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Evaluation of hepatoprotective agents - Hemant Kanase

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1. Introduction
2. Hepatotoxicity: Mechanism
3. Therapeutic strategies available – their limitations
4. In vivo models of liver damage

- Non-invasive model
a. Chemically induced hepatotoxicity
b. Drug-induced hepatotoxicity
c. Radiation-induced hepatotoxicity
d. Metal-induced hepatotoxicity
e. Diet-induced hepatotoxicity

 Models of Acute Hepatitis
 Models of chronic hepatitis
 Models of fibrosis
 Models of cholestasis
 Models of steatosis

4. Problems faced with animal studies
5. In vitro models of liver damage
6. Advantages and disadvantages of in vitro models
7. Parameters of evaluation
8. Clinical Assessment

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Evaluation of hepatoprotective agents - Hemant Kanase

  1. 1. Evaluation of Hepatoprotective Agents Dr. Hemant R Kanase 1
  2. 2. Flow of the seminar - Introduction - Ideal hepatoprotective agent - Limitations of current treatment - Preclinical test: • In-vitro methods • In-vivo methods - Parameter of evaluation - Clinical Assessment 2
  3. 3. Introduction • Largest and the most important organ (weighing 1–1.5 kg and representing 1.5–2.5% of the body mass.) • Principle site for metabolism, secretory and excretory functions. • Distinguishing fact: potential of hepatocyte to proliferate and regenerate along with its dual blood supply 3
  4. 4. 5 Liver Toxicity The main causes of liver damage: Chemicals like carbon tetrachloride CCL4, phosphorous , aflatoxins, chlorinated hydrocarbon etc Drugs i.e. DILI ( drugs induced liver injury ) eg. Halothane, Paracetamol. Alcohol Autoimmune disorders : PBC, PSC, Wilson’s disease Infections: Viral , bacterial, parasites
  5. 5. 6  Caruaru tragedy – dialysis tragedy (1996) • 126 pts on hemodialysis • Intoxicatced Microcystis sp. Blue algae – Microcystin LR – Hepatoxic agent  Case report: • 76 yr female • Presented with blister on foot, later died with sepsis, bleeding and hepatic faillure • Diagnosis : colitis • Treatment : Mecaptopurine (thiopurine) with allopurinol Drug interaction - hepatoxicity
  6. 6. 7
  7. 7. On depletion of glutathione Leads to accumulation of NAPQI Causes Toxicity 8
  8. 8. An ideal hepatoprotective agent should be able to:  control infection or counter the infective noxious and injurious elements, if these are the etiological factors,  protect the liver parenchyma, promote healing of damaged parenchyma to revert back to a structurally normal or near normal state,  help liver parenchymal cells to regain their functional capabilities,  prevent complications both short term and long term leading to mortality and morbidity,  an ideal antifibrotic would be one that is liver specific, well tolerated and effective in attenuating excessive collagen deposition without affecting normal extra cellular matrix synthesis. 9
  9. 9. Currently available modalities of hepatoprotection:  Removal of causative agent • iron and copper overload, alcohol consumption, secondary biliary obstruction, drug induced liver disease  Inhibiting the inflammatory response of liver • anti-inflammatory agents : Corticosteriods (lack of anti fibrotic activity) Colchicine (Anti inflammatory + Anti Fibrotic )  Inhibition of activation and proliferation of hepatic stellate cells • Antioxidants: Vitamin E (-tocopherol), phosphatidylcholine, S-adenosyl-L-methionine (SAMe) • Prevent hepatic stellate cell proliferation and collagen formation: Interferon-, pentoxifylline and amiloride: • Antifibrotic activity: Vasodilators like PGE2 and nitric oxide donors 10
  10. 10. Limitations of currently available drugs • Most of them don’t have proven efficacy conclusively. • There is high risk of relapse • Many of them produce side effects. Treatment largely been supportive and symptomatic. Shift in focus to alternative forms of therapy based on drugs derived from plants. Thus there is a need for evaluation of new treatment modalities by using modern scientific methodology 11
  11. 11. Models of hepatoxicity Pre clinical test : • In vivo • In vitro Clinical assessment 12
