Hepatoprotective agents

Drugs protecting Liver, its mechanism of action and its utility.

1. Hepatoprotective agents Dr Priyank shah, Jr 1, Dept of pharmacology 1

2. Introduction Liver is one of the largest organs in human body and the chief site for intense metabolism and excretion. 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. 2

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4. Liver Toxicity The main causes of liver damage are The major 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 CCL 4 , phosphorous , aflatoxins, chlorinated hydrocarbon etc Drugs i.e. DILI ( drugs induced liver injury ) Autoimmune disorders Infections like viral hepatitis 4

5. Drugs Liver Disease Drug Metabolites (the good, the bad and the ugly) Drug Elimination Drugs and the Liver Drug-Drug Interactions LIVER 5

6. Drugs causing DILI Anti tuberculosis drugs All drugs causes hepatotoxicity except ethambutol Anti convulsant drugs Carbamazepine and 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 6

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8. Clinical Presentations Asymptomatic elevation in hepatic enzymes No progress despite Continued use of the Medication. (Drug tolerance) INH Phenytoin Chlopromazine Progression to Hepatic injury with Continued use of the medication AST & ALT 3-5 times Upper limit of normal May progress to Hepatic failure 8

9. Acute Hepatocellular Injury by Drugs Characterized by Marked elevation in ALT and AST Normal or minimally elevated alkaline phosphatase Bilirubin variably increased-----›worse prognosis. Comprise 1/3 of all cases of fulminant hepatic failure in the US. 20% due to Acetominophen 12%-15% due to other drugs 9

10. Acute Hepatocellular Injury Alcohol AST is always 2-3 times higher than ALT AST remains less than 300 IU. ALT is almost always less than 100 IU. Towering elevation of ALT&AST(5000-10000 IU) Drugs (acetaminophen) Differential: Chemical toxins Toxic Mushrooms Shock liver Unusual with other causes of liver diseases including Viral Hepatitis. 10

11. Mechanism of Hepatotoxicity 11

12. 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 12

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15. Mechanism of hepatotoxicity Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid per oxidation and other oxidative damages in liver. By forming the reactive free oxygen radicals which directly induces hepatotoxicity Increasing the apoptosis Reducing Glutathione stores an antioxidant of human body 15

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17. DILI 17

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19. Hepatoprotective agents 19

20. List of Hepatoprotective agents 20 N acetylcysteine vitamins Penicillamine Anti oxidants melatonin cardiotropin 1 glutathione Herbal medications beta carotene S adenosyl methionine (SAM) many more

21. N acetyl cysteine 21

22. Description N-acetyl derivative of L-cysteine Pharmacologic category Antidote Mucolytic 22

23. Indications 23 FDA labeled indications Acetaminophen (APAP) overdose Adjunctive mucolytic therapy Diagnostic bronchial studies Off-label use Prevention of contrast-induced nephrotoxicity (CIN) Helicobacter pylori infection

24. Pathophysiology of APAP Overdose 24 APAP primarily metabolized via Glucoronidation or sulphation Secondary metabolism by CYP 450 system In OD, primary route saturated → CYP 450 system → NAPQI production NAPQI converted to non-toxic form by glutathione In OD, glutathione stores consumed → excess NAPQI → covalent binding to hepatocytes

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26. 26 NAPQI causes Protein binding cytoplasm loss /damage of protein Apoptosis pro apoptotic factors 2. Formation ROS lipid peroxidation Liver necrosis DNA damage

27. 27 N-acetylcysteine (NAC) has been used for several decades and has proven to be the antidote of choice in treating acetaminophen-induced hepatotoxicity. oral and intravenous NAC are equally efficacious in the prevention of hepatotoxicity. An important factor in assessing the efficacy of NAC is the timing of therapy initiation in relation to the ingestion. Pt should receive NAC within 8hours of APAP poisoning.

