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Hepatic Micrisomal Enzyme System

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CYP, UGT, Enzyme induction / inhibition, Clinical relevance

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Hepatic Micrisomal Enzyme System

  1. 1. Dr. Ajinkya Rodge JR-1 Dept Of Pharmacology TNMC, Mumbai Date : 17th September 2016
  2. 2. Concepts Biotransformation Microsomal Enzymes Cytochrome P450 Enzymes UDP Glucuronosyl Transferases Clinical Relevance Of Drug Metabolism Conclusion
  3. 3. • 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
  4. 4. • 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
  5. 5. Biotransformation of drugs may lead to the following- A. Active drug  Inactive metabolite Eg. 1) Lidocaine 2) Ibuprofen 3) Chloramphenicol 4) Propranolol
  6. 6. Biotransformation of drugs may lead to the following- B. Active drug  Active metabolite
  7. 7. Biotransformation of drugs may lead to the following- C. Inactive drug (Prodrug)  Active metabolite
  8. 8. PHASE I Reactions PHASE II Reactions Reactions • Nonsynthetic / Functionalisation Reactions • Synthetic / Conjugation Reactions Metabolite • Active or Inactive • Mostly Inactive
  9. 9. PHASE-I PHASE-II • Oxidation • Reduction • Hydrolysis • Cyclisation • Decyclisation • Glucoronidation • Acetylation • Methylation • Sulfate conjugation • Glycine conjugation • Glutathione conjugation • Ribonucleoside/Nucleotide synthesis
  10. 10. Liver Intestine Kidney Brain Lung Plasma
  11. 11. 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
  12. 12. • Located in Endoplasmic Reticulum • Microsomes – ER isolated by homogenisation & fractionation of cell reform into vesicles known as Microsomes
  13. 13. • Flavin Mono Oxygenases • Cytochrome P450 • UDP Glucoronosyl Transferases (UGT) • Glutathione-S-Transferases • Epoxide Hydrolases • Carboxyl Esterases
  14. 14. • 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
  15. 15. • 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 )
  16. 16. • 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
  17. 17. • About 100 different isoforms identified in humans • Using Gene arrays, immunoblotting analyses, selective functional markers & P450 inhibitors • Divided into Families & Subfamilies
  18. 18. CYP 3 A 4 FAMILY INDIVIDUAL ENZYME SUB-FAMILY
  19. 19. • 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
  20. 20. 50% 20%
  21. 21. Some P450 substrate drugs ↓ Repeated administration ↓ Induce P450 expression ↓ ↑ Synthesis / ↑ Expression Or ↓ Degradation / Enzyme stabilisation
  22. 22. Enzyme induction ↓ ↑ Substrate metabolism ↓ ↑ Non-active metabolite or ↑ Reactive metabolism ↓ ↓ ↓ Pharmacological action ↑ Pharmacological action / toxic effects
  23. 23. A) 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
  24. 24. 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
  25. 25. B) Enzyme Stabilisation/↓ Degradation Inducer binds to active site of enzyme ↓ Enzyme stabilised ↓ ↓ Degradation of enzyme ↓ Accumulation of enzyme ↓ ↑ Enzyme activity
  26. 26. • 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
  27. 27. • 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
  28. 28. • 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
  29. 29. • 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
  30. 30. • 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
  31. 31. • 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
  32. 32. Competitive inhibition of co- administered drugs Suicide inhibition
  33. 33. A) 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
  34. 34. B) Suicide Inhibition Drug Metabolised by P450 Reactive Intermediate Covalent interaction with heme moiety Irreversible inhibition of the metabolising P450 Inhibit the metabolism of- Eg. • Chloramphenicol – 2B1 • Phencyclidine – 2B6 • Clopidogrel – 2B6 • Ritonavir – 3A4, 2C19
  35. 35. 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)
  36. 36. 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
  37. 37. Substrates • SSRI’s • Flecainide • Propafenone • TCA’s • Haloperidol • ẞ Agonists • Codeine Inducers • Not known Inhibitors • Quinidine • Amiodarone • SSRI’s • Haloperidol • Clomipramine
