This document summarizes several models for screening hepatoprotective drugs. It describes in vitro models including hepatic stellate cell culture, liver slice systems, and assays measuring proline hydroxylation and collagen synthesis. It also outlines several in vivo rat models, including those using CCl4, allyl alcohol, bile duct ligation, and galactosamine to induce liver fibrosis, necrosis, or damage and evaluate potential hepatoprotective effects of test compounds. Parameters measured include liver enzymes, hydroxyproline content, histology, and serum markers of collagen synthesis. These preclinical models allow evaluation of drug candidates for protection against liver injury.

1. S C R E E N I N G M O D E L S O F
H E P A T O P R O T E C T I V E D R U G S
Presented by Under the Guidance of
P N.Savitha Mrs.V.Rajani M.Pharm(Ph.D)
M.Pharmacy 1yr1 st semester Associate Professor
Dept of Pharmacology Dept.of Pharmacology
SKU COLLEGE OF PHARMACEUTICAL SCIENCES, S.K.UNIVERSITY,
ANANTAPURAMU

2. INTRODUCTION
 Liver is large organ that found in vertebrates which
detoxifies various matabolites ,synthesizes
proteins,produces biochemicals necessary for digestion.
 It has main role in maintenance,performance and
regulating homeostasis of body.
 Liver produces bile,a compound needed to digest fat
and to absorb vit A,D,E,K.
 Liver involved with biochemical pathways to
growth,fight against disease,nutrient supply,energy
provision& reproduction.

3. HEPATO TOXICITY
 Hepatotoxicity is the injury or liver damage
caused by exposure to drugs.
 The hepatic injury can be classified into:
Hepatocellular
Cholestatic
Mixed
 It is caused by increase in alanine,
aminotransferases, alkaline phosphates
than upper limit of normal.
 Hepatotoxicity caused majorly by drugs,
autoimmune disorders, and infection like viral
hepatitis.

4. MARKERS OF HEPATOTOXICITY
1)Aspartate serum transferase(AST)
2)Alanine amino transferase(ALT)
3)Alkaline phosphatase(APP)
4)Lactate dehydrogenase(LDH)
5)Total bilirubin(TB)
6)Total protein(TP)
7)Total triglycerides(TG)
8) γ -Glutamyl transferase(GGT)
M EC H A NI S M O F ACTI ON
 Oxidation in liver produce reactive free oxygen
radicles.These cause hepatotoxicity because of
release of free radicles cause apoptosis in liver
reduces Glutothiolevels.

5. LIST O F HEPATOPROTECTIVE
AGENTS
 N-Acetylcysteine
 Pencillamine
 Cardiotropin 1
 S-Adenosyl Methionine
 Herbal medications eg:Silymarine
 Antioxidants
eg: Vitamins,
Glutathione,
Melatonin,
β Carotene.

6. Anti tubercular
drugs
• Rifampacin
• Isoniazid
• Pyrazinamide
• Ethionamide
• Levofloxacin
• Thioacetazone
• Cycloserine
NSAIDS
• Paracetamol
• Diclofenac
• Ibuprofen
• Indomethacin
• Oxicam groups
Antimicrobials
• Dapsone
• Ketoconazole
• Sulphonamides
• Erythromycin
• Azithromycin
• Streptomycin
• Anti retrovirals
D R U G S INDUCED LIV ER INJUR Y

7. Anticonvulsants
• Carbamazepine
• Phenobarbitol
• Valproic acid
Anaesthetics
• Halothane
• Isoflurane
• Enflurane
Miscellaneous
• Nicotinic acid
• Flutamide
• Statins
• Labetlol
• Disulfuram
• Propylthiouracil

8. SCREENING METHODS
1) Inhibition of Proline hydroxylation.
2) Influence on collagen synthesis in Human skin fibroblasts.
3) Influence on collagen synthesis in Chicken Calvaria.
4) Liver Cirrhosis and Necrosis.
5) Primary hepatocyte Cell culture.
6) Hepatic stellate cell culture(HSC).
7) Kuffer Cell Culture.
8) Liver Slice System.

