Screening of hepatoprotective drugs

Screening of hepatoprotective drugs

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  SCREENING OF HEPATOPROTECTIVE DRUGS PRESENTEDBY- MS.DIPANJALI D KAMTHE M.PHARM PHARMACOLOGY STES’ KASHIBAI NAVALE COLLEGE OF PHARMACY KONDHWA(BK),PUNE 1
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  CONTENT:  Introduction andfunction of liver  Hepatotoxicity and drug causing DILI  Hepatoprotective agent  Methods of Screening of hepatoprotectives 2
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  INTRODUCTION:  Liver islarge, 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. 3
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  LIVER TOXICITY  Themajor 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 4
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  DRUGS CAUSING DILI 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 OC pills  Markers of Hepatotoxicity-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) levels 5
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  LIST OF HEPATOPROTECTIVEAGENTS  N acetylcysteine  Penicillamine  Anti oxidants  cardiotropin 1  Herbal medications e.g. silymarine  S adenosyl methionine (SAM) Vitamins Melatonin Glutathion Beta-carotene 6
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  METHODS FOR SCREENINGOF HEPATOPROTECTIVES: 7 1.primary hepatocyte cell culture 2.Stellate cell culture 3.kupffer cell culture. 4.Liver cirrhosis and necrosis 5.Inhibition of proline hydroxylation 1.Hepatitis in Long Evans Cinnamon rats 2.Temporary hepatic ischemia 3.Carbontetrachloride induced liver fibrosis in rats 4.Bile duct ligation induced liver fibrosis in rats 5.Galactosamine induced liver necrosis 6.Thiocetamide induced necrosis of liver 7.Paracetamol induced liver damage in rats 8.Rifampicin+Isoniazid induced hepatotoxicity in rats IN VITRO METHOD IN VIVO METHODS:
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  IN VITRO METHODS PRIMARYHEPATOCYTE 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. 8
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  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. 9
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  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 10
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  LIVER CIRRHOSIS ANDNECROSIS  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. 11
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  INHIBITION OF PROLINEHYDROXYLATION  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. 12
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  IN-VIVO METHODS Hepatitis inlong 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 rats,making this animal a model for Wilson’s disease in human.  Chelation therapy or feeding copper deficient diet decreases symptoms in Long Evans Cinnamon rat and Wilson’s disease. 13
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  PROCEDURE Long evans cinnamonrats of age of 5 weeks are housed in temprature and humidity controlled room. Group of 6-10 rats are given different diets on a 15% purified egg protein diet and supplemented with vitamins or drugs.drugs are supplied via minipumps intraperitoneally implanted under ether anesthesia. The occurance of jaundice is esasily observable as the time when the ears and tails tuen yellow and the urine becomes bright ending in death of aminal within about a week. 14
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  TEMPORARAY HEPATIC ISCHEMIA: Hepatocellularfunction is altered by temporary hepatic ischemia as occuring 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 the common bile duct.  Procedure: Male albino rats(300–350 g )are fasted for 16 h prior to the experiment but are allowed water ad libitum. The rats are anesthetized lightly with ether and the abdominal cavity is opened through a midline incision. 15
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  Portal vein asthe hepatic artery and the bile duct are occluded by placing a tourniquet around the vessels. Blood pressure is measured via a catheter inserted into the right femoral artery.During the ischemic period, 0.7 ml of saline is given i.v. at 20-min intervals for volume replacement. At the end of the 60-min ischemic period, the tourniquet around the portal vein, hepatic artery and the bile duct is removed in order to reestablish blood flow to the liver. The abdominal incision is then closed and the animals receive either saline (nontreated) or the drug. 16
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  ALLYL ALCOHOL INDUCEDLIVER 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 17
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  EVALUATION  The parietalsides 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 18
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  CARBON TETRACHLORIDE INDUCEDLIVER FIBROSIS IN RATS  PURPOSE AND RATIONALE Chronic administration of tetrachloride to rats induces severe disturbances of hepatic function together with histologically observable liver fibrosis.  PROCEDURE Groups of 20 female Wistar rats(100–150g)are used. The animals are treated orally twice a week with 1 mg/kg carbon tetrachloride, dissolved in olive oil 1 : 1, over a period of 8 weeks. Animals are fed on chow diet with water ad libitum.Control group receives only olive oil. Test and standard drugs given twice daily except on Sunday when only one dose is given. The animals weighed every week, at the end of 8th week animals are sacrificed using anesthetic ether . 19
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   In serum,parameters determined includes-Total bilirubin,total bile acids,7S fragment of type IV collagen,procollagen III N-peptide.  Hydroxyproline is determined in Liver, kidney, aortic wall, and tail tendons.  The specimens of the organs are weighed and completely hydrolyzed in 6 N Hal. Hydroxyproline is measured by HPLC and expressed as mg/mg wet weight of the organs.  For histological analysis, 3–5 pieces of the liver weighing about 1 g are fixed in formalin and 3 – 5 sections of each liver are embedded, cut and stained with azocarmine aniline blue (AZAN) and evaluated for the development of fibrosis using a score of 0–IV. Grade 0: Normal liver histology. Grade I: Tiny and short septa of connective tissue. Grade II: Large septa of connective tissue. Grade III: Nodular transformation of the liver. Grade IV: Excessive formation and deposition of connective tissue. 20
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   EVALUATION  Fordetection of significant differences (p < 0.05), the unpaired t-test is used. For comparison of the scores in the histological evaluation, chi-square test is used.  MODIFICATIONS OF THE TEST  Instead of chronic intoxication with carbon tetrachloride resulting in liver fibrosis, acute Hepatocellular damage can be achieved by short term application of carbon tetrachloride.  The rats are treated daily for 5 days with various oral doses of the compound under investigation. From day 4 to day 5 the rats receive 1 mg/kg carbon tetrachloride dissolved in olive oil (1 : 1).  Blood is withdrawn every day and the aminotransferases and total bilirubin determined in the serum. 21
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  BILE DUCT LIGATIONINDUCED 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 ketamineHCL.Laparatomy is performed under antiseptic conditions. 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. 22
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  A blunt aneurysmneedle 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. 23
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  GALACTOSAMINE INDUCED LIVERNECROSIS  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 months. 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. 24
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  EVALUATION  The liversare 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. 25
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  THIOCETAMIDE INDUCED HEPATOTOXICITYIN 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. 26
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  Hepatotoxicity is inducedby 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 27
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  PARACETAMOL INDUCED LIVERDAMAGE IN RATS:  Paracetamol is one of the most commonly and widely used analgesic anti-pyretic drug used in higher dose lead to hepatic damage.  It gets metabolised to an active metabolite N-acetyl-p-benzoquinone imine by the cytocromeP-450 microsomal enzyme system which results in an oxidative stress producing liver glutathione and glycogen depletion.  Procedure: Wistar rats of either sex weight(150-200g)are used.Paracetamol 2g/kg body weight is administered orally as a single dose The animal are given the test drug foe 6 days prior to paracetamol administration and on the seventh day along with paracetamol The animal are sacrificed after 24h and the blood/serum is used for biochemical analysis and the liver for histopathological studies 28
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  RIFAMPICIN+ISONIAZID INDUCED HEPATOTOXICITY INRATS:  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. RMP 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. 29
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  REFERENCES:  H. GerhardVogel 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