Hepatic Micrisomal Enzyme System

CYP, UGT, Enzyme induction / inhibition, Clinical relevance

1. Dr. Ajinkya Rodge JR-1 Dept Of Pharmacology TNMC, Mumbai Date : 17 th September 2016 Hepatic Microsomal Enzyme System

2. OUTLINE

3. ENZYMES Biocatalysts synthesized by living cells Protein in nature (exception - RNA acting as ribozyme) Colloidal and thermolabile in character Specific in their action

4. XENOBIOTICS Substances absorbed across lungs or skin Ingested either – -Unintentionally as compounds present in food & drinks -Deliberately as drugs for therapeutic purposes Taken for recreational purposes

5. BIOTRANSFORMATION The processes by which biochemical reactions alter the drugs/ xenobiotics within the body Chemical alteration of the drugs in body Renders Non-polar(Lipid Soluble) compounds  Polar (Lipid Insoluble) So that they are not reabsorbed & are excreted

6. Biotransformation of drugs may lead to the following - Active drug  Inactive metabolite Eg . 1) Lidocaine 2) Ibuprofen 3) Chloramphenicol 4) Propranolol BIOTRANSFORMATION

7. Biotransformation of drugs may lead to the following - B. Active drug  Active metabolite BIOTRANSFORMATION

8. BIOTRANSFORMATION Biotransformation of drugs may lead to the following - C. Inactive drug ( Prodrug )  Active metabolite

9. PHASE I Reactions PHASE II Reactions Reactions Nonsynthetic / Functionalisation Reactions Synthetic / Conjugation Reactions Metabolite Active or Inactive Mostly Inactive BIOTRANSFORMATION

10. PHASE-I PHASE-II Oxidation Reduction Hydrolysis Cyclisation Decyclisation Glucoronidation Acetylation Methylation Sulfate conjugation Glycine conjugation Glutathione conjugation Ribonucleoside /Nucleotide synthesis BIOTRANSFORMATION

11. BIOTRANSFORMATION

12. XENOBIOTIC METABOLIZING ENZYMES ENZYMES REACTIONS Phase I “ Oxygenases "     Cytochrome P450s (P450 or CYP) C & O oxidation, dealkylation , Flavin -containing monooxygenases (FMO) N, S, and P oxidation Epoxide hydrolases ( mEH , sEH ) Hydrolysis of epoxides Phase II “ Transferases "     Sulfotransferases (SULT) Addition of sulfate UDP- glucuronosyltransferases (UGT) Addition of glucuronic acid Glutathione-S- transferases (GST) Addition of glutathione N- acetyltransferases (NAT)  Addition of acetyl group Methyltransferases (MT) Addition of methyl group Other enzymes     Alcohol dehydrogenases Reduction of alcohols Aldehyde dehydrogenases Reduction of aldehydes

13. HEPATIC MICROSOMAL ENZYMES

14. MICROSOMAL ENZYMES Located in Endoplasmic Reticulum Microsomes – ER isolated by homogenisation & fractionation of cell reform into vesicles known as Microsomes

15. Flavin Mono Oxygenases Cytochrome P450 UDP Glucoronosyl Transferases (UGT) Glutathione-S- Transferases Epoxide Hydrolases Carboxyl Esterases MICROSOMAL ENZYMES

16. CYTOCHROME P450 ENZYMES Abbreviated as CYP or P450 The CYPs are a superfamily of enzymes, all of which contain a molecule of heme non-covalently bound to the polypeptide chain  Hemoproteins

17. CYTOCHROME P450 ENZYMES The term P450 because the reduced hemoprotein binds with CO to form a complex that absorbs light maximally at 450 nm Located in Endoplasmic Reticulum ( In its lipid bilayer )

18. CYP’s are involved in synthesis and metabolism of some endogenous substances Eg . Synthesis of steroid hormones, bile acids Metabolism of retinoic acid, fatty acids (PG’s & eicosanoids) CYP’s carry out the Oxidative reactions (Phase I) of the drug metabolism Along with CYP’s, microsomal drug oxidations also require- P450 reductase , NADPH, and molecular oxygen CYTOCHROME P450 ENZYMES

19. About 100 different isoforms identified in humans Using Gene arrays, immunoblotting analyses, selective functional markers & P450 inhibitors Divided into Families & Subfamilies CYTOCHROME P450 ENZYMES

