Biological Standardization and Screening of Drug

Activities (PCL-533)
Presented by
Asmaa Ahmed
Lecturer of pharmacology
Fall 2021
LECTURE 10
 OBJECTIVES OF LECTURE

 By the end of this lecture, the student should be able to:

Screening of drugs that have a beneficial role in liver disease via several models as:

I. Allyl Alcohol Induced Liver Necrosis in Rats

II. Carbon tetrachloride Induced Liver Fibrosis in Rats .
III. Bile Duct Ligation Induced Liver Fibrosis in Rats .
IV. Galactosamine Induced Liver Necrosis.
LIVER FUNCTIONS
 ANIMAL MODELS FOR SCREENING OF
HEPATOPROTECTIVE DRUGS

I. Allyl Alcohol Induced Liver Necrosis in Rats

II. Carbon tetrachloride Induced Liver Fibrosis in Rats .

III. Bile Duct Ligation Induced Liver Fibrosis in Rats .

IV. Galactosamine Induced Liver Necrosis

 I. Allyl Alcohol Induced Liver Necrosis in Rats

Administration of allyl alcohol induces focal liver necrosis in rats which can be partially
prevented by treatment with several drugs such as antibiotics.

Allyl alcohol
Alcoholic
Acrolein (Highly reactive compound)
???
dehydrog
enase GSH
enzyme

Execration
Female Wistar rats weighing 120–150 g are withdrawn from food with access to water

Then the compounds to be tested for protective activity are administered i.p. or orally.

One hour later, the animals are dosed orally with 0.4 ml/kg of a 1.25% solution of allyl alcohol in water.

On the second day, the treatment with the potentially protective drugs is repeated.

on the third day, the animals are sacrificed and the liver removed.

The livers 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.
✓ Using animals for controls and for treatment group, the mean of necrosis index is
calculated and compared.

✓ The protective effect is expressed as percentage decrease of the necrosis index versus
controls.

The diameter of the

necrotic areas is
determined and added for
each animal

Drugs have Necrotic index

hepatoprotective effect
% inhibition of necrosis
 II. CARBON TETRACHLORIDE INDUCED LIVER
FIBROSIS IN RATS

Chronic administration of tetrachloride to rats induces severe disturbances of hepatic function

together with histologically observable liver fibrosis.
??? Serum samples was
collected for liver
function tests

Group1 Rat weight of 100-150 g
Group 2
Olive oil
Treated orally twice a
week with 1mg/kg CCL4,
dissolved in olive oil 1:1,
over a period of 8 weeks
Animals were
euthanized and liver
fixed in formalin and
Group 3
stained with
Masson's trichrome
Administered the test stain to EVALUATE
drug + CCL4 for period the degree of liver
of 8 weeks fibrosis
 III. BILE DUCT LIGATION INDUCED LIVER
FIBROSIS IN RATS

• Bile duct ligation (BDL) in mice and rats is one of the most widespread
experimental models to investigate the intervening therapies in cholestatic
liver disease.

• BDL results in the gradual development of different stages of cholestatic-

induced liver disease, as the induction of cholestasis, subsequently
accompanied by liver inflammation and finally liver fibrosis
Male rats weighing approximately 250 g are anesthetized.

Mid-line incision in the abdomen is made.

Push the intestines downwards and the liver upwards to get a clear
vision to expose the bile duct.

Bile duct is ligated obstructed using a surgical knots

Stitch the peritoneum and abdominal skin separately with sutures

Groups of animals receive the test compound twice daily for 6 weeks.

Then, they are sacrificed and the liver is used for histological studies
EVALUATION Histological structure
 GALACTOSAMINE INDUCED LIVER
NECROSIS

▪ Galactosamine has great liver specificity because hepatocytes have high

levels of galactokinase and galactose-1-uridyltransferase.

▪ Single dose or a few repeated doses of D-galactosamine causes acute

hepatic necrosis in rats while prolonged administration leads to
cirrhosis.
▪ This model of liver damage most closely resembles the changes
observed during human viral hepatitis.

▪ Its toxicity mainly related to depletion of uridine triphosphate in

liver resulting in suppression of protein synthesis with
deterioration of cell membrane.
 Acute Chronic
Hepatotoxicity Hepatotoxicity

Rats injected i.p.

Divided doses of 400 mg/kg Three times weekly with

D-galactosamine during one 500mg/kg D-galactosamine over
day. a period of 3 months.
Tested substances are administered orally with
the food or by gavage every day.

The rats are sacrificed and the livers obtained for histological studies
 GENERAL ASPECTS FOR EVALUATION OF
HEPATOPROTECTIVE DRUGS

The magnitude of the protective effect can be measured by :

1 Liver Enzymes 2 Histologically

GGT
Bilirubin ALT AST

3 Survival Rate
 REFERENCES

 Drug Discovery and Evaluation: Pharmacological Assays. Hock, Franz J.,

4th Edition, 2016.

 Najmi, A. K., Pillai, K. K., Pal, S. N., & Aqil, M. (2005). Free radical
scavenging and hepatoprotective activity of jigrine against galactosamine
induced hepatopathy in rats. Journal of ethnopharmacology, 97(3), 521-525.

 Van Campenhout, S., Van Vlierberghe, H., & Devisscher, L. (2019).

Common bile duct ligation as model for secondary biliary cirrhosis.
In Methods in Molecular Biology.