9. 1. Bile duct ligation induced Liver fibrosis in Rats.
2. CCL4 Induced Liver fibrosis in Rats.
3. Allyl alcohol induced Liver necrosis in rats.
4. Galactosamine induced Liver necrosis.
5. Temporary Hepatic ischemia.
6. Hepatitis in Long-Evans Cinnamon Rats.
7. Model for direct Transhepatic studies in Dogs.
8. Thioacetamide induced Liver necrosis.
9. Paracetomol induced liver necrosis.
10. Rifampacin+Isoniazid induced Hepatotoxicity in rats.
11. Dimethylnitrosamine induced fibrosis & necrosis.

10. INVITRO METHOD
1) Hepatic stellate cell culture(HSC)
Purpose:To asess the antifibrotic efficacy of exp drugs,these primarly cultured
cells are useful in assesing specific effects on HSC activities.
Procedure:HSC are isolated from the livers of normal rats weighing at least
500g in order to achieve good separation from the other hepatic cells.The liver
is digested with pronase,cillagenase,Dnase by insitu perfusion
After several centrifugation steps,cell suspension is subjected to a Nycodenz
gradient to collect the HSC on top of the Nycodenz layer.
The separation is based on low density of HSC as compared to other liver cells,
as a consequence of their high cellelar lipid content.
About 5 mice have to be used at a same time to yield more amount of HSC from
liver.
Isolated cells are cultured in DMEM containing 10% FCS,100U/ml
pencillin,100mg/ml streptomycin.
Evaluation:After 10-14 days in culture,the cells display an activated
phenotype as assessed by light microscopy & acquire the presence of alpha
smooth muscle actin.

11. 2) Liver slice system
Purpose:It developed to assess effects of antifibrotic drugs in liver slice
preaparation.
Procedure:Liver slices are prepared as follows:
Livers are excised from rats & stored.Slices were prepared in Krebs-
Henseleit buffer saturated with95% O2, 5%CO2 & containing 25mM
glucose,25mM NaHCO3,10mM hepes using Krumdieck tissue slicer.
To equillibrate the tissue,slices are preincubated for 1h in Williams medium
E-Glutamax with 25mM glucose,50mg/ml gentamicin under carbogen
atmosphere at 37ºC in 6 well culture plates while gently shaken.
Again slices transferred to fresh medium & incubated.
Evaluation:.HSC & extracellular matrix components & resident hepatic cells
which allow testing of Multicellular interactions.
Advantages:
1. Easily prepared from normal, fibrotic,cirrhotic tissue.
2. This system easily allows drug testing in human liver material.
Disadvantages:
1. This method is limited time period of study.
2. Absence of blood flow through the slice.

12. 3) Inhibition of proline hydroxylation
Purpose:The thermal stability of triple helix of collagenase is depend on
intramolecular H bond synthesized by the enzyme prolyl 4 hydroxylase.
Procedure:
Reaction volume of 1ml contains
50mM Tris buffer(ph-7.5),10-100mM 2oxo glutarate, 1-50mMFeso4,
1mMAscorbate, 10-100mg(ProPro-Gly)10, 0.1mg catalase, 2mg bovine serum
albumin, 100mmdithiotretiol, 0.2mgenzyme & inhibitors.
After incubation at37ºC for 30 min, the generated C02 is trapped and
determined.
Evaluation:
Inhibition modes are determined by plotting 1/v versus 1/c of variable
substrate(Lineweaver burk plot).
The Ki values are derived from secondary transformation(slopes or
intercepts).
The lines of best fit for primary & secondary transformations are calculated
using the method of ‘least square’.