  12. 12. In vitro models 13
  13. 13. In vitro models of liver damage 1. Primary screening of the potential therapeutic agents 2. Primary hepatocyte cell culture 3. Stellate cell culture 4. Kupffer cell culture 5. Rat liver slice 6. Assays for measuring total cell population 7. Assays for measuring cell proliferation 14
  14. 14. 1. Primary screening of the potential therapeutic agents  Different in vitro concentrations of the test drug are incubated with the culture and effect of the drugs on the cell line is studied.  Parameters : viability and proliferation of the cells in presence of the test drugs.  Viability - cytotoxic effect of the drug.  Effect on proliferation of cells - Antifibrotic effect, evaluated by the modulation of monocyte macrophage system.  The drugs can be incubated in various in vitro concentrations with macrophages 15
  15. 15. 2. Primary hepatocyte 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 the transaminases released into the medium by the hepatocytes Hepatotoxins: CCl4, paracetamol, d-galactosamine, tert-Butyl hydroperoxide and ethanol. 16
  16. 16. 3. 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 isolated from rat liver by collagenase / pronase digestion  The test drugs are incubated with the activated stellate cell culture by hepatotoxins  Parameters studied :morphology of the cells, -SMA expression, cell count with thymidine incorporation and inhibition in synthesis of collagen type I and III. 17
  17. 17. 4. 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 to 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. 18
  18. 18. 5. Rat liver slice  A liver slice consists of the various cell types of the organ where the cells maintain normal architecture, acinar localization and cell-cell communications.  The functional heterogeneity and biochemical capacity of the whole organ is retained.  Histopathological assessment of the exposed tissue can be done.  It may be possible to assess action of hepatotoxin on the liver even if only few milligram of test drug is available. 19
  19. 19. 6. Assays for measuring total cell population To determine the total cell population in the assay system Dye exclusion test : dye is retained by dead cells and excluded by live cells DiSC assay (Differential Staining Cytotoxicity) : live cells and dead cells are counted by two different colours against an internal standard MTT assay : colorimetric assay which depends upon the reductive capacity of live cells to metabolize the MTT dye to a highly coloured formazan crystals. • The UV absorbance of the solubilized crystals is measured spectrophotometrically and directly correlates with the number of living cells in suspension. 20
  20. 20. 7. Assays for measuring cell proliferation Clonogenic assay (Human tumour stem cell assay)  Culturing of cells in petri dishes, capillaries or wells with or without drug.  At the end of the culture period, the number of colonies is counted.  The difference between the number with and without drug is taken as a measure of drug effect.  Thymidine incorporation assay: Actively growing and multiplying cells Cells are incubated with radiolabelled thymidine, they use radioactive thymidine for their DNA synthesis.  The amount of incorporated thymidine is proportional to the proliferation. 21
  21. 21. Advantages & disadvantages of in vitro models  Advantages • Ability to screen numerous samples at one time. • Convenient • Less variation in results. • Good reproducibility. Disadvantages • Damage to hepatocytes may be mediated through mechanisms different from those operating in vivo, and results may differ. • The test drugs may not cross the GI barrier to reach the liver tissue in exactly the same proportions as that added to the cell culture. • Extrapolation of these findings to human beings is more difficult. 22
  22. 22. In vivo models of hepatotoxicity 23
  23. 23. In vivo models of hepatotoxicity A. Non-invasive model a. Chemically induced hepatotoxicity b. Drug-induced hepatotoxicity c. Radiation-induced hepatotoxicity d. Metal-induced hepatotoxicity e. Diet-induced hepatotoxicity B. Invasive model Bile duct ligation C. Genetic model Transgenic animal model Animals – rodents (wistar rats, spargue dawley, Swiss albino mice) 24
