Hepatoprotective screening methods

Hepatoprotective screening methods

• 1.
  NOIDAINSTITUTEOFENGINEERINGANDTECHNOLOGY (PHARMACY INSTITUTE) Presented by: ShobhiniChandel M.Pharm 1stsemester Submitted to: Dr. Saumya Das Associate Professor NIET(Pharmacy Institute) Hepatoprotective Screening Methods
• 2.
  TABLE OF CONTENT Introduction Livertoxicity Drugs causing DILI Hepatoprotective drug classification Invitro models Invivo models References
• 3.
  Introduction:  The liveris the body's largest internal organ that sits on the right-hand side of the belly.  The liver is an essential organ that has many functions in the body, including making proteins and blood clotting factors, , it breaks down and detoxifies substances in the body, and it also acts as a storage unit, manufacturing triglyceride and cholesterol, glycogen synthesis, and bile production.  Many different disease processes can occur in the liver, including infections such as hepatitis, cirrhosis (scarring), cancers, and damage by medications or toxins.
• 4.
  Introduction:  Hepatoprotective agentsare those compounds, which mitigate the liver injury caused by hepatotoxic agents thus can prevent damage to the liver. Examples: Ursodeoxycholic acid (UDCA), silibinin, and N-acetyl cysteine (NAC).
• 5.
  Liver toxicity: Main causesof liver or hepatic toxicity are as follows:  Alcohol consumption (ethanol)  Chemicals (carbon tetrachloride, phosphorus, alfatoxin, chlorinated hydrocarbon, etc)  Drug-Induced Liver Injury  Autoimmune disorder  Infection (hepatitis A, B, C)  Cancer and other growths (Liver cancer, Bile duct cancer, Liver adenoma)
• 6.
  Drugs causing DILI: Anti tuberculosis drugs Except ethambutol(1st line oral anti-TB drug) all drugs causes hepatotoxicity  Anticonvulsant drugs Carbamazepine and valproic acid  NSAIDs Paracetamol, diclofenac, indomethacin, oxicam  Antimicrobial drugs Depson, ketoconazole, sulfonamide, antiretroviral  Anesthetics Enflurane, Isoflurane  Miscellaneous drug Disulfiram, flutamide, statins, labetalol, nicotinic acid, OC pills, propylthiouracil
• 7.
  Hepatoprotective drugs classification: https://www.slideshare.net/DipanjaliKamthe/screening-of-hepatoprotective-drugs
• 8.
  Screening methods ofhepatoprotectives:  In vitro models 1. Primary hepatocytes cell culture 2. Stellate cell culture 3. Kupffer cell culture 4. Liver cirrhosis and necrosis 5. Inhibition of proline hydroxylation  In vivo methods 1. Hepatitis in Long Evans Cinnamon Rat 2. Temporary hepatic ischemia 3. Allyl alcohol induced liver necrosis in rat 4. Carbon tetrachloride induced liver fibrosis in rat 5. Galactosamine induced liver necrosis 6. Thiocetamide induced necrosis of liver 7. Paracetamol induced liver damage in rat 8. Rifampicin and isoniazid induced hepatotoxicity in rats
• 9.
  In vitro models: A.Primary hepatocytes cell culture-  Fresh hepatocyte preparation and primary culture hepatocyte are used. The basic method  Isolation of hepatocyte by perfusion of liver with collagenase or utilization primary cultured hepatocyte  Incubation of the cell culture with hepatotoxin and with or without the test drug.  Determination of the viability of the hepatocyte Evaluation parameters Determination of the activity of transaminases released into the medium by the hepatocyte
• 10.
  B. Stellate cellculture  In chronic injury there is stellate cell activation  There is secretion of matrix by activated stellate cell results in liver fibrosis and ultimately cirrhosis  Stellate cell are isolated from rat liver by collagenase or pronase digestion  Test drugs are incubated with activated stellate cell culture by hepatotoxin Evaluation parameters Morphology of cell, Alpha SMA expression, cell count with thymidine incorporation and inhibition in synthesis of collagen type 1 and 3
• 11.
  C. Inhibition ofproline hydroxylation  The thermal stability of the triple helix of collagen is depend on intramolecular hydrogen bond synthesized by the enzyme propyl-4-hydroxylase Procedure  Reaction volume of 1 ml contain tris buffer, glutarate ,ferrous oxide, ascorbate, catalase, BSA, dithiotreitol inhibitor  After incubation at 37 degrees celsius for 30 minutes the generated CO2 is trapped and determined
• 12.