13. 4) Influence on collagen synthesis in human skin fibroblasts
Purpose: Secretion of collagen by fibroblasts & other cells capable of
synthesizing extracellular matrix is dependent on the hydroxylation of proline
residues by propyl 4-hydroxylase.
Procedure: Culture of human skin fibroblasts are preincubated for 24hr at37ºC
in Dulbeccos medium supplemented with 50mg/ml sodium ascorbate,60mg/ml
3-aminopropionitrile,10U/ml pencillin G.
The cells are then exposed to potential inhibitor at various concentration for 20
min,followed by addition of radioactive(C14) 2m proline/ml.
The incubation is continued for 5h at 37ºC. Then the cells are separated from
the medium.
After removal of proline,the proteins from medium, the cells are hydrolysed &
the hydroxyproline content is determined by aminoacids analysis.
The total incorporation of radioactivity serves as marker for protein synthesis.
Evaluation: Proline incorporation is expressed as % of control Radioactivity.
Formula: Hyp/Pro Sample
= *100
Hyp/Pro Control

14. 5) Influence on collagen synthesis in chicken calvaria
Purpose:To find fibrosuppresive agents for therapeutic use,drugs which
converted intracellularly to the active agent.Proinhibitors which are cleaved
hydrolytically can suppress collagen synthesis in chicken calvaria.
Procedure:
Calvaria are removed from chicken embroys,age 15 days&washed.They are
tranferred to pyrex tubes containing medium supplemented with 2mM
glutamine,6mCi radioactive proline&various conc of inhibitors.
The samples are incubated.Calvaria removed,washed,pooled with incubation
medium.BSA& phenyl methyl sulphonyl flouride are added.
The calvaria are extracted for 16h with 25ml of acetic acid.Aliquots are
withdrawn for SDS-PAGE & the triple helix stability assay performed.
By using remaining material, hydroxyproline content is determined.
To study the degree of collagen hydroxylation&proportion of collagen
biosynthesis,calvaria are incubated in presence of 10mCi and 2mCi
radioactive proline/ml for 3h.
After lyophilization,aliqouts of media&calvaria samples are digested with
collagenase.

15. The degree of hydroxylation is calculated from the 3H/C₁₄ ratio in digested
material.
The amount of collagen as a proportion of total protein synthesis is
determined by :
The relation of collagenase degradable v/s collagenase resistant activity.
Evaluation:
IC50 values of hydroxyproline synthesis are read graphically from
concentration response curves.
Total protein synthesis is estimated as the incorporation of proline;the
mean±standard deviation is calculated from 4 samples.

16. 6) Liver Cirrhosis & Necrosis
Purpose & Rationale:
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 triple helix & 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.
Evaluation:
Fibrosuppressive effects by inhibition of proline hydroxylation can be
screened invitro method.
But the desired organ specificity has to be tested in invivo models.

17. 7) Kuffer cell culture
Isolation:
Kuffer cells are isolated from the liver by perfusion of liver with pronase
followed by differential centrifugation.
The isolated cells are isolated are maintained in culture where their
phagocytic properties are retained.
On exposure of cells phagocytic stimuli like zymosan particles, ther is
increased O2 consumption & superoxide production.
The inhibitory effects of various drugs on kuffer cells are tested in the
invitro cultures.

18. 8) Primary Hepatocyte cell culture
Fresh hepatocyte preparations & primary cultured hepatocytes are used.
Common method:
Isolation of hepatocytes by perfusion of liver with collagenase or utilization
of primary cultured hepatocytes.
Determination of viability of the hepatocytes.
Incubation of cell culture with hepatotoxin with or without drug test drug.
Determination of activity of transaminase released into medium by the
hepatocytes.
Hepatotoxins include:CCL4
D-galactosamine
Tert butyl hydroperoxide
Ethanol.