28. Metabolism of NAC N-Acetylcysteine 12/2002 Society For Free Radical Biology and Medicine Ercal & Gurer-Orhan 28 Oral NAC administration Extensive first-pass metabolism in liver and intestine 3% of NAC excreted in feces Rapid absorption LIVER INTESTINE

29. 29 N Acetyl cysteine Prevents hepatic injury primarily by restoring hepatic glutathione  N acetyl cysteine improves hemodynamics and oxygen use,  Decreases cerebral edema. It may involve scavenging of free radicals or changes in hepatic blood flow

30. Adverse Reactions Oral Drowsiness Chills/fever Nausea / Vomiting Bronchospasm Rhinorrhea Unpleasant odor IV Anaphylactoid reactions Vomiting 30

31. Penicillamine 31

32. 32 Penicillamine is a degradation product of penicillin but has no antimicrobial activity. It was first isolated in 1953 from the urine of a patient with liver disease who was receiving penicillin.

33. Mechanism of action 33 Penicillamine chelates several metals including copper, lead, iron, and mercury, forming stable water soluble complexes that are renally excreted. It also combines chemically with cystine to form a stable, soluble, readily excreted complex. It may also have antifibrotic effects as it inhibits lysyl oxidase , an enzyme necessary for collagen production.

34. 34 It also directly binds to collagen fibrils, preventing cross-linking into stable collagen fibers . Penicillamine may have immunomodulatory effects and has been demonstrated to reduce IgM rheumatoid factor in humans with rheumatoid arthritis.

35. S Adenosyl Metheonine (SAM) 35

36. Methionine & SAMe Methionine is an essential amino acid that is primarily metabolized in the liver. SAM biosynthesis is the first step in methionine metabolism in a reaction catalyzed by methionine adenosyltransferase (MAT) In mammals, this reaction in the liver catabolizes nearly half of the daily intake of methionine The liver is the main source of SAM biosynthesis and metabolism, turning over nearly 8 gm per day in a normal adult SAM is used in transmethylation reactions and is converted to S-adenosylhomocysteine (SAH)

37. Hepatic methonine metabolism and SAME biosynthesis

38. S Adenosyl Methionine SAM is the principal biological methyl donor required for methylation of DNA, RNA, biogenic amines, phospholipids, histones, and other proteins It is the precursor for synthesis of polyamines, which are required for cell proliferation and the maintenance of cell viability In the liver, SAM is a precursor for glutathione, a major endogenous antioxidant that protects cells against injury by scavenging free radicals. Thus, SAM deficiency can impair many vital functions of liver, which render it susceptible to injury by toxic agents, including alcohol

39. SAMe (S-adenosylmethionine) SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death

40. SAM functions as an antioxidant Additionally, SAM functions as an antioxidant not only because of its role as a precursor for GSH biosynthesis, but also because of its capacity to interact directly with reactive oxygen species Evidence to support this role comes from studies on the capacity of SAM to diminish ischemia/ reperfusion injuries in clinical trials during liver transplantation These studies demonstrated that SAM was more effective than GSH in scavenging hydroxyl radicals and in chelating iron ions to inhibit generation of these radicals and, unlike GSH, SAM did not stimulate formation of scavenging hydroxyl radicals at lower concentrations

41. Other mechanisms 41 Abnormal inflammatory cytokine metabolism is a major feature of both alcoholic and non-alcoholic steatohepatitis. Apoptotic cell death of hepatocytes is emerging as an important mechanism contributing to the progression of human liver diseases. SAM effectively reduces Apoptosis and inflammatory cytokines.