  24. 24. Chemically induced hepatoxicity Commonly used hepatotoxins • Carbon tetrachloride (CCL4) • Thioacetamide • Dimethyl or diethyl nitrosamine • Alfatoxin B1 25
  25. 25. CCl4 induced  Grain fumigant, dry cleaning fluid, fire extinguisher fluid The formation and release of free radicals and lipid peroxides is mechanisms of injury. used for inducing both acute and chronic liver disease 26
  26. 26. Principle: CCl4 --- Cytochrome P-450/2E1 (CYPE2E1) in hepatocytes ---- CCl3 (Toxic metabolite) The free radical (trichloromethylperoxyl radical) ------ attacks lipids on the membrane of endoplasmic reticulum, induces an acute centrolobular necrosis. Procedure : • Wistar rats (120 – 150 gm) • Before experiment, animals are deprived from food. • Hepatotoxicity is produced in animals by different concentration of CCl4 by different route (i.p., s.c., oral)and different time period . • On the last day of the study the animals are anesthetized and killed for studying biochemical parameter and Histopathological tests. 27
  27. 27. Model of acute hepatitis: Model of fibrosis: • 1 mg/kg carbon tetrachloride twice a week orally dissolved in olive oil (1:1), over a period of 8 weeks. Dose mg/kg Duration Route of admin 1 mg/kg 0.5 ml/kg 0.2 ml/kg 0.125 ml/kg 1.0 ml/kg 2 ml/kg 2-5 days 3 days 2 weeks 7 days Every 72 hrs for 10 days Every 72 hrs for 10 days Oral i.p. i.p. i.p. i.p. s.c. 28
  28. 28. Evaluation: 1. The following parameters are determined in the serum: • Total bilirubin • ALT, AST • GGT • 7S fragment of type IV collagen • Procollagen III N-peptide 2. Histological analysis of the liver using a score from 0 to IV Grade 0: Normal histology Grade I: Tiny short septa of connective tissue without influencing the hepatic lobule structure Grade II: Large septa of connective tissue with penetration into parenchyma and tendency to develop nodules Grade III: Nodular liver architecture with lost hepatic lobule structures Grade IV: Excessive connective tissue deposition subdividing the regenerating lobules and development of scars 29
  29. 29. Effect of CCl4 Morphological parameters Normal liver Post - CCl4 liver 30
  30. 30. SilymarinDistilled water CCl4 Liver histopathology post- CCl4 (Masson Trichome Stain) 31
  31. 31. Modification: Kawaura et al. (1993) produced liver cirrhosis with ascites in dogs by administration of 2 ml CCl4 per kg body weight once a week for 4 weeks. Wirth et al. (1997) studied the effects of a bradykinin B1 receptor antagonist in rats with CCl4-induced liver cirrhosis. 32
  32. 32. D – Galactosamine Single dose or a few repeated doses of D-galactosamine cause acute hepatic necrosis in rats. • Prolonged administration leads to cirrhosis Accumulation of uridine sugar nucleotide causing disruption of metabolism of glycoproteins and glycolipids leading to increased cytosolic Ca+ Procedure Wistar rats (100-180 gm) are used. Single or divided doses of 100-400 mg/kg D-galactosamine are injected i.p. or i.v. during one day. Model of fibrosis: 500mg/kg of D-galactosamine is injected i.p. thrice weekly for 1-3 months Evaluation: Liver - HPE Blood - Biochemical parameters 33
  33. 33. Thioacetamide (TAA)induced hepatotoxicity.  White crystalline solid, serves as source of sulphide ions in the synthesis of organic and in organic compounds.  TAA – not toxic, Thio acetamide S – oxide binds to hepatic macromolecules, increases intracellular Ca+ concentration and causes cellular damage and necrosis.  Widely used to induce fibrosis  Procedure: Wistar rats 100-150 gms. I.P. dose of thioacetamide: 200 mg/kg - thrice weekly for 8 weeks 200 mg/kg - for 2 weeks Evaluation: Liver - HPE Blood - Biochemical parameters 34
  34. 34. Diethyl nitrosamine (DEN) induced  Carcinogenic: used in the manufacture of some cosmetics, pesticides, rubber products  DEN is hydroxylated to ethyldiazonium ion by CYP2E1 causes DNA damage and induces hepatotoxicity  This model is important in studying progression of fibrosis to HCC.  Procedure • Wistar Rats • Free access to standard rat pellet food and tap water.. • Animals treated with diethyl nitrosamine having different concentration (50–200 mg/kg, i.p. for 4–12 weeks). Evaluation: Liver - HPE Blood - Biochemical parameters 35
