SCREENING MODELS FOR HEPATOPROTECTIVE DRUGS.pptx

The liver is the largest internal organ and plays a vital role in metabolism, detoxification, protein synthesis, and bile production.
Hepatotoxicity refers to liver damage caused by chemicals, drugs, or toxins. Types of Hepatotoxicity are hepatocellular injury, cholestatic injury and mixed injury.
In Vivo Screening (Animal Models) are :
CCl₄-induced Hepatotoxicity
Paracetamol-induced Hepatotoxicity
Alcohol-induced Injury
Thioacetamide-induced Fibrosis
D-galactosamine Model

1. SCREENING MODELS FOR HEPATOPROTECTIVE DRUGS Prepared BY: VAISHNAVI J. AWARE m. Pharm (pharmacology) GUIDED BY: DR. A. V. KULKARNI Assignment seminar for 50 marks DR. D. Y. PATIL COLLEGE OF PHARMACY, AKURDI, PUNE 411044 2024-25

2. Content Liver anatomy Hepatotoxicity Classification of hepatotoxicity Screening models of hepatotoxicity 2

3. LIVER ANATOMY The liver is the largest internal organ and plays a vital role in metabolism, detoxification, protein synthesis, and bile production. Structure of the Liver Lobes: Right and left lobes (larger) with caudate and quadrate lobes (smaller). Hepatic lobule: Functional unit of the liver, consisting of hepatocytes arranged in plates around a central vein. Cells of the liver: Hepatocytes – Main functional cells, responsible for metabolism and detoxification Kupffer cells – Macrophages involved in immune defense Stellate cells – Store vit. A and contribute to fibrosis Endothelial cells – Lining cells that regulate exchange between blood and hepatocytes 3

4. Hepatotoxicity Hepatotoxicity refers to liver damage caused by chemicals, drugs, or toxins. Causes Of Hepatotoxicity Drugs: Acetaminophen, NSAIDs, Antitubercular Drugs (Isoniazid, Rifampicin). Alcohol: Chronic Alcohol Consumption Leads To Cirrhosis. Viruses: Hepatitis A, B, C, And E. Metabolic Disorders: Wilson’s Disease, Hemochromatosis. Herbal & Environmental Toxins: Aflatoxins, Pyrrolizidine Alkaloids. 4

5. Type Features Hepatocellular Injury Damage to hepatocytes, elevated ALT & AST (e.g., viral hepatitis, drug-induced). Cholestatic Injury Bile flow obstruction, elevated ALP (e.g., gallstones, primary biliary cholangitis). Mixed Injury Features of both hepatocellular and cholestatic damage (e.g., drug-induced liver injury). 5 Types of Hepatotoxicity

6. Classification of Hepatoprotective Drugs Natural Hepatoprotective Agents Flavonoids : Silymarin (Milk Thistle), Quercetin Terpenoids : Andrographolide (Andrographis Paniculata) Polyphenols : Curcumin (Turmeric), Resveratrol Alkaloids : Berberine (Berberis Species) Glycosides : Glycyrrhizin ( Licorice ) Synthetic Hepatoprotective Agents Antioxidants : N-acetylcysteine (NAC) For Acetaminophen Toxicity Cytoprotective Agents : Ursodeoxycholic Acid (UDCA) Enzyme Inducers : Phenobarbital For Improving Liver Detoxification 6

7. Screening Methods for Hepatoprotective Drugs 7

8. In Vivo Screening (Animal Models) CCl ₄-induced Hepatotoxicity Paracetamol-induced Hepatotoxicity Alcohol-induced Injury Thioacetamide-induced Fibrosis D-galactosamine Model 8

9. CCl ₄-induced Hepatotoxicity Carbon tetrachloride ( CCl ₄) is one of the most commonly used hepatotoxins to induce liver damage in experimental studies. It mimics oxidative stress-mediated liver injury , making it ideal for evaluating hepatoprotective drugs. Equipments : Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser . Drugs: Liquid paraffin, Test drug, Carbon tetrachloride, 10% formalin, Silymarin. Animals: Wistar Albino rats 9

10. Principle of CCl ₄- InduceD Hepatotoxicity Metabolism of CCl ₄ in the Liver: CCl ₄ is metabolized in hepatocytes by cytochrome P450 (CYP2E1) to generate trichloromethyl radical ( CCl ₃•). This free radical interacts with lipids in hepatocyte membranes, leading to lipid peroxidation and oxidative stress. Results in cell membrane damage, inflammatory responses, and hepatic necrosis. 10