42. Therapeutic potential of SAM in ALD The therapeutic potential of SAM was tested in a 24-month randomized, placebo-controlled, double-blind, multicenter clinical trial in patients with alcoholic cirrhosis SAM treatment improved survival or delayed the need for liver transplantation in alcoholic liver cirrhosis, especially those with less advanced liver disease Mechanism of protection was not investigated in this trial, increased hepatic concentrations of glutathione may have contributed to beneficial effect of SAM This notion is supported by another study in which oral 1.2 g SAM/d for 6 months significantly increased hepatic glutathione concentrations in ALD patients Authors evaluated whether SAMe infusion improved methionine clearance in patients with stable ALD

43. Herbal medication 43

44. List of herbal medication used in hepatic diseases 44 Silybum marinum Eclipta alba Foeniculum vulgare Trigonella foenum graecum Jatropha curcas Garcinia mangostana Linn Chamomile capitula

45. Silybum marianum 45 Silybum marianum, commonly known as ‘milk thistle ’ (Family: Asteraceae/Compositae) is one of the oldest and thoroughly researched plants in the treatment of liver diseases. The extracts of milk thistle is being used as a general medicinal herb from as early as 4 th century B.C. and first reported by Theophrastus. Silymarin, a single herbal drug formulation which is mostly used in liver diseases amounts to about 240 million US dollars in Germany alone.

46. 46 Silymarin is a complex mixture of four flavonolignan isomers, namely silybin, isosilybin, silydianin and silychristin with an empirical formula C 25 H 22 O 10 Among the isomers silybin is the major and most active component and represents about 60-70 per cent, followed by silychristin (20%), silydianin (10%), and isosilybin (5%). The structural similarity of silymarin to steroid hormones is believed to be responsible for its protein synthesis facilitatory actions

47. Mechanism of action 47 Stimulation of protein synthesis:- Silymarin can enter inside the nucleus and act on RNA polymerase enzymes resulting in increased ribosomal formation. This in turn hastens protein and DNA synthesis. This action has important therapeutic implications in the repair of damaged hepatocytes and restoration of normal functions of liver.

48. 48 Anti-inflammatory actions: The inhibitory effect on 5-lipoxygenase pathway resulting in inhibition of leukotriene synthesis is a pivotal pharmacological property of silymarin. Strong inhibitory effect on LTB 4 but not on TNF alpha or on prostaglandin formation In vivo study in mice treated with silymarin suggested that parenteral exposure to silymarin results in suppression of T-lymphocytes at low doses and stimulation of inflammatory process at higher doses.

49. 49 Antifibrotic action: Liver fibrosis can result in remodeling of liver architecture leading to hepatic insufficiency, portal hypertension and hepatic encephalopathy. The conversion of hepatic stellate cells (HSC) into myofibroblast is considered as the central event in fibrogenesis. Silymarin inhibits HSC activation It also inhibits protein kinases and other kinases involved in signal transduction and may interact with intracellular signaling pathways .

50. 50 Drug and toxin related liver damage : Hepatocellular injury due to drugs seems to be the primary event. This is rarely due to the drug itself and a toxic metabolite is usually responsible. The drug metabolizing enzymes activate chemically stable drugs to produce electrophilic metabolites. All this leads to reduction in glutathione stores, lipid peroxidation, and membrane damage.

51. 51 Silymarin has a regulatory action on cellular and mitochondrial membrane permeability in association with an increase in membrane stability against xenobiotic injury. prevent the absorption of toxins into the hepatocytes by occupying the binding sites as well as inhibiting many transport proteins at the membrane

52. 52 As an antioxidant : Free radicals, including the superoxide radical, hydroxyl radical (.OH), hydrogen peroxide (H2O2), and lipid peroxide radicals have been implicated in liver diseases. The cytoprotective effects of silymarin are mainly attributable to its antioxidant and free radical scavenging properties. Silymarin can also interact directly with cell membrane components to prevent any abnormalities in the content of lipid fraction responsible for maintaining normal fluidity .

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54. Adverse Drug Reactions 54 Silymarin is reported to have a very good safety profile. Both animal and human studies showed that silymarin is non toxic even when given at high doses (>1500 mg/day). gastrointestinal tract side effects like bloating, dyspepsia, nausea, irregular stool , diarrhoea It also produced pruritus, headache, exanthema, malaise, asthenia, and vertigo.