20. CYP 3 A 4 NOMENCLATURE OF CYP 450 ENZYMES FAMILY INDIVIDUAL ENZYME SUB-FAMILY

21. CYP isoforms found in human liver are CYP1A2 CYP2A6, CYP2B6, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 CYP3A4, CYP3A5 CYP4A11 CYP7 The most active CYPs for drug metabolism are those in the CYP2C, CYP2D, and CYP3A sub-families CYTOCHROME P450 ENZYMES

22. Fraction of drugs metabolised by major Phase I enzymes 50% 20%

23. Some P450 substrate drugs ↓ Repeated administration ↓ Induce P450 expression ↓ ↑ Synthesis / ↑ Expression Or ↓ Degradation / Enzyme stabilisation ENZYME INDUCTION

24. Enzyme induction ↓ ↑ Substrate metabolism ↓ ↑ Non-active metabolite or ↑ Reactive metabolism ↓ ↓ ↓ Pharmacological action ↑ Pharmacological action / toxic effects ENZYME INDUCTION

25. Increased Expression Inducer drug binds a cytoplasmic/nuclear receptor ↓ Translocation of inducer-receptor complex to nucleus ↓ Dimerisation of complex with other nulclear receptor (RXR, Arnt ) ↓ This heterodimer binds to response elements in promoter regions of specific P450 genes ↓ Gene expression induced ↓ ↑ Enzyme levels  ↑ Enzyme expression ENZYME INDUCTION : MECHANISM

26. RECEPTORS INVOLVED IN ENZYME INDUCTION RECEPTOR LIGANDS ENZYME INDUCED Aryl hydrocarbon receptor (AHR) Omeprazole, Tobacco smoke, Charcoal broiled meat, Cruciferous vegetables CYP 1A1, 1A2 Constitutive androstane receptor (CAR) Phenobarbital CYP 2B6, 2C8, 2C9, 3A4 Pregnane X receptor (PXR) Rifampin , Hyperforin CYP 3A4 Peroxisome proliferator activated receptor (PPAR) Fibrates CYP 3A4

27. B) Enzyme Stabilisation/↓ Degradation Inducer binds to active site of enzyme ↓ Enzyme stabilised ↓ ↓ Degradation of enzyme ↓ A ccumulation of enzyme ↓ ↑ Enzyme activity ENZYME INDUCTION : MECHANISM

28. Also known as Ethanol type induction Inducers are known as Ethanol type inducers INDUCERS ENZYME INDUCED Ethanol CYP 2E1 Troleandomycin Clotrimazole CYP 3A4 Isosafrole CYP 1A2

29. Enzyme induction increases the rate of metabolism by 2-4 fold Reaches its peak by 4-14 days Maintained till inducing agent is administered Enzymes return to their original value over 1-3 weeks ENZYME INDUCTION

30. ENZYME INDUCTION : CONSEQUENCES Decreased intensity and/or duration of action of drugs inactivated by metabolism, eg . oral contraceptives failure Increased intensity of action of drugs activated by metabolism, eg . Acute paracetamol toxicity due to its metabolite N-acetyl-p- benzoquinoneimine (NAPQI) Tolerance due to auto induction , eg . carbamazepine, rifampin Endogenous substrates (steroids, bilirubin) are metabolized faster

31. Precipitation of acute intermittent porphyria : enzyme induction increases porphyrin synthesis Adjustment of dose of drugs taken regularly given with intermittent use of inducer drugs, eg . oral anticoagulants, oral hypoglycaemics , antiepileptics , antihypertensives Interference with chronic toxicity testing in animals ENZYME INDUCTION : CONSEQUENCES

32. Congenital nonhaemolytic jaundice due to deficient glucuronidation of bilirubin – Phenobarbitone hastens clearance of jaundice Cushing’s syndrome : phenytoin reduces manifestations by ↑ degradation of adrenal steroids produced in excess Chronic poisonings : by faster metabolism of the accumulated poisonous substance Liver disease ENZYME INDUCTION : USES

33. Enzyme inhibition takes place by action of inhibitor drugs directly on enzymes As the inhibitors act directly on the enzymes, it has a fast time course (within hours) compared to enzyme induction Inhibitor drugs functionally inactivate the enzymes ↓ Inhibition of drugs metabolism ↓ Toxicity of the object drug ENZYME INHIBITION