  35. 35. Alfatoxin B1 induced hepatotoxicity  Mycotoxin – acute hepatotoxicity and liver carcinomas  Metabolized by cytochrome P450 to highly reactive 8, 9-epoxide that binds to cellular macromolecules, primarily in the periportal region of the liver.  Liver injury manifests as periportal parenchymal cell necrosis, haemorrhage, and injury to intrahepatic bile ducts.  Used to establish models of cholestasis  Procedure: Wistar Rats Disease is induced by administration of different concentration of alfatoxin (200 micro gm/kg – 6 mg/kg PO) for 2days – 52 weeks.  Evaluation: Blood – Biochemical analysis Liver - HPE 36
  36. 36. Drug induced hepatotoxicity 37
  37. 37. Acetaminophen induced  Paracetamol administration causes necrosis of the centrilobular hepatocytes  Procedure: • Wistar rats 150-200 gms. • Single oral dose of 2gm/kg p.o. body weight • Test compound is given for 6 days prior to PCM administration and on 7th day along with PCM administration • On last day animal are sacrificed. • different doses (2-3 mg/kg, p.o. for 7 days–3 weeks)  Evaluation: Blood – Biochemical analysis Liver - HPE 38
  38. 38. Anticancer drugs induced…  Mechanism: Secondary to production of reactive oxygen species (ROS), intended to induce tumor cell apoptosis. Cisplatin induced hepatoxicity:  Mechanism: Elevated CYP2E1 enhances production of ROS and oxidative stress which induces hepatotoxicity Procedure: • Wistar rats (100-150 gms) • Free access to food and water. • Dosage and duration: 3.5–7 mg/kg i.p. for 1–5 days  Evaluation: Blood – Biochemical analysis Liver - HPE 39
  39. 39. Anti-TB drugs induced…  INH , Rifampicin  Principle Isoniazid is chemically isonicotinyl hydrazine which is used to treat tuberculosis. N - acetyltransferase CYP2E1/Cyt chrome p450 INH --------------------- acetyl hydrazine -------------------------------- reactive acylating sp. Rif (+) Rif (+)  Reactive species covalently with liver cell macromolecules cause hepatocyte injury.  Procedure: • Wistar rats 150–200 g, free access to food and water. • Rats are treated with isoniazid (INH) 50mg/ kg, co-administered with rifampicin (RIF) 100mg/kg i.p.of 10–28 days  Evaluation: Blood – Biochemical analysis Liver - HPE 40
  40. 40. Radiation induced hepatotoxicity: Principle :  Activation of the coagulation cascade leading to accumulation of fibrin and formation of clots in the central veins and hepatic sinusoids.  Cause hepatic dysfunction by death of centrilobular hepatocytes and atropy of the inner hepatic plate. Procedure: • Animals allowed free access to a standard diet and clean drinking water. • Animals are irradiated • Single dose of 5 Gy of gamma-radiation for 2 days • single dose level of 3 - 6 Gy for 7 days • Single dose of gamma rays 6 Gy for 15 consecutive days  Evaluation: Blood – Biochemical analysis Liver - HPE 41
  41. 41. Metal induced hepatoxicity  Toxic and carcinogenic metal compounds.  It promotes the formation of reactive oxygen species (ROS) such as hydrogen peroxides. These ROS enhance the production of lipid peroxides and the highly reactive hydroxyl radical Procedure: • Animals allowed free access to a standard diet and clean drinking water. • Animals are treated by mercury, • Mercury chloride (HgCl2) (80 mg/l) as drinking water for 4 weeks. • 5 mg/kg s/c inj of HgCl3 on the 7th day of experiment  Evaluation: Blood – Biochemical analysis Liver - HPE 42
  42. 42. Diet induced hepatoxicity Alcohol induced..  Acetaldehyde: is toxic and may cause membrane damage and cell necrosis via generation of highly reactive oxygen species (ROS) (alpha-hydroxyethyl) Procedure: • Animals allowed free access to a standard diet and clean drinking water. • Ethanol 2.0 ml/l00 g p.o. for 21 days • Ethanol 7.9 g/kg p.o. daily for 45 days • Ethanol 5 g/kg/day p.o. for 60 days • Ethanol 3.76 gm/kg twice a day p.o. for 25 days  Evaluation: Blood – Biochemical analysis Liver - HPE 43