55. Liv 52 55 Key ingrediants The caper Bush ( Himsra ) It contains p- methoxy benzoic acid which is a potent Hepatoprotective. It prevents the elevation of malondialdehyde (biomarker for oxidative stress) It also inhibits the elevation of ALT and AST Chicory ( Kasani ) Potent antioxidant It suppresses the oxidative degradation of DNA

56. 56 Mechanism of action Potent Hepatoprotective against chemical induced hepatotoxicity It protects liver parenchyma and promotes hepatocellular regenration It maintains cytochrome p450, hastens the early recovery period and maintains functional integrity Rapid clearance of aldehyde and protects from alcohol liver disease

57. 57 Side effects Only mild GI symptoms bloating, dyspepsia, nausea, irregular stool , Diarrhoea When taken at prescribed dose it is totally side effects free

58. As a supplementary therapy Anti oxidants 58

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60. 60 The mechanism of free radical damage include ROS- induced peroxidation of polyunsaturated fatty acid in the cell membrane bilayer, which causes a chain reaction of lipid peroxidation, thus damaging the cellular membrane and causing further oxidation of membrane lipids and proteins. Subsequently cell contents including DNA, RNA, and other cellular components are damaged.

61. Antioxidants Prevents the transfer of electron from O 2 to organic molecules Stabilizes free radicals Terminates free radical reactions

62. Cardiotrophin 1 63

63. 64 Phase I study in healthy volunteers completed Orphan Drug status granted for solid organ transplantation and acute liver failure by FDA and EMA IP protection granted for liver diseases, and filed for solid organs transplantation and kidney diseases

64. 65 Cardiotrophin 1 (CT-1) belongs to the IL-6 family of cytokines. CT-1 receptor activation ( gp 130-LIF-R) induce cell survival through signal transduction mechanisms mediated by Stat3, MAPK y PI3K. CT-1 is induced in the context of cell damage in several tissues. Strong anti-apoptotic effects on hepatocytes . Reduce the cellular damage cause by ischemia/reperfusion. Decrease oxidative damage. Potent anti-inflammatory agent.

65. 66 Cardiotropin 1 has FDA and EMA orphan drug designation for transplantation and acute liver failure. It has been granted for its use in hepatic regenration .

66. Vaccines 67

67. HEPATITIS VACCINES HEPATITIS A Inactivated viral vaccine Not given < 1 year 2 doses, 6 – 18 months apart Combination Vaccine Inactivated Hep – A + Recombinant Hep – B 3 doses, 0, 1 & 6 months, > 1 year older.

68. HEPATITIS B rDNA – Yeast derived Vaccine Adults – 10 -20 mcg IM at 0, 1, 6 months < 10 yr – 5 – 10 mcg IM 95 % immunity till 40 yrs Not contraindicated in pregnancy For Immunodeficient : Recombivax HB(1 dose, 40 g ) Engerix B (2 dose 20 g ) New Vaccines Status HBc VLP+ cochleates, nasal - Phase I HBV polyepitope vaccine - Phase I DNA coding for T-cell epitopes - Phase I

69. HEPATITIS C No Vaccine yet available New Vaccine Status Vaccine Stage of Development Recombinant E1/E2 + MF59 subunit vaccine Phase I Recombinant E1glycopro-tein subunit vaccine Phase II HCcAg /E1/E2 VLPs Preclinical Multiepitope synthetic peptides in virosomes Preclinical Multigene recombinant Adenovirus Preclinical Live recombinant MVA-NS3-NS4-NS5 antigens vaccine Preclinical

70. HEPATITIS E New Vaccines Status Vaccine Stage of Development 56 kD ORF-2 protein VLPs ( baculovirus ) Phase II/III DNA vaccine (ORF-2) Preclinical

71. Evaluation of hepatoprotective drugs 72

72. In vitro metho Hepatocytes are isolated and placed in HEPES (N-2-hydroxyethylpiperazine – N- 2 ethanesulphonic acid) Hepatocytes are exposed to test samples and toxins CCL4, alcohol or paracetamol. Degree of protection is assesed by viability tests (Trypan blue dye exclusion method) and measurement of enzymes levels. Ex vivo method After completion of in vivo test, hepatocytes are isolated and % of viable cells and biochemical parameters are determined Better correlated to clinical models than in vitro or in vivo 73

73. 74 Paracetamol model Ethanol model Ccl4 model Thioacetamide model D- galactosamine model Experimental rat models

74. Future direction 75

75. 76 Hepatotoxicity will remain clinical and controlling significance. With increasing the incidence of hepatotoxicity the need for new , potent and efficacious Hepatoprotective agents arises.