19. INVIVO METHOD
1) CCl4 induced liver fibrosis in rats
Purpose:Chronic administration of CCl4 to rats liver fibrosis with severe
disturbances of hepatic function.
Procedure: Groups of 20 wistar rats weighing 100-150g.
CCL4 Dissolved in olive oil (1:1) ,given orally twice a week of 1mg/kg-over a
period of 8 weeks.
Animals are kept in standard conditions:22ºC temp,std diet,water ad libitum
20 rats Contol-olive oil,
20 rats Carbon tetrachloride only
20 rats ccl4 along with various doses of test drug twice a
week ,at weekend ingle dose given.
The animals are weighed every week,after 8 weeks animals are sacrified
using anaesthetic ether.
In serum,parameters determined include:
Total bilirubin
Total bile acids
7S fragment of typeⅣcollagen
Procollagen Ⅲ N peptide.

20. Organs for determination of hydroxyproline are:
Liver
Kidney
Aortic wall
Tendons
specimens of organs are weighed & completely hydrolysed in 6N
HCL.Hydroxyproline is measured by HPLC& expressed in mg/mg wet weight
of organs.
Histology:3 to 5 pieces of 1g liver fixed in formalin& carnoy solution
3to5 sections of each liver are embedded,ct,stained with Azocarmine aniline
blue & evaluted for development of fibrosis using scores:
0-Normal liver history
Ⅰ-Tiny & short septa of connective tissue
ⅠⅠ-Large septa of connective tissue
Ⅲ-Nodular transformation of liver
Ⅳ-Excessive formation &deposition of connective tissue
Evaluation:
For detection of significant differences,the unpaired t-test used.
For comparison of scores in histological evalution ,chi-square test used.

22. 2) Allyl alcohol induced liver necrosis in rats
Purpose:Allyl alcohol induces focal liver necrosis in rats, which can be
prevented by treatment with antibiotics.
Procedure:Female wistar rats(150-200g)are fasted overnight
1 day-morning test drug given orally/Ip,
after 1h 0.4ml/kg of 1% Allyl alcohol soln orally.
2 day-again test drug given.
3 day-animal sacrified,liver is removed.
Evaluation:
The parietal sides of liver are checked in stereomicroscope in
25Xmagnification.
Focal necrosis is observed as white green/yellowish haemorrhagic area.
 Using contol & treatment group,mean of Necrosis index is calculated &
compared with students t-test.
The protective effect is expressed as% decrease of Necrosis index v/s controls.

23. 3) Bile duct ligation induced liver fibrosis in rats
Purpose:Bile duct ligation in rats induces Liver fibrosis which can be
evaluted by histological means & determination of serum collagen
parameters.
Procedure:
Male sprague dawley rats(250g) anesthetized with ketamine ,xylazine
Laparactomy is performed under aseptic condition.
Expose the common bile duct,from hilum of liver to opening into
duodenum&duct is embedded for greater part of its length in
pancreas,which opens into it by numerous small duct.
A blunt needle is passed under duct,stripping the panceas away& duct is
divided b/w double ligatures of thread.peritonium,muscle layers,skin
wounds are closed by stiching.
Groups of 5-10 animals receive test compound in various doses twice a day
over 6 weeks & sacrified.

24. Evaluation:
In serum,parameters to be determined are:
Total bilirubin,
Total bile acid,
7S fragment of typeⅣcollagen,
ProcollagenⅢ N peptide.
 The liver is used for Histological studies & Hydroxyproline determinations.
 Contol animals show excessive bile duct proliferation&formation of fibrous
septa.

25. 4) Galactosamine induced liver Necrosis
Purpose:Single dose or repeated dose of D-galactosamine cause acute hepatic
necrosis. Prolonged administration leads to cirrhosis.
Procedure:
Male wistar rats weighing 150-200g are used.
For induction of Acute hepatotoxicity:Divided doses of 100-400mg/kg GS is
given in Ip route for 1day.
For induction of liver cirrhosis:500mg/kg Galactosamine is given ip
route,thrice a week over 1-3 months.
Potential protective drugs are given oral route with food ,everyday.
Rats are sacrified and the livers obtained by autopsy.
Evaluation:
 The livers are evaluted by light microscopy&immunohistology using
antibodies against macrophages,lymphocytes, extracellular matrix
components:e.g:laminin, fabronectin, desmin&collagen typesⅠ,Ⅲ&Ⅳ.
 Serum enzyme activities,such as GOT & GPT determined.
The extent of liver necrosis&immunoreactivity is graded on scale as:
0-Absent
1-Trace
2-Weak
3-Moderate
4-Strong