  43. 43. Models of acute hepatitis: 1. CCl4 – 1 mg/kg for 5 days 2. D- galactosamine – 100 – 400 mg/kg single / divided dose on single day 3. Paracetamol induced – 2mg/kg for 6 days Limitations: • Lesions produced in above mentioned models resemble lesions produced in viral hepatitis, i.e. necrosis, the underlying mechanism may be totally different. • The viruses may not act in the same way as hepatotoxins. There are models of acute hepatitis using virus: • MHV A 59, MHV 3 (focal necrosis), woodchuck hepatitis virus (Hep B like hepatitis) • Induce hepatitis by macrophage virus interaction 44
  44. 44. Models of chronic hepatitis Liver appearances in chronic hepatitis of viral and non-viral origin are similar. Features: infiltration of mononuclear cells into the liver and signs of parenchymal damage. Various models: i.p. injection of Corynebacterium parvum (0.7 mg/mouse i.v.). i.p. injection of LPS from Escherichia coli (25 g per mouse) weekly i.p. injection with syngenic liver homogenate fractions containing a self antigen liver specific proteins (LSP) and Freund’s adjuvant (FCA) for 8 weeks  Evaluation: Blood – Biochemical analysis Liver - HPE 45
  45. 45. Models of fibrosis  Persistant exposure to inducing agent may lead to fibrosis and serves as a good model for anti-fibrotic drugs.  Models for such injury should meet the following criteria: • It must be reproducible. • Mortality rate of animals should be low. • If recovery is to be studied the injury should be reversible. • Stages from the onset of acute injury to the development of fibrosis must be distinct  The models of chronic liver injury can be induced by • 1 mg/kg carbon tetrachloride twice a week orally 8 weeks • 500mg/kg of D-galactosamine is injected i.p. thrice weekly for 1-3 months 46
  46. 46. Using surgical/ invasive techniques: Bile duct ligation (BLD) induced fibrosis in rats.  Useful experimental model to study fibrosis.  BDL stimulates the proliferation of biliary epithelial cells, resulting in proliferating bile ducts, cholestasis, portal inflammation, and fibrosis, causing secondary biliary cirrhosis  Procedure:  Male Sprague Dawley rats (250 g) are anesthetized. • midline abdominal incision • isolation of the common bile duct above the duodenum • two ligatures were placed to the proximal and on distal part of the bile duct. 47
  47. 47. • Animals are given normal diet and water ad libitum throughout the experiment. • Test compound is administered twice daily for 6 weeks. • After 6 weeks animals are sacrificed • Blood: Hepatic enzymes, bile acids, 7S fragment of type IV collagen, and procollagen III N-peptide. • The liver: histological studies and for hydroxyproline determinations  At 4 – 6 weeks: features resemble biliary cirrhosis in humans 48
  48. 48. Models of Cholestasisis A. Endotoxin induced cholestasis:  Mechanism: inhibits the uptake and secretion of bile acids and organic anions.  7.5 micro gm /kg dose is administered i.p. B. Ethinyl estradiol induced cholestasis: 5 mg/kg body weight orally. The cholestatic effect is due to the endogenous estrogen metabolite estradiol-17-D-glucoronide (leads to impairment of transport mechanism in basolateral and canalicular hepatocyte membrane.) Other drugs:  Cyclosporine A, Rifamycin, rifampicin glibenclamide, Alpha napthyl isothiocynate inhibit bile salt transport and induce cholestasis. 49
  49. 49. Model of obstructive cholestasis:  Physical obstruction to the biliary system  Total: Impaired excretion and retention of bile within the hepatocytes increased ductular proliferation and periportal necrosis  Partial: Incomlete obstruction to the bile duct causing mild ductular proliferation and portal fibrosis Good model for chronic fibrosing cholangitis.  Selective obstruction: Selected bile ducts draining selected lobes. For studying reserve secretory capacity. 50
  50. 50. Models of steatosis: Lipid content of hepatocyte regulated by: Uptake, synthesis, oxidation and export Hepatic steatosis : input of fat exceeds the capacity for fatty acid oxidation i.e output For studying human nonalcoholic fatty liver disease 1. Increased hepatic `input’ of fats  Genetically modified mice with increased hepatic lipogenesis  Environmental causes of increased fatty acid input 2. Decreased hepatic `output’ of fats  Genetically modified mice with decreased hepatic elimination of fats  Environmental causes of decreased hepatic elimination of fats 51