34. ENZYME INHIBITION : MECHANISM

35. Competitive Inhibition Of Co-administered Drugs Drugs or their metabolites tightly bind to P450 heme iron ↓ Competitive enzyme inhibition ↓ ↓ metabolism of co- administerd drugs 1. Cimetidine, Ketoconazole (Drugs themselves inhibit enzymes) 2. Macrolide antibiotics like Troleandomycin , Erythromycin (Drug metabolites inhibit Enzymes) 3. Proadifen (SKF-525 A) – Quasi-irreversibly inactivates enzyme

36. B) Suicide Inhibition Eg . Chloramphenicol – 2B1 Phencyclidine – 2B6 Clopidogrel – 2B6 Ritonavir – 3A4, 2C19

37. CYP 3A4 Substrates Acetaminophen Ethinyl estradiol Terfenadine Astemizole Cisapride Felodipine Erythromycin Statins Buspirone Quinidine Sildenafil Inducers Carbamazepine Phenytoin Phenobarbital Efavirenz Rifampicin Glucocorticoids St. John’s wort Inhibitors Antifungal Azoles Protease Inhibitors Macrolides (except Azithromycin) Cimetidine Grapefruit juice ( furanocoumarins )

38. Due to CYP 3A4 induction – Oral contraception failure St. John’s Wort reduce the plasma level of cyclosporin to subtherapeutic levels  rejection of a transplant St. John’s Wort also decreases the statin levels  raised cholesterol levels Due to CYP 3A4 inhibition – QT prolongation due to inhibition of metabolism of substrate drugs – Terfenadine , Astemizole , Cisapride Grapefruit juice - Hypotension due to inhibiton of metabolism of substrate drug Felodipine Dizziness & Serotonin syndrome due to ↑ levels of Buspirone DRUG INTERACTIONS

39. Substrates SSRI’s Flecainide Propafenone TCA’s Haloperidol ẞ Agonists Codeine Inducers Not known Inhibitors Quinidine Amiodarone SSRI’s Haloperidol Clomipramine CYP 2D6

40. Due to CYP 2D6 inhibition – Inhibition by Quinidine  No pain relief with Codeine Adverse effects of TCA’s are increased DRUG INTERACTIONS

41. CYP 2C9 Substrates Celecoxib Diclofenac Ibuprofen Flurbiprofen S-Warfarin Losartan Phenytoin Glimepiride Inducers Barbiturates Carbamazepine Rifampicin Inhibitors Fluconazole Miconazole Amiodarone Phenylbutazone

42. DRUG INTERACTIONS Due to CYP 2C9 induction – Doses of substrate drugs like phenytoin, losartan, glimepiride need to be increased Due to CYP 2C9 inhibition – Risk of bleeding due to enhanced Warfarin activity

43. CYP 2C19 Substrates Omeprazole Lansoprazole Diazepam Phenytoin Naproxen Propranolol Inducers Barbiturates C arbamazepine Rifampin Inhibitors Omeprazole Fluoxetine Ritonavir Sertraline Drug interactions : Inhibitors like Ketoconazole, INH, Omeprazole co-administered with substrate drugs like Anticonvulsants, Diazepam, TCA, Omeprazole leads to more ADR’s of substrate drugs

44. CYP 2E1 Substrates Ethanol Halothane Paracetamol Inducers Ethanol INH Inhibitors Disulfiram Drug interactions : Induction of enzyme by alcohol leads to increased formation of N-acetyl-p- benzoquinoneimine (NAPQI ), hepatotoxic metabolite of Paracetamol

45. CYP 1A Substrates Theophylline Caffeine P aracetamol Carbamazepine R-Warfarin Inducers Smoking Charcoal-broiled meat Rifampicin Carbamazepine Inhibitors Fluvoxamine Compared to its drug metabolising role, its role in activation of procarcinogens is more important

46. UDP GLUCURONOSYL TRANSFERASES (UGT’s) Catalyse the Phase II reaction – ‘ Glucuronidation ’ Glucuronidation is the only conjugation reaction that takes place in ER (other conjugation  in cytosol) Substrate drug Glucuronide metabolite

47. Glucuronides excreted via – 1. Kidney in urine (minor) 2. Intestines with bile (major) Glucuronides are cleaved by ẞ- Glucuronidase which is found in bacteria of lower GI tract  Enterohepatic circulation of drugs Eg . OCP’s UGT’s

48. ENTEROHEPATIC CIRCULATION

49. UGT proteins encoded by 19 human genes UGT 1 locus  Chr. 2  9 Genes UGT 2 locus  Chr. 4  10 Genes UGT 1 Family  Drug’s metabolism UGT 2 Family  Endogenous substances metabolism UGT’s