26. 5) Thioacetamide induced hepatotoxicity in rats
Purpose:Thioacetamide acts as hepatocarcinogen & hepatotoxicant.This
action is ,mediated by formation of S-oxide,which covalently binds with liver
cell macromolecules like protein,nucleic acid,lipids which increases
intracellular Ca+ concentration & causes necrosis.
Procedure:
Wistar/sprague dawley rats weighing 200-250g are used.
 They are maintained on a standard chow diet ,water &libitum at 23ºC
under 12h dark/12h light.
Animals are starved for 18h,before experiment starts.
 Hepatotoxicity is induced by thioacetamide 200mg/kg oral or 100mg/kg S.C
route thrice weekly over 8 weeks.
Animal is sacrified,liver is separated&serum collection.
Evaluation:
Serum is used for determination of Aminotransferase marker.
Liver is used for Histopathological studies.

27. 6) Paracetamol induced liver damage in rats
Purpose:
Paracetamol in higher dose induce Hepatic damage.
It gets metabolized to N-acetyl p benzoquinone imine by cytp450 enzyme
which results in oxidative stress causing liver glutathione & glycogen
depletion.
Procedure:
Wistar rats of either sex weighing 150-200 g are used.
Paracetamol 2g/kg body wt is administered orally as single dose.
Test drug- given for 6days in (oral).
Inducing agent-Paracetamol given on 7th day.
Animals are sacrified after 24hr.Blood is collected.Liver is separated.
Evaluation:
Blood/serum is used for biochemical analysis.
Liver is used for Histopathological stidies.

28. 7) Rifampicin+Isoniazid induced Hepatotoxicity in rats
Purpose:
Rifampicin&Isoniazid are used together as 1st line antitubercular drugs.
In higher doses,they show toxic effects to liver of rats.
Procedure:
Wistar/sprague dawley rats of either sex weighing 150-200g are used.
Rifampicin&Isoniazid are given in dose of 50mg/kg body wt each by Ip inj
once daily for 15 days along with test drug.
At the end of experiment rats are sacrified by decapitation.
Evaluation:
Blood/serum is used for biochemical analysis.
Liver is used for Histopathological studies.

29. 8) Dimethylnitrosamine induced liver Fibrosis(DMN)
Purpose:DMN induces liver injury by intiating damage to the hepatocyte.
It is metabolised in hepatocytes by cytP450 to more toxic compounds with
formation of reactive O2 species which leads to lipid peroxidation.
Procedure:
Albino wistar rats of either sex weighing 200-250g are use.
Animlas are maintained in standard Chow diet,water ad libitum at 23ºC.
 To induce fibrosis,DMN given in Ip route,dose of 10μl body wt ,thrice a week
for 3weeks.
At end of experiment animals are sacrified.Liver is used for Histopathological studies.
Evaluation:
After giving DMN, haemorrhagic necrosis is evident in centrolobular part (zoneⅢ)of
liver.
 Incomplete septa appear after 7 days.Micronodular cirrhosis is developed after 3
weeks treatment with DMN.
Increased no.of HSC & myofibroblasts are found in formed septa.Influx of
inflammatory cells mainly lymphocytes,is noted early.
Advantages:
1. Disease introduction is quite reproducible in the animals.
2. This model is associated with prominent inflammatory reaction.
3. Used to study the transition from cirrhosis to hepatocellular carcinoma&influence of
drugs on this process.