  51. 51. 1. Targeted over expression of genes that promote lipogenesis Eg. A-BZIP/F mice Adipose specific C/EBPKO aP2-Diptheria Toxin mice • Enlarged, fatty livers at a very young age • Deficient in the adipocyte hormone leptin 2. Naturally occurring mutations that promote lipogenesis Eg. • ob/ob mice - naturally occurring mutation that prevents the synthesis of leptin • db/db mice - mutation of leptin receptors • fa/fa mice - exhibit mutation of the gene for leptin receptor. • Resistance to leptin I. Increased hepatic `input’ of fats  Genetically modified mice with increased hepatic lipogenesis 52
  52. 52. Environmental causes of increased fatty acid input (i) Diet-Induced Obesity High sucrose/ fructose diet (70 % sucrose, 10% fructose) High fat diet (60% fat) given for 8 weeks (ii) Diets that induce hepatic lipogenesis without Obesity Arginine-deficient diet (abnormal orotic acid metabolism) 53
  53. 53. II. Decreased hepatic `output’ of fats A. Genetic causes of decreased hepatic elimination of fats (i) Mice with targeted deletion of genes that regulate fatty acid oxidation PPARK-/- mice AOX KO (Acyl CoA oxidase) Aromatase KO (female) (ii) Mice with naturally occurring mutations that inhibit fatty acid oxidation Juvenile visceral steatosis (carnitine deficiency) B. Environmental causes of decreased hepatic elimination of fats • Methionine and or choline deficient diet (0.05%) 54
  54. 54. Problems faced with animal studies 1. Most of the animal models are based on chemical injury whilst the most common form of clinical disease is viral in etiology. 2. Liver damage caused by toxins may be different from effect of natural hepatotoxins. 3. Chronic liver disease is hard to mimic in laboratory. 4. The drug used is usually administered along with or before injection of toxin, and hence focus is on preventing damage due to toxin. However, patients present after liver injury has set in. 5. Costly 6. Inconsistency in serum indicators used in studies. 55
  55. 55. Parameters of evaluation In a particular model system of liver injury induced by any agent,  the extent of functional and structural damage to the liver  the regenerative capacity of the liver following such damage This can be assessed by  gross and histopathological examination of the liver using various stains,  biochemical methods which measure various enzymes or substances in serum liberated after injury to cells,  evaluation of the clearance of substances from the blood. 56
  56. 56. Gross and Histopathological Examination  Gross examination : Done by by isolating the organ from the body. • Gives an idea about changes in margin, consistency and colour of the liver e.g. in cirrhosis the liver is shrunken, nodular where as in obstructive jaundice it is enlarged.  Histopathological examination: liver is sectioned,fixed and,stained  The different stains: • Haematoxylin-Eosin stain - nucleus and cytoplasm • Masson trichome or Sirius red stain - collagen fibers • Reticulin stain - reticulin fibers of the liver 57
  57. 57. Advantages of HPE Information on nature of injury. Eg. hepatocytes (degeneration, necrosis, and regeneration), inflammatory reaction, structure of bile ducts and vascular distortion can be studied. The structural changes can be graded using scores: knodell score, NASH activity score The most widely used is the histological activity index (HAI) also known as the Knodell score. • Periportal +/- Bridging necrosis • Intralobular degeneration and focal necrosis • Portal inflamation • Fibrosis • Total score of 22. 58
  58. 58. Limitations: • Histological grades sometimes poorly correlate with clinical and biochemical assessment. • May be due to sampling error, and a tendency for histological features to change more slowly in response to therapy than biochemical ones 59
  59. 59. Biochemical parameters  Assessment of liver function 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 • Endogenous substances that are liberated in the serum after injury to the liver • Tests are always done in ‘batteries’ to minimize the false negative results. Therefore, the predictive accuracy is very high, 95 % or even better. • The conventional tests are of prognostic help. 60