11. Group Treatment Normal control Liquid paraffin (3ml/kg) Inducer control Liquid paraffin (3ml/kg, s.c. ) + CCl4 (1ml/kg, s.c. ) Test drug Test drug + CCl4 (1ml/kg, s.c. ) Reference standard Silymarin (100 mg/kg, p.o. ) daily + CCl4 (1ml/kg, s.c. ) 11

12. Procedure Weigh the animal and into groups and label them. Administer vehicle to normal control and inducer group, test group with test drug and silymarin to the reference standard group for 1week. 60 minutes after the treatment with drugs administer CCl4 (1ml/kg, s.c. ) In liquid paraffin. On 8 th day withdraw blood from retroorbital plexus and collect it in sterilized centrifuge tubes. Allow the blood to coagulate for 30 mins at room temperature and then separate the clear serum by centrifugation at 2500 rpm for 10mins. Estimate the levels of markers in serum AST, ALT, SGPT, SGOT, ALP, TBL and CHL using serum diagnostic and an autoanalyzer. 12

13. Thereafter, sacrifice the animals under anaesthesia then dissect them and take out liver, wash with water, dry with filter paper and preserve in 10% formalin solution for histopathological studies including cell necrosis, fatty change, hyaline degeneration, ballooning degeneration and infiltration of Kupffer cells and lymphocytes. Compare the results of test drug and reference standard group against the results of inducer control group using suitable statistical analysis method. Reduction in levels of various enzymes, as a marker of liver damage in rats of test drug as compared to induced control group supported by positive results in histopathological examination indicated hepatoprotective activity of test drug. 13

14. Paracetamol-induced Hepatotoxicity Paracetamol-induced hepatotoxicity occurs when excessive doses of paracetamol (acetaminophen) cause liver damage due to the formation of toxic metabolites. It is a major cause of drug-induced liver injury (DILI), leading to acute liver failure (ALF) in severe cases. Equipments : Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser . Drugs: Distilled water, Test drug, Paracetamol, 10% formalin, Silymarin. Animals: Wistar Albino rats 14

15. Principle of Paracetamol-induced hepatotoxicity Metabolism in the Liver Paracetamol is primarily metabolized by glucuronidation (60%) and sulfation (35%) pathways for safe elimination. A small fraction (5-10%) is converted by cytochrome P450 (CYP2E1) into N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive toxic metabolite. Role of Glutathione (GSH) Under normal doses, NAPQI is detoxified by glutathione (GSH) and excreted. In overdose, GSH stores are depleted, leading to the accumulation of NAPQI, which binds to cellular proteins and causes oxidative stress and hepatocyte necrosis. 15

16. Group Treatment Normal control Distilled water (1ml/kg, p.o. ) Inducer control Distilled water ( 1ml/kg, p.o. ) + Paraetamol (500 mg/kg, p.o. ) Test drug Test drug + Paracetamol (500 mg/kg, p.o. ) Reference standard Silymarin (100 mg/kg, p.o. ) daily + Paracetamol (500 mg/kg, p.o. ) 16

17. Procedure Weigh the animal and into groups and label them. Administer vehicle to normal control and inducer group, test group with test drug and silymarin to the reference standard group for 15 days. 60 minutes after the treatment with drugs administer paracetamol (500 mg/kg, p.o. ) In distilled water. On 16 th day withdraw blood from retroorbital plexus and collect it in sterilized centrifuge tubes. Allow the blood to coagulate for 30 mins at room temperature and then separate the clear serum by centrifugation at 2500 rpm for 10mins. Estimate the levels of markers in serum AST, ALT, SGPT, SGOT, ALP, TBL and CHL using serum diagnostic and an autoanalyzer. 17

18. Thereafter, sacrifice the animals under anaesthesia then dissect them and take out liver, wash with water, dry with filter paper and preserve in 10% formalin solution for histopathological studies. Dehydrate the liver samples fixed for 48 hours in 10% formalin by passing in different mixtures of ethyl alcohol-water. Clean the sample in xylene and embed them in paraffin. Prepare 4-5mm thick sections and then stain with hematoxylin and eosin dye for microscopic observation of cell necrosis, fatty change, hyaline degeneration, ballooning degeneration and infiltration of Kupffer cells and lymphocytes. Compare the results of test drug and reference standard group against the results of inducer control group using suitable statistical analysis method. Reduction in levels of various enzymes, as a marker of liver damage in rats of test drug as compared to induced control group supported by positive results in histopathological examination indicated hepatoprotective activity of test drug. 18