  In vivo methods A.Hepatitis and Long Evans Cinnamon Rat Purpose and rationale  The Long Evans Cinnamon Rat is useful model to study genetically transmitted hepatitis and chronic liver disease  Due to excessive copper accumulation in the liver of Long Evans Cinnamon Rat making this animal a model for Wilson’s disease in human(excess copper accumulation in body) Procedure  Long Evans Cinnamon Rat of age of 5 weeks are housed in temperature and humidity controlled room  Group of 6 to 10 rats are given different diets on a 15% purified protein diet and supplemented with vitamin or drugs  Drugs are supplied via mini pumps intraperitoneal implanted under anesthesia  The occurrence of jaundice is easily observable as the time when the ear and tail turn yellow and the urine become bright ending in death of animal within about a week.
• 13.
  B. Temporary hepaticischemia  Hepatocellular function is altered by temporary hepatic ischemia as occurring during surgical management of acute hepatic trauma and being essential during hepatic transplantation. Purpose and rationale  Total hepatic ischemia in rats is produced by placing a ligature around the hepatic artery portal vein and common bile duct Procedure  Male albino rat 300 to 350 g is fasted for 16 hours prior to the experiment but allow water ad libitum  The rats are anesthetized lightly with ether and abdomen cavity is opened through a midline incision  Portal vein as a hepatic artery and the bile duct are occluded by placing a tourniquets around the vessels
• 14.
   Blood pressureis measured by a catheter is inserted into the right femoral artery. During the ischemic period, 0.7ml of saline is given intravenous at 20 minute interval for volume replacement  At the end of 60 minute ischemic period the, tourniquet around the portal vein, hepatic artery and the bile duct is removed in order to re stabilize blood flow to the liver  The abdominal incision is then closed and the animal receive either saline or the drug
• 15.
  C. Allyl alcoholinduced liver necrosis in rat Purpose and rationale  Administration of allyl alcohol induced liver necrosis in rat which can be partially prevented by treatment with several drugs such as antibiotic . Procedure  Female Wistar rat weighing 120-150 g are fasted overnight with water ad libitum  On following morning test drug is given orally or intraperitoneally to 10 rats. After 1 hour 0.4 ml per kg of 1.25% allyl alcohol solution is given orally  Test drug given again on second day and sacrifice on 3rd day liver is removed
• 16.
  Evaluation parameter  Theparietal sides of the liver are checked using a stereo microscope with 25 times magnification  Focal necrosis is observed as white green or yellowish hemorrhagic area clearly separated from an infected tissue  The diameter of the necrotic area is determined using ocular micro meter  The value added for each animal to obtain an index of necrosis  Mean of necrosis index is calculated and compared with student t test  The protective effect is expressed as percentage decrease of the necrosis index versus control
• 17.
  D. Carbon tetrachlorideinduced liver fibrosis in rat Purpose and rationale  chronic administration of tetrachloride to rat induced severe disturbance of hepatic function together with histologically observable liver fibrosis. Procedure  Group of 20 female Wistar rat is used weighing 100- 150g  The animal are treated orally twice a week with 1 mg per kg carbon tetrachloride dissolved in olive oil 1:1 over a period of 8 week  Animal are fed on chow diet with water ad libitum. Control group receive only olive oil. Test and standard drug given twice daily except on Sunday when only one dose is given  Animal weighed every week and at the end of 8 week animal sacrifice using and anesthetic ether Evaluation parameter  Total bilirubin  Total bile acid  7S fragment of type 4 collagen  Procollagen 3 N- peptide
• 18.
  E. Galactosamine inducedliver necrosis  Single dose or few repeated dose of D galactosamine cause acute hepatic necrosis in rat  Prolonged administration lead to cirrhosis Procedure  Divided doses of 100 to 400 mg/ kg D galactosamine are injected to rat IP or IV during day one  for induction of liver cirrhosis male Wistar rat weighing 110-180 gm are injected IP three time weekly with 500 mg/ kg D galactosamine over a period of 1 to 3 months  Potential protective substance are administered orally with the food every day  The rat sacrifice at various time interval and the liver obtained by autopsy
• 19.
  Evaluation  The liverare evaluated by light microscope and immunohistology using antibodies against macrophages, lymphocytes and the extracellular matrix component  The extent of liver cell necrosis and Immuno reactivity for macrophages, lymphocytes and extracellular matrix component is graded semi-quantitatively on 0-4 scale  0= absent  1 –trace  2- weak  3- moderate  4- strong
• 20.
  References -  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265599/ https://www.spandidos-publications.com/ijmm/42/4/2020  https://www.sciencedirect.com/science/article/abs/pii/S03788741 20336552?via%3Dihub  https://pubmed.ncbi.nlm.nih.gov/?term=hepatoprotective+agents  https://www.sciencedirect.com/topics/medicine-and- dentistry/liver-toxicity  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202426/  https://www.slideshare.net/DipanjaliKamthe/screening-of- hepatoprotective-drugs