SCREENING MODELS OF HEPATOPROTECTIVE DRUGS

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
Ms. I. Sai Reddemma.
(Reg. No. 20L81S0101)
Department of Pharmacology
Under the guidance/Mentorship of
Dr. K. Somasekhar Reddy. M. Pharm., Ph.D.
Associate Professor and Head Department of Pharmacology.
SCREENING MODELS OF
HEPATOPROTECTIVE DRUGS

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• Introduction
• Liver toxicity
• Drugs causing DILI
• Markers of hepatotoxicity
• List of hepatoprotectives
• Functions of liver
• Screening models of hepatoprotective drugs
Contents

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Introduction :
• Liver is large, meaty organ that located in the right upper quadrant
of the abdomen below the diaphragm.
• It is the only organ found in vertebrates which detoxifies various
metabolites, synthesizes proteins and produces biochemical's
necessary for digestion.
• It has a surprising role in the maintenance, performance and
regulating homeostasis of the body.
• It is involved with almost all the biochemical pathways to growth,
fight against disease, nutrient supply, energy provision and
reproduction.

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• The major cause in India is ethanol and it is suspected that more
than half of the cases of Hepatotoxicity is caused by alcohol.
• Chemicals like carbon tetrachloride CCL4, phosphorous ,
aflatoxins,chlorinated hydrocarbon etc
• Drugs i.e. DILI ( drugs induced liver injury )
• Autoimmune disorders
• Infections like viral hepatitis
LIVER TOXICITY

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• Anti tuberculosis drugs-All drugs causes hepatotoxicity except
ethambutol.
• Anti convulsant drugs-Carbamazepine, valproic acid.
• NSAIDS-Paracetamol , diclofenac ,indomethacin , oxicam group
• Anti-microbials-Dapsone,ketoconazole,Sulfonamides, anti retrovirals
• Anaesthetics- Enflurane , Isoflurane.
• Miscellaneous drugs-
• Disulfuram, Flutamide, Statins, Labetlol, Nicotinic acid,
Propylthiouracil and OC pills.
DRUGS CAUSING DILI

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• 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)
Markers of Hepatotoxicity

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• N acetylcysteine
• Penicillamine
• Anti oxidants
• cardiotropin
• Herbal medications e.g. silymarine
• S adenosyl methionine (SAM)
• Vitamins
• Melatonin
• Glutathione
• Beta-carotene.
LIST OF HEPATOPROTECTIVE AGENTS

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IN – VITRO METHODS
• 1.primary hepatocyte cell culture
• 2.Stellate cell culture
• 3.kupffer cell culture.
• 4.Liver cirrhosis and necrosis
• 5.Inhibition of proline hydroxylation.
SCREENING MODELS OF
HEPATOPROTECTIVE DRUGS

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1. 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 transaminases released into the medium
by the hepatocytes.
• Hepatotoxins: ccl4 , paracetamol , d- galactosamine , tert- Butyl
hydroperoxide and ethanol.

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2. 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 isolate from rat liver by collagenase / pronase
digestion
• The test drugs are incubated with the activeted stellate cell culture
by hepatotoxins
• Parameters studied: morphology of the cells, alfa- SMA expression,
cell count with thymidine incorporation and inhibition in synthesis of
collagen type I and III.

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3.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 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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4. LIVER CIRRHOSIS AND NECROSIS
• Excessive formation of connective tissue with collagen over production
reduces hepatic blood flow.
• Collagen is formed as a response to chronic injury.The collagenous fibers
consist of triple helical molecules.
• Their formation depends on the presence of hydrogen bonds.
• If the number of hydrogen bonds is reduced,the resulting collagen can not
form the triple helix and is degraded instead of being deposited in the
extracellular matrix.
• The aim of fibrosuppressive compounds is to reduce only the excessive
formation of insoluble collagen.
• Fibrosuppressive effects by inhibition of proline hydroxylation can be
screened with in vitro methods, however, the desired organ specificity has
to be tested in models of liver cirrhosis and fibrosis in vivo.

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5. INHIBITION OF PROLINE HYDROXYLATION
• The thermal stability of the triple helix of collagen is depend on
intramolecular hydrogen bond synthesized by the enzyme prolyl 4-
hydroxylase.
PROCEDURE
• Reaction volume of 1 ml contains Tris buffer pH 7.5, glutarate,
FeSO4,ascorbate,catalase,bovine serum albumin,dithiotreitol,
inhibitors.
• After incubation at 37 °C for 30 min, the generated CO2 is trapped
and determined.

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1. Chemically induced hepatoxicity
2. Galactosamine induced liver necrosis
3. Allyl alcohol induced liver necrosis in rats
4. Bile duct ligation induced liver fibrosis in rats
5. Thiocetamide induced necrosis of liver.
6. Rifampicin+Isoniazid induced hepatotoxicity in rats.
IN – VIVO METHODS

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• Commonly used hepatotoxins:
• Carbon tetrachloride (CCL4)
• Thioacetamide
• Dimethyl or diethyl nitrosamine
• Alfatoxin B1
1. CHEMICALLY INDUCED HEPATOXICITY

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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.

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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

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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.

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• Single dose or a few repeated doses of D-galactosamine cause acute hepatic
necrosis in rats .
• Prolonged administration leads to cirrhosis.
PROCEDURE:
• Divided doses of 100 to 400 mg/kg D-galactosamine are injected to rats i.p. or i.v.
during one day.
• For induction of liver cirrhosis, male Wistar rats weighing 110–180 g are injected
intraperitoneally three times weekly with 500 mg/kg D- galactosamine over a
period of one to 3 month.
• Potential protective substances are administered orally with the food or by
gavages every day.
• The rats are sacrificed at various time intervals and the livers obtained by autopsy.
2. GALACTOSAMINE INDUCED LIVER
NECROSIS

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• EVALUATION
• The livers are evaluated by light microscopy and immuno- histology
using antibodies against macrophages, lymphocytes and the
extracellular matrix components.
• e.g., laminin, fibronectin, desmin, collagen type I, III, and IV.
• The extent of liver cell necrosis and immuno reactivity for
macrophages, lymphocytes and the extracellular matrix components
is graded semiquantitatively on a 0 to 4 scale (0 = absent, 1+ = trace,
2+ = weak,3+ = moderate, and 4+ = strong).
• Furthermore, serum enzyme activities, such as GOT and GPT are
determined.

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3. ALLYL ALCOHOL INDUCED LIVER NECROSIS IN RATS
PURPOSE AND RATIONALE
• Administration of allyl alcohol induces liver necrosis in rats which can
be partially prevented by treatment with several drugs such as
antibiotics.
Procedure:
• Female Wistar rats weighing 120–150 g are fasted overnight with
water and libitum.
• On following morning test drug is given orally or i.p to 10 rats.
• After 1hour 0.4ml/kg of 1.25% allyl alcohol solution is given orally.
• Test drug given again on 2nd day and sacrificed on 3rd day,liver is
removed.

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EVALUATION
• The parietal sides of the liver are checked using a stereomicroscope with
25 times magnification.
• Focal necrosis is observed as white-green or yellowish hemorrhagic areas
clearly separated from unaffected tissue.
• The diameter of the necrotic areas is determined using a ocular
micrometer.
• These values are added for each animal to obtain an index for necrosis.
• Mean of necrosis index is calculated and compared with Student’s t-test.
• The protective effect is expressed as percentage decrease of the necrosis
index versus controls.

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4. BILE DUCT LIGATION INDUCED LIVER FIBROSIS IN RATS
PURPOSE AND RATIONALE
• Bile duct ligation in rats induces liver fibrosis which can be evaluated by
histological means and by determination of serum collagen parameters.
Procedure:
• Male Sprague Dawley rats weighing approximately 250 g are anesthetized
with ketamine HCL.
• Expose the common bile duct, which pursues an almost straight course of
about 3 cm from the hilum of the liver to its opening into the duodenum.
• There is no gallbladder, and the duct is embedded for the greater part of its
length in the pancreas, which opens into it by numerous small ducts.

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• A blunt aneurysm needle is passed under the part of the duct selected,
stripping the pancreas away with care, and the duct is divided between
double ligatures of cotton thread.
• The peritoneum and the muscle layers as well as the skin wound are closed
with cotton stitches.
• The animals receive normal diet and water ad libitum throughout the
experiment.
• Groups of 5–10 animals receive the test compound in various doses or
vehicle twice daily for 6 weeks.
• Then, they are sacrificed and blood is harvested for determination of bile
acids, 7S fragment of type IV collagen, and precollegen III N- peptide.
• The liver is used for histological studies and for hydroxyproline
determinations.
• Control animals show excessive bile duct proliferation as well as formation
of fibrous septa.

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Evaluation:
• Blood – Biochemical analysis
• Liver - HPE

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5.THIOCETAMIDE INDUCED HEPATOTOXICITY IN
RATS:
• Thiocetamide actcs as a hepatocarcinogen and hepatotoxicant
• This action is mediated by formation of S- oxide, which covalently
binds with liver cell macromolecules like protein, nucleic acid and
lipids.
Procedure:
• Wistar/Sprague dowel rats of either sex weighing between 180-250 g
are used.
• They are maintained on a standard chow diet with water and libitum
at 21±2°c room temprature and under 12h dark/12h light cycle .
• The food is withdrawn 18h before the experiment but water is
allowed ad libitum.

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• Hepatotoxicity is induced by 200mg/kg body weight of dissolved in
saline and administered orally or 100mg/kg given S.C
• The serum is used for determination of aminotransferases and the
liver is used for histopathological studies.
Evaluation:
• Liver - HPE

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6. RIFAMPICIN+ISONIAZID INDUCED HEPATOTOXICITY
IN RATS
• Rifampicin and isoniazid are used together as 1st line anti-T.B drug.
• Procedure:
• Wistar/sprague dowley rats of either sex weighing between 150-200
g are used.
• Rifampicin and INH both are given in the dose of 50mg/kg body
weight of each by i.p injection once daily for 15 days.
• The test drug were also administered along with RMP+INH
combination daily for 15 days.
• At the end of experiment the rats are killed by decapitation and used
for further biochemical analysis and histopathology of the liver.

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Evaluation:
• Liver - HPE

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• H. Gerhard Vogel Drug Discovery and Evaluation Pharmacological
Assays ,Second Edition .Page no: 936-944
• N.S Parmar, Shiv PrakashʻʻScreening methods in pharmacology.”Page
No:281-286 30
References

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SCREENING MODELS OF HEPATOPROTECTIVE DRUGS

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