19. Alcohol-induced hepatotoxicity Alcohol-induced hepatotoxicity refers to liver damage caused by excessive alcohol consumption, primarily due to the toxic effects of ethanol and its metabolites on liver cells. The main mechanism involves oxidative stress, inflammation, and metabolic dysregulation, leading to liver injury, fibrosis, and cirrhosis. Equipment: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser . Drugs: Distilled water, Test drug, 30% Alcohol, 10% Formalin, Anaesthetic ether, Silymarin. Animals: Wistar albino rats (150-200 gm) 19

20. Principle of Alcohol-induced hepatotoxicity Alcohol-induced hepatotoxicity occurs due to the toxic effects of ethanol and its metabolites on liver cells. Ethanol is metabolized into acetaldehyde, a highly reactive compound, which, along with reactive oxygen species (ROS), leads to oxidative stress, mitochondrial damage, and inflammation. Chronic alcohol consumption disrupts lipid metabolism, causing fatty liver (steatosis) and activates hepatic stellate cells, leading to fibrosis and cirrhosis. This progressive liver damage impairs liver function and may result in liver failure. 20

21. Thioacetamide induced hepatotoxicity Thioacetamide (TAA) is a hepatotoxic compound widely used in experimental models to induce liver injury. Its toxicity is mediated through its metabolic activation in the liver, leading to reactive metabolites, oxidative stress, mitochondrial dysfunction, inflammation, and fibrosis. TAA primarily affects hepatocytes by disrupting cellular structures, causing necrosis, apoptosis, and ultimately leading to cirrhosis. Equipment: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser . Drugs: Distilled water, Test drug, Thioacetamide, 10% Formalin, Anaesthetic ether, Silymarin. Animals: Wistar albino rats (150-200 gm) 21

22. Principle of thioacetamide induced hepatotoxicity Thioacetamide (TAA) causes liver toxicity through its metabolic activation in the liver. It is converted by cytochrome P450 enzymes (CYP2E1, CYP3A4) into reactive metabolites (TASO and TASO₂), which induce oxidative stress, mitochondrial dysfunction, and inflammation. This leads to hepatocyte necrosis, apoptosis, and activation of hepatic stellate cells, resulting in fibrosis and cirrhosis. Chronic exposure to TAA ultimately impairs liver function, leading to liver failure. 22

23. D-galactosamine induced hepatotoxicity D-galactosamine (d- galn ) induces hepatotoxicity by disrupting hepatic RNA and protein synthesis, primarily due to the depletion of uridine triphosphate (UTP). This leads to mitochondrial dysfunction, oxidative stress, inflammation, hepatocyte apoptosis, and necrosis, resulting in liver damage similar to viral hepatitis. Equipment: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser . Drugs: Distilled water, Test drug, D-galactosamine, 10% Formalin, Anaesthetic ether, Silymarin. Animals: Wistar albino rats (150-200 gm) 23

24. Principle of D-galactosamine induced hepatotoxicity D-galactosamine (d- galn ) induces liver toxicity by depleting uridine triphosphate (UTP), which inhibits RNA and protein synthesis, leading to mitochondrial dysfunction, ATP depletion, and oxidative stress. This triggers kupffer cell activation, inflammation ( tnf - α, IL-6), and hepatocyte apoptosis/necrosis, resulting in severe liver injury resembling viral hepatitis. 24

25. references Vyawahare N. S., Karathara V. G., Pujari R. R., “ Preclinical Screening Of Drugs”, Nirali Prakashan, Chapter 7, Page No. 7.13 – 7.22. Klaassen, C.D. (2019). " Casarett And Doull’s Toxicology: The Basic Science Of Poisons" (9th Ed.). Mcgraw-hill Education. Brunton, L.L., Knollmann , B.C., & Hilal-dandan, R. (2017). "Goodman & Gilman’s: The Pharmacological Basis Of Therapeutics" (13th Ed.). Weber, L.W.D., Boll, M., & Stampfl, A. (2003). "Hepatotoxicity And Mechanism Of Action Of Carbon Tetrachloride ( CCl ₄)." Toxicology , 189(1-2), 1-12. Jaeschke, H., Xie, Y., & Mcgill , M.R. (2014). "Acetaminophen-induced Liver Injury: From Animal Models To Humans." Journal Of Clinical And Translational Research , 1(1), 5-17. 25

26. Thank you !!! 26