76. NAC for anti TB hepatotoxicity 77

77. NAC for statins 78

78. 79 It also showed Hepatoprotective activity against Carabamazepine All in preclinical phase N-N dimethylformaide Anti retroviral agents

79. 80 Cannabinoids A very attractive target for modulating hepatic fibrosis. CB1 receptor – up regulated in myelofibroblast and promote fibrosis CB2 receptor – antifibrotic but may amplify inflammation. As a result of these findings, CB1 antagonism is a more promising strategy than CB2 agonism

80. 81 Rimonabant CB1 antagonist showed significant weight loss and improved metabolic function, decreased triglycerides and improved insulin resistance. But it lead to increased neuropsychiatric complication so was withdrawn from market. As a result, current efforts are directed towards CB1 antagonists that do not cross the bloodbrain barrier .

81. 82 Endothelin (ET1) receptor antagonist High levels of ET-1 and endothelin receptors are present during cirrhosis. The blockade of ET-1 type A receptor and the administration of vasodilators (prostaglandins E2 and NO donor) exert an antifibrotic activity in rodents and in humans also improves portal hypertension.

82. Hormones 83 Adipokines Metabolic abnormalities are considered the ‘first hit ’ of liver injury in obesity-related liver disease, followed by oxidative stress and inflammation. Leptin and its natural antagonist adiponectin are key adipokines secreted by adipose tissue and stromal cells, especially HSC. Elevated leptin levels signal through their specific receptors to promote fibrogenesis by JAK/STAT signalling. While adipokines are inversely related to body fat and inhibit inflammation Gherlin ( a gut hormone) has also showed anti inflammatory activity in rats

83. Tyrosine kinase receptors inhibitors 84 Many proliferative cytokines, including PDGF, fibroblast growth factor (FGF), and TGF-a signal through tyrosine kinase receptors. Antagonism of pathways that mediate PDGF or VEGF signals reduces HSC proliferation. Sorafenib, a multiple receptor tyrosine kinase inhibitor approved for therapy in hepatocellular carcinoma, targets the PDGF receptor and Raf/ERK signaling pathways

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86. References 87 Manouchehr Khoshbaten et.al, N- Acetylcysteine Improves Liver Function in Patients with Non-Alcoholic Fatty Liver Disease, Hepatitis Monthly 2010; 10(1): 12-16. Abajo F.J., Montero D., Madurga M ., Garcia Rodriguez L.A. Acute and clinically relevant drug-induced liver injury: a population based case–control study. Br J Clin Pharmacol . 2004; 58: 71–80. Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 2004; 5: 836-847 Taniguchi M, Takeuchi T, Nakatsuka R, Watanabe T, Sato K. Molecular process in acute liver injury and regeneration induced by carbon tetrachloride. Life Sci 2004; 75: 1539-1549. Natalia A Osna , Hepatoprotective effects of S - adenosyl -L- methionine against alcohol- and cytochrome P450 2E1-induced liver injury; World J Gastroenterol 2010 March 21; 16(11): 1366-1376 Lieber CS. S- adenosyl -L- methionine : its role in the treatment of liver disorders. Am J Clin Nutr 2002; 76: 1183S-1187S. S- adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol Ther 1997; 73: 265-280 www.dignabiotech.com Sharma SK, Ali M and Gupta J. Phytochemistry and Pharmacology, 2002; 2: 253-70. S . Luper , (1998).A review of plants used in the treatment of liver diseases: Part 1, Altren Med Rev 3: 410-421.

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