  60. 60. Conventional liver function tests Pathological feature Endogenous substance Comments Cellular injury + cholestasis Bilirubin Confirmatory evidence for the presence of liver disease Hepatocellular damage AST, ALT Rough estimate of necrosis Prothrombin time Coagulation proteins Reflect status of synthetic capacityAlbumin, prealbumin Pseudocholinesterase Inflammatory reaction Acute phase protein -  globulin, CRP, MDG, SOD, GSH Non-specific for chronic liver disease Cholestasis Alkaline phosphatase Non-specific marker 5ʹ nucleotidase Specific marker  glutamyl transpeptidase Marker for alcohol abuse Vascular distortion Bile acids (total and conjugated) Indicate porto-systemic anastomosis 61
  61. 61. Quantitative liver function test Measure the functional capacity of the liver and quantify the important functions like metabolic, excretory, and vascular functions Principle: Known amount of an endogenous compound which is normally processed by the liver and whose fate in the body is well characterized is administered. Its clearance from the body is determined by taking plasma, urine or salivary samples. Any change in its disposition can be quantified which reflects a specific functional defect in perfusion, metabolism and/or excretion. 62
  62. 62. Quantitative liver function tests MEGX test is useful as a real- time method for quantitatively assessing pre- and post- transplant liver function LiMAx test (maximum liver function capacity) - based on the metabolism of 13C- methacetin Costly and complex to perform Not used routinely Substrates Sampling sites 1. Vascular function Indocyanine green Blood 2. Metabolic capacity Antipyrine Aminopyrine Galactose( ditingush between HC and bil. obs) Caffeine Saliva, blood Breath Blood Breath Saliva, blood Breath 3.Excretory functions BSP Bile acids Blood Blood 63
  63. 63. Assessment of liver fibrosis Conventional LFT : provide little info on presence of fibrosis and no means of following its progression For exact quantification we use liver tissue. Measured parameters: • the content of total peptide bound hydroxyproline • the activity of prolyl hydroxylase, lysl oxidase and -N-acetyl glucosaminidase • collagenase 64
  64. 64. Specific tests have been developed to measure circulating collagens, procollagen peptides, extra cellular matrix glycoproteins and their fragments. Non invasive and can be performed repeatedly, thus offering potential for monitoring anti-fibrotic therapy. Markers reflect hepatic fibrogenesis, fibrolysis or both? They serve as supplements to the routinely used parameter for assessment of fibrosis. Connective tissue polypeptide Fibrogenesis Fibrolysis Liver specificity PICP + - + PIIINP + +, Acute + PIVCP - (?) + + PIVNP(7-S) - (?) + + CVI - + + Lam + (?) + (?) + Undulin - +, remodelling + 65
  65. 65. Immunological parameters  In liver disease there is increased antigenic influence that is responsible for increased antibody titers in cirrhotic patients  It may be because of decreased ‘filter’ effect of the liver which is done by the macrophage system of the liver.  Experimental evaluation of Kupffer cell activity : injection of particulate matter and studying its clearance  Particulate matter: Colloidal carbon, human serum albumin lipid emulsions, radioactively tagged bacteria, denatured serum protein fraction labelled with I131, magnetic iron colloid 66
  66. 66. Nitric oxide (NO)  Ubiquitous messenger molecule involved in diverse cellular process such as neurotransmission, immune regulation, and vascular homeostasis  NO plays a important role in liver homeostasis  NO protects hepatocytes involve induction of heat shock proteins, suppression of caspase activity, activation of soluble guanylyl cyclase and modulation of mitochondrial respiration.  The intrahepatic synthesis of nitric oxide is markedly decreased in advanced human and rat liver fibrosis. 67
  67. 67. 68 Pre clinical test Screening of lead compound Clinical phase In vitro In vitro • Cell culture techniques Hepatoxin induced • Various models Hepatitis, fibrosis, cholestasis, steatosis, diet induced.