50. Substrates – Endogenous compounds – Bilirubin, Steroid hormones, Thyroxine Drugs - Chloramphenicol, Aspirin, Paracetamol , Diazepam, Lorazepam , Morphine, Metronidazole, Digitoxin UGT’s

51. Involved in Phase I reactions (minor contribution) 6 families of FMO’s are present FMO3 is the most abundant in human liver Substrates -Nicotine, Cimetidine, Ranitidine, Clozapine FMOs are not induced or inhibited by any clinically used drugs ↓ Not involved in drug-drug interactions FLAVIN MONO OXYGENASES

52. Carry out hydrolysis of epoxides, most of which are produced by CYPs Types – Soluble Epoxide Hydrolase ( sEH ) Microsomal Epoxide Hydrolase ( mEH ) mEH metabolises very few drugs Substrate – Carbamazepine Inhibitor - Valnoctamide, Valproic acid Drug interaction – mEH inhibition  Toxic effects of Carbamazepine EPOXIDE HYDROLASES (EH)

53. Found in both ER & cytosol Catalyse the hydrolysis of ester- and amide-containing chemicals Detoxification or Metabolic activation - drugs , environmental toxicants, carcinogens Eg . Chemotherapeutic Prodrug Irinotecan  Active drug SN-38 CARBOXYL ESTERASES

54. Found in both ER & cytosol 20 isoforms identified Microsomal Forms – Metabolism of endogenous leukotrienes & prostaglandins Cytosolic forms - conjugation, reduction, isomerization reactions of drug metabolism GLUTATHIONE – S – TRANSFERASES (GST’s)

55. Dose and frequency of administration required to achieve effective therapeutic levels vary in individuals Individual differences in rates of drug metabolism Depend on genetic and non-genetic factors CLINICAL RELEVANCE

56. Genetic Factors – Genetic Polymorphism Phase I Enzyme Polymorphism Phase II Enyme Polymorphism Non-genetic Factors - Commensal gut microbiota Diet & environmental factors Age & sex Concurrent exposure to inhibitors or inducers Diseases FACTORS AFFECTING DRUG METABOLISM

57. Genetic Polymorphism – Definition - occurrence of a variant allele of a gene at a population frequency of ≥ 1%, resulting in altered expression or functional activity of the gene product, or both Clinically significant genetic polymorphisms seen in both phase I & II drug metabolising enzymes ↑/↓ catalytic activity of enzyme Results in altered efficacy of drug therapy or adverse drug reactions GENETIC FACTORS

58. Genetic Polymorphism – Based on Metabolic Ratio, individuals are divided into – Poor metabolisers (PM) Extensive metabolisers (EM) Ultra rapid metabolisers (UM) [Metabolic Ratio: defined as percent of dose excreted as unchanged drug divided by the percent of dose excreted as metabolite in urine collected over a time period after oral ingestion of drug] GENETIC FACTORS

59. P450 genetic polymorphism – Three P450 genetic polymorphisms have been particularly well characterized – CYP 2D6 CYP 2C19 CYP 2C9 PHASE I ENZYME POLYMORPHISM

60. Debrisoquin-Spartein Oxidation type of polymorphism PM – 3-10% Caucasians UM – 33% Ethiopians & Saudi Arabians Inherited as autosomal recessive trait F aulty expression of the P450 protein due to either defective mRNA splicing or protein folding CYP2D6 dependent oxidations of debrisoquin and other drugs are impaired CYP2D6 POLYMORPHISM

61. In PM - ↓ CYP2D6-dependent metabolic activation of Tamoxifen to Endoxifen  ↑ relapse in breast cancer In UM – ↓ plasma levels of Nortriptyline - No therapeutic effect, ↑suicide rates ↑ prodrug Codeine  Morphine - ↑ s/e of Morphine, morphine-induced death of breast-fed infant of mother taking excess Codeine CYP 2D6 POLYMORPHISM

62. 3–5% Caucasians and 18–23% Japanese Inherited as autosomal recessive trait Independent of CYP 2D6 polymorphism PM genotype – Due to splicing defects, Allelic variants - CYP2C19*2, CYP2C19*3 EM genotype – Due to increased transcription, Allelic variant – CYP2C19*17 CYP 2C19 POLYMORPHISM

