker for alcohol abuse
Vascular distortion Bile acids (total and conjugated) Indicate porto-systemic
anastomosis
61

Quantitative liver function test
Measure the functional capacity of the liver and quantify the important functions like
metabolic, excretory, and vascular functions
Principle: Known amount of an endogenous compound which is normally processed by
the liver and whose fate in the body is well characterized is administered.
Its clearance from the body is determined by taking plasma, urine or salivary samples.
Any change in its disposition can be quantified which reflects a specific functional
defect in perfusion, metabolism and/or excretion.
62

Quantitative liver function tests
MEGX test is useful as a real-
time method for quantitatively
assessing pre- and post-
transplant liver function
LiMAx test (maximum liver
function capacity) - based on
the metabolism of 13C-
methacetin
Costly and complex to perform
Not used routinely
Substrates Sampling sites
1. Vascular function
Indocyanine green Blood
2. Metabolic capacity
Antipyrine
Aminopyrine
Galactose( ditingush
between HC and bil.
obs)
Caffeine
Saliva, blood
Breath
Blood
Breath
Saliva, blood
Breath
3.Excretory functions
BSP
Bile acids
Blood
Blood 63

Assessment of liver fibrosis
Conventional LFT : provide little info on presence of fibrosis and no means of following
its progression
For exact quantification we use liver tissue.
Measured parameters:
• the content of total peptide bound hydroxyproline
• the activity of prolyl hydroxylase, lysl oxidase and -N-acetyl glucosaminidase
• collagenase
64

Specific tests have been developed
to measure circulating collagens,
procollagen peptides, extra cellular
matrix glycoproteins and their
fragments.
Non invasive and can be performed
repeatedly, thus offering potential
for monitoring anti-fibrotic therapy.
Markers reflect hepatic fibrogenesis,
fibrolysis or both?
They serve as supplements to the
routinely used parameter for
assessment of fibrosis.
Connective
tissue
polypeptide
Fibrogenesis Fibrolysis Liver
specificity
PICP + - +
PIIINP + +, Acute +
PIVCP - (?) + +
PIVNP(7-S) - (?) + +
CVI - + +
Lam + (?) + (?) +
Undulin - +,
remodelling
+
65

Immunological parameters
 In liver disease there is increased antigenic influence that is responsible for increased
antibody titers in cirrhotic patients
 It may be because of decreased ‘filter’ effect of the liver which is done by the
macrophage system of the liver.
 Experimental evaluation of Kupffer cell activity : injection of particulate matter and
studying its clearance
 Particulate matter: Colloidal carbon, human serum albumin lipid emulsions, radioactively
tagged bacteria, denatured serum protein fraction labelled with I131, magnetic iron colloid
66

Nitric oxide (NO)
 Ubiquitous messenger molecule involved in diverse cellular process such as
neurotransmission, immune regulation, and vascular homeostasis
 NO plays a important role in liver homeostasis
 NO protects hepatocytes involve induction of heat shock proteins, suppression of
caspase activity, activation of soluble guanylyl cyclase and modulation of
mitochondrial respiration.
 The intrahepatic synthesis of nitric oxide is markedly decreased in advanced human
and rat liver fibrosis.
67

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Pre clinical test
Screening of
lead
compound
Clinical phase
In vitro In vitro
• Cell culture
techniques
Hepatoxin induced
• Various models
Hepatitis, fibrosis,
cholestasis, steatosis,
diet induced.

70
 To evaluate safety
 To select appropriate dose based on the preclinical studies
 To determine the pharmacokinetic profile.
 To evaluate efficacy & potency
Clinical Assessment

Phase 1 and 2
 Objective :
• Assessment of safety and tolerance of the drug.
• Generation of preliminary PK data, duration of action and general PD effects.
• To determine adverse events in human subjects
• Pharmacokinetics- AUC, t1/2 , Ke, Tmax
• Study group: Normal healthy volunteers.
• Dosing : Multiple ascending dose study in elderly volunteers.
• Safety and tolerability assessed by : Clinical examination, biochemical test
71

Phase II/III studies
Objective :
 Confirm efficacy in patients/ risk of hepatic damage
 To determine safe dose range and maximum tolerable dose
 Monitor side effects
 Comparator – Placebo? Silymarin
72

Key Inclusion Criteria:
• Males and females >= 18 years of age.
• Diagnosis of acute viral hepatitis (A,B,C,E, EBV, CMV) (<1 month) as manifested by a
combination of the following symptoms: jaundice, dark-coloured urine, light-colored
stools, pruritus, fever, nausea, vomiting, anorexia, and right upper abdominal
discomfort, pain or feeling of pressure.
• Serum ALT level > 2.5 times the upper limit of normal.
• Albumin level >3.5 gm/dl
• Negative anti-HCV antibody
• Subject has given written informed consent.
73

Exclusion Criteria:
• Pregnant or breastfeeding women
• Suspected hypersensitivity
• Advanced liver disease (e.g. ascites,
bleeding esophageal varices and hepatic
encephalopathy)
• Chronic liver disease as cirrhosis
• History or alcohol abuse
• Subjects with positive anti-HCV antibody
• Simultaneous elevation of bilirubin > 10
mg/dl along with an ALT level between 100
and 150 U/L
• Platelets count <150,000
• Morbid obesity i.e. BMI > 40
• Subjects with severe illness, e.g.,
multisystem failure, cancer or poorly
controlled diabetes i.e. known diabetic with
Hemoglobin A1C (HbA1C)>7%, CCF
• h/o of drug-induced acute hepatitis.
• recent use Hepatoprotective agent within
past two weeks.
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Primary safety objective:
• To assess safety and tolerability of the test drug in patients with AVH.
Primary efficacy objective:
• To assess the percentage of subjects who normalize their SGOT, SGPT, ALP, Sr.
Albumin, globulin, Prothrombin time.
Secondary efficacy Objective:
• To assess the percentage of subjects in each group who show
• Symptomatic improvement- Assess quality of life (QOL) - physical examinations and
standardised questionnaire.
• Differences in response in different AVH etiologies (i.e. HAV, HBV, HCV, HEV) using
subgroup analyses.
• Total duration of recovery
75

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Phase IV
 Post marketing survelliance
 ADR monitoring
 Rare but serious adverse reactions or chronic toxicity
 Drug interactions

Evaluation of hepatoprotective agents - Hemant Kanase

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