  68. 68. 69
  69. 69. 70  To evaluate safety  To select appropriate dose based on the preclinical studies  To determine the pharmacokinetic profile.  To evaluate efficacy & potency Clinical Assessment
  70. 70. Phase 1 and 2  Objective : • Assessment of safety and tolerance of the drug. • Generation of preliminary PK data, duration of action and general PD effects. • To determine adverse events in human subjects • Pharmacokinetics- AUC, t1/2 , Ke, Tmax • Study group: Normal healthy volunteers. • Dosing : Multiple ascending dose study in elderly volunteers. • Safety and tolerability assessed by : Clinical examination, biochemical test 71
  71. 71. Phase II/III studies Objective :  Confirm efficacy in patients/ risk of hepatic damage  To determine safe dose range and maximum tolerable dose  Monitor side effects  Comparator – Placebo? Silymarin 72
  72. 72. Key Inclusion Criteria: • Males and females >= 18 years of age. • Diagnosis of acute viral hepatitis (A,B,C,E, EBV, CMV) (<1 month) as manifested by a combination of the following symptoms: jaundice, dark-coloured urine, light-colored stools, pruritus, fever, nausea, vomiting, anorexia, and right upper abdominal discomfort, pain or feeling of pressure. • Serum ALT level > 2.5 times the upper limit of normal. • Albumin level >3.5 gm/dl • Negative anti-HCV antibody • Subject has given written informed consent. 73
  73. 73. Exclusion Criteria: • Pregnant or breastfeeding women • Suspected hypersensitivity • Advanced liver disease (e.g. ascites, bleeding esophageal varices and hepatic encephalopathy) • Chronic liver disease as cirrhosis • History or alcohol abuse • Subjects with positive anti-HCV antibody • Simultaneous elevation of bilirubin > 10 mg/dl along with an ALT level between 100 and 150 U/L • Platelets count <150,000 • Morbid obesity i.e. BMI > 40 • Subjects with severe illness, e.g., multisystem failure, cancer or poorly controlled diabetes i.e. known diabetic with Hemoglobin A1C (HbA1C)>7%, CCF • h/o of drug-induced acute hepatitis. • recent use Hepatoprotective agent within past two weeks. 74
  74. 74. Primary safety objective: • To assess safety and tolerability of the test drug in patients with AVH. Primary efficacy objective: • To assess the percentage of subjects who normalize their SGOT, SGPT, ALP, Sr. Albumin, globulin, Prothrombin time. Secondary efficacy Objective: • To assess the percentage of subjects in each group who show • Symptomatic improvement- Assess quality of life (QOL) - physical examinations and standardised questionnaire. • Differences in response in different AVH etiologies (i.e. HAV, HBV, HCV, HEV) using subgroup analyses. • Total duration of recovery 75
  75. 75. 76 Phase IV  Post marketing survelliance  ADR monitoring  Rare but serious adverse reactions or chronic toxicity  Drug interactions
  76. 76. 77

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