63. S- Mephenytoin  Hydroxylation  Glucuronidation  Inactive metabolite excreted in urine R- Mephenytoin  N-demethylation  Active metabolite Nirvanol In PM’s - Hydroxylation of S- Mephenytoin ↓ N- demethylation to Nirvanol ↑ ↑ adverse effects like sedation, ataxia PM phenotype can significantly improve therapeutic efficacy of Omeprazole in gastric ulcer & GERD CYP 2C19 POLYMORPHISM

64. EM’s with allele CYP2C19*17 – higher expression and higher function Higher metabolic activation of prodrugs – - Tamoxifen ↓relapse of breast cancer - Clopidogrel ↑risk of bleeding Higher elimination of drugs – -Antidepressants like Imipramine -Antifungals like Voriconazole CYP 2C19 POLYMORPHISM

65. Alleles encode single amino acid mutations which are responsible for altered metabolic activity of the enzyme CYP2C9*2 ↔ Arg144Cys mutation CYP2C9*3 ↔ Ile359Leu mutation Individuals with these mutations have lower tolerance for Warfarin  adverse effect like bleeding Also low tolerance for drugs like Phenytoin, Losartan CYP 2C9 POLYMORPHISM

66. > 50 genetic lesions in UGT1A1 gene Lead to inheritable unconjugated hyperbilirubinemia – Crigler-Najjar syndrome I (AR)- Complete absence of bilirubin glucuronidation Crigler-Najjar syndrome II (AR)- Decreased bilirubin glucuronidation Gilbert’s syndrome (AD)- circulating bilirubin levels are 60-70% higher than normal subjects UGT POLYMORPHISM

67. Gilbert’s Syndrome – M ost common genetic polymorphism - mutation in the UGT1A1 gene promoter, UGT1A1*28 allele Patients may be predisposed to ADRs resulting from a reduced capacity of UGT1A1 to metabolize drugs – Toxicities of Irinotecan , Atazanavir UGT POLYMORPHISM

68. Commensal Gut Microbiota – Metabolism of drug by intestinal microorganisms – nonoxidative , predominantly reductive and hydrolytic reactions; decarboxylation , dehydroxylation , dealkylation , dehalogenation , and deamination Co-treatment with antibiotics like erythromycin, tetracycline leads to death of commensal bacteria ↓bacterial enzymes; this leads to – Increased levels of drugs metabolised in intestine, Eg . Digoxin Decreased entero -hepatic circulation of glucuronidated drugs NON GENETIC FACTORS

69. Age – Increased susceptibility to the pharmacologic or toxic activity of drugs has been reported in very young & very old patients compared with young adults Differences in absorption, distribution, excretion and drug metabolism play a role Both Microsomal & Non-microsomal enzymes deficient in newborns More susceptible to drugs like chloramphenicol , opiods Sulfate conjugation is rather well developed in the newborn Glururonide conjugation and oxidation occurs at a lower rate Paracetamol metabolised by Sulfate conjugation in newborn but by Glucuronide conjugation in adults NON GENETIC FACTORS

70. Sex – Sex-dependent variations in drug metabolism well documented in rats Young adult male rats metabolize drugs much faster than mature female rats or prepubertal male rats Sex-dependent differences in drug metabolism exist in humans for ethanol, propranolol, some benzodiazepines, estrogens , and salicylates NON GENETIC FACTORS

71. Diet & Environmental Factors – Charcoal-broiled meat, Smoking, Cruciferous vegetables – Inducers Grapefruit Juice – Inhibitor Alcohol - Few days of relatively high doses of Alcohol cause inhibition of various CYP 450 enzymes - Regular long term intake (50 gms /day) cause induction of these CYP 450 enzymes Industrial workers exposed to some pesticides metabolize certain drugs more rapidly than unexposed individuals NON GENETIC FACTORS

72. Diseases – Acute & chronic liver diseases affecting liver architecture or function impare hepatic metabolism of drugs Alcoholic hepatitis, active or inactive alcoholic cirrhosis, hemochromatosis , chronic active hepatitis, biliary cirrhosis, viral hepatitis In liver, metabolism of drugs Propranolol, Verapamil, Amitriptyline, Isoniazid, Lidocaine etc , is blood-flow limited  metabolism impaired in cardiac diseases NON GENETIC FACTORS

73. Understanding drug metabolism & drug interactions within the body allows principles of biotransformation to be applied in better designing & therapeutic usage of drugs Increased understanding of biotransformation based on pharmacogenomics will also render pharmacologic treatment of disease more individualised, efficacious and safe CONCLUSION

74. Thank You