  38. 38. Due to CYP 2D6 inhibition – • Inhibition by Quinidine  No pain relief with Codeine • Adverse effects of TCA’s are increased
  39. 39. Substrates • Celecoxib • Diclofenac • Ibuprofen • Flurbiprofen • S-Warfarin • Losartan • Phenytoin • Glimepiride Inducers • Barbiturates • Carbamazepine • Rifampicin Inhibitors • Fluconazole • Miconazole • Amiodarone • Phenylbutazone
  40. 40. 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
  41. 41. Substrates • Omeprazole • Lansoprazole • Diazepam • Phenytoin • Naproxen • Propranolol Inducers • Barbiturates • Carbamazepine • 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
  42. 42. 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
  43. 43. Substrates • Theophylline • Caffeine • Paracetamol • Carbamazepine • R-Warfarin Inducers • Smoking • Charcoal-broiled meat • Rifampicin • Carbamazepine Inhibitors • Fluvoxamine o Compared to its drug metabolising role, its role in activation of procarcinogens is more important
  44. 44. • 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
  45. 45. • 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
  46. 46. • 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
  47. 47. Substrates – • Endogenous compounds – Bilirubin, Steroid hormones, Thyroxine • Drugs - Chloramphenicol, Aspirin, Paracetamol, Diazepam, Lorazepam, Morphine, Metronidazole, Digitoxin
  48. 48. • 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
  49. 49. • 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
  50. 50. • 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
  51. 51. • Found in both ER & cytosol • 20 isoforms identified • Microsomal Forms – Metabolism of endogenous leukotrienes & prostaglandins • Cytosolic forms - conjugation, reduction, isomerization reactions of drug metabolism
  52. 52. • 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
  53. 53. Genetic Factors – • Genetic Polymorphism i. Phase I Enzyme Polymorphism ii. Phase II Enyme Polymorphism Non-genetic Factors - • Commensal gut microbiota • Diet & environmental factors • Age & sex • Concurrent exposure to inhibitors or inducers • Diseases
  54. 54. 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
  55. 55. Genetic Polymorphism – • Based on Metabolic Ratio, individuals are divided into – 1) Poor metabolisers (PM) 2) Extensive metabolisers (EM) 3) 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]
  56. 56. P450 genetic polymorphism – • Three P450 genetic polymorphisms have been particularly well characterized – 1) CYP 2D6 2) CYP 2C19 3) CYP 2C9
  57. 57. • Debrisoquin-Spartein Oxidation type of polymorphism • PM – 3-10% Caucasians • UM – 33% Ethiopians & Saudi Arabians • Inherited as autosomal recessive trait • Faulty expression of the P450 protein due to either defective mRNA splicing or protein folding • CYP2D6 dependent oxidations of debrisoquin and other drugs are impaired
  58. 58. • In PM - ↓ CYP2D6-dependent metabolic activation of Tamoxifen to Endoxifen  ↑ relapse in breast cancer • In UM – i. ↓ plasma levels of Nortriptyline - No therapeutic effect, ↑suicide rates ii. ↑ prodrug Codeine  Morphine - ↑ s/e of Morphine, morphine-induced death of breast-fed infant of mother taking excess Codeine
  59. 59. • 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
  60. 60. 1) 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 2) PM phenotype can significantly improve therapeutic efficacy of Omeprazole in gastric ulcer & GERD
  61. 61. 3) 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
  62. 62. • 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
  63. 63. • > 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
  64. 64. Gilbert’s Syndrome – • Most 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
  65. 65. 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 – i. Increased levels of drugs metabolised in intestine, Eg. Digoxin ii. Decreased entero-hepatic circulation of glucuronidated drugs
  66. 66. 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
  67. 67. 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
  68. 68. 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
  69. 69. 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
  70. 70. • 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

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