CYTOCHROME P450 for physicians....a ppt. that give you a brief overview about the much talked about enzyme system.

1. Cytochrome P450 The P450 enzymes have unique spectral properties, and the reduced forms combine with carbon monoxide to form a “PINK” compound (hence 'P') with absorption peaks near 450nm (range 447-452nm). Dr. Pavitra Raj Dewda 1

2. Dr. Pavitra Raj Dewda 2 Contents

3. 1.BIOTRANSFORMATION What is it? What are the Components? What is the Process? Dr. Pavitra Raj Dewda 3 xenobiotics metabolism substances absorbed across the lungs or skin or, more commonly, ingested either unintentionally as compounds present in food and drink or deliberately as drugs for therapeutic or "recreational" purposes. Organs of Biotransformation LIVER (hepatocytes) kidney (proximal tubule) lungs (type II cells) testes (Sertoli cells) skin (epithelial cells) intestines (microbial flora)

4. Dr. Pavitra Raj Dewda 4

5. Dr. Pavitra Raj Dewda 5

6. Clinical CorrelateMetabolites can alter the pharmacological action of drug qualitatively TerfenadineFexofenadine FluoxetineNorfluoxetine Dr. Pavitra Raj Dewda 6 METABOLITE CYP3A4 Blocks histamine H 1 receptors only & not cardiac K+ channels Non sedating antihistaminic Can Block K+ Channel Cause Arrhythmias METABOLITE -demethylated T1/2 – 50 hrs T1/2 – 10 days , COMPETES for hepatic oxidases with TCA ect..days after administration of the parent drug has been stopped. CYP2D6

7. 2.Cytochrome P450 isoenzymes (CYPs) What are they? Nomenclature CYP reaction Dr. Pavitra Raj Dewda 7

9. Of these CYP 1, 2 and 3 constitute 70% of total hepatic CYPs which responsible for 94% of drugs metabolism

10. Xenobiotic metabolising - 12 CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5)

11. Some show genetic polymorphism while others can be induced or inhibited>55 amino acid sequence homology & 57 such isotype 40-55% amino acid sequence homology & about 20 subfamilies are imp in man

12. What are CYPs ? Superfamily of haemoprotein enzymes found on the membrane of SER The heme is a cofactor which contains iron-protoporphyrin IXthat functions to bind & activate Oxygen and transfer electrons. CYPs are also known as Mixed function oxidases Poly substrate Mono-oxygenases Location liver , intestine , lungs , kidneys , brain etc. Dr. Pavitra Raj Dewda 9

13. CYP Oxidation Reaction Dr. Pavitra Raj Dewda 10

14. Dr. Pavitra Raj Dewda 11 NADPH +H++ O2+RH NADP++H2O+R-OH

15. Microsomal drug oxidations Reaction step 1 Oxidized (Fe3+) P450 combines with a drug substrate to form a binary complex. step 2 NADPH donates an electron to the flavoprotein P450 reductase, which in turn reduces the oxidized P450-drug complex . step 3 A second electron is introduced from NADPH via the same P450 reductase, which serves to reduce molecular oxygen and to form an "activated oxygen"-P450-substrate complex” step 4 This complex in turn transfers activated oxygen to the drug substrate to form the oxidized product Sometimes the oxidised product is a Epoxide (highly reactive electrophiles that can bind to cellular nucleophiles found in protein, RNA, and DNA, resulting in cell toxicity and transformation) EpoxideHydrolaseshydrolyse such epoxides microsomalepoxidehydrolase (mEH) soluble epoxidehydrolase (sEH) Dr. Pavitra Raj Dewda 12

17. Headaches

18. Migraines

19. Motor coordination impairment

20. Upset stomach Valnoctamide Valproic acid

21. 3.Genetic Polymorphism What is it? Phenotypic (allelic) variants Examples New approach to personalized Rx PRINCIPLE Testing for Polymorphisms (genotyping) could predict therapeutic failures or ADRs therefore used to optimize drug choice, avoid serious ADRs and decrease medical costs! Dr. Pavitra Raj Dewda 14

22. Genetic Polymorphism of CYP450 Polymorphism - www.imm.ki.se/CYPalleles/ Is Genetic variations in a population when both gene or allelic variants exist with a frequency of at least one percent CYP enzymes in families 1-3 are polymorphic CYP2D6, CYP2C19 and CYP2C9 –main polymorphic forms responsible for almost 40% of P450 metabolism Homozygous mutations in the CYP1B1 gene are associated with primary congenital glaucoma Dr. Pavitra Raj Dewda 15

23. P450 Phenotypes based on the allelic variation & clinical importance Dr. Pavitra Raj Dewda 16

24. Dr. Pavitra Raj Dewda 17

25. From impossible to possible Dr. Pavitra Raj Dewda 18

27. According to FDA labeling, "Information about CYP2D6 genotype may be used as an aid to clinicians in determining therapeutic strategy and treatment doses for therapeutics that are metabolized by the CYP2D6 product"Dr. Pavitra Raj Dewda 19

28. CYP450 Enzyme Induction What is enzyme induction? What are the consequences of enzyme induction? What is the mechanism of enzyme induction? Examples Dr. Pavitra Raj Dewda 20

29. What is enzyme induction? Increase in the CYP450 metabolizing capacity Requires repeated drug administration Onset & Duration of induction Depends on ‘kinetics ’ & ‘half life’ of the drug (ranges from 1-6 days) Peak induction4-14 days On withdrawing inducer the CYP returns to their original level in 1-3 weeks Mechanism Enhancing the rate of its synthesis* Increasing transcription Reducing its rate of degradation /substrate stabilization Increasing translation Dr. Pavitra Raj Dewda 21

30. What is the mechanism of enzyme induction? Is incompletely understood but is similar to a steroid that bind to nuclear receptors (induction/regulatory receptors). Drugs bind to the ligand binding domain of a soluble protein, termed the aromatic hydrocarbon (Ah) receptor which is cytosolic in nature. This binding leads to conformational change in receptor Transportation of this complex to the nucleus by an Ah-receptor nuclear translocater In the DNA binds Ah-receptor response elements thereby promoting transcription of the gene Dr. Pavitra Raj Dewda 22 Non-transcriptional mechanisms Protein / Substrate stabilization or increased protein translation i.e. decreased degradation. e.g. CYP2E 1 by ethanol and isoniazide CYP3A by clotrimazole

32. Anticonvulsants Induction CYP3A4

33. Pharmacological induction/Herbal Drug Cyclosporin St. Johns Wort Metabolite Result Impaired Immune response Rejection Dr. Pavitra Raj Dewda 24 Induction CYP3A4

34. Pharmacological induction Paracetamol Ethanol(chronic) INH Toxic Metabolite(N-acetylbenzoiminoquinone) Result Hepatotoxicity Dr. Pavitra Raj Dewda