30. 9) Hepatitis in Long-Evans cinnamon rats
Purpose:
To study genetically transmitted fulminat heapatitis&chronic liver
disease.Excessive copper accumulation in the rats liver, making model best
for Wilsons diseasein humans.Chelation therapy/feeding copper deficient diet
can ameliorate symptoms in rats & wilsons disease.
Procedure:
Male long cinnamon rats obtained from commercial breeder.
Groups of 6-10 rats are given different diets based on a 15%purified egg
protein,vitaminds or drugs.
Drugs are applied via minipumps Ip implanted under ether anaesthesia.
 The occurrence of jaundice-ears & tail turn yellow,urine becomes bright
orange,staining the fur in lower abdominal region.
Usually jaundice progressively worsens,ending in death of animal within
about week.Incidence of jaundice&mortality v/s time used as parameters.
Evaluation:
Statistics are performed for students T-test,ANOVA,Scheffe F-test for
comparison b/w means.
P-value<0.05 is used as the thresold of significance.

31. 10) Models for Direct Transhepatic studies in Dogs
Purpose:
To study hepatic effects of pancreatic hormone secretion&glucose metabolism.
To study hepatic mechanisms associated with high first pass metabolism&
food interaction of drugs.
To study the insulin balance in dogs that have undergone previous
pancreactomy&islet cell autotransplantation.
Procedure:
Male dogs weighing 20-25kg are anaesthetised by isoflourane inhalation.
 Skin interface sites & subcutaneous pockets for placement of catheters are
prepared.
After skin closure the external ends of catheters are sealed.
 Then the catheters & flow probes are placed into abdomen retriving them
from subcutaneous pockets.
a.Hepatic artery,Portal venous flow probes inserted.
b.The portal vein&hepatic vein catheters are placed & abdomen is closed.
c. The carotid artery & jugular vein catheters are placed.
Evaluation:
Blood samples are withdrawn from catheters.
Blood flow & Plasma flow is measured by flow probes in hepatic artery &
hepatic portal vein.
Drug conc measured in portal vein,hepatic artery,hepatic vein,right
external jugular vein.

32. 11)Temporary hepatic ischemia
Purpose:
Total hepatic ischemia in rats is produced by placing a ligature around the
hepatic artery,portal vein,bile duct.
Procedure:
Albino rats300-350g are fasted 16h prior to experiment allowed water libitum.
Rats anesthetized with ether,abdominal cavity is opened.
Hepatic artery,portal vein,bile duct are occluded by by placing torniquet
around vessels.BP is measured in femoral artery via catheter.
During ischemic period 0.7ml saline given at 20min intervals for volume
replacement.
At the end of 60min ischemic period,tourniquet is removed to reestablish blood
flow to liver.Abdominal incision closed&Animals receive either saline or drug.
Measurement of Indocyanine green:
In exp animals,femoral artery &vein of each aniamal cannulated.Sodium
heparin(400units) is given in Ip route,animals allowed to awake.

33. Indocyanine geen is given Iv low 5mg/kg & high doses 25mg/kg.Arterial blood
samples are taken at 5,6,8,10,12,15,18 & 20 min later.
Blood samples are mixed with 0.8ml of 1%BSA in saline & centrifuged at 6000
rpm for 20min at 4ºC.
The spectrophotometric absorbance of supernatant is read at 800nm &
finally indocyanin green conc determined from standard curve.
Evaluation:
 The t½ of indocyanin green clearance is computed for each animal using
computarised program,which calculates ‘Least Square Line’ of log
indocyanin green v/s time.
Mean Standard errors for each group are compared using Students t-Test.

34. RE F E RE N C E S :
H Gerhard Vogel, Drug discovery & Evalution pharmacological assays,
2nd edition.page no:936-944.
SK Gupta,Drug screening methods preclinical evalution of new drugs,2nd
edition,page no:193-200.
N S Parmar,Shiv prakash ,Screening methods in Pharmacology,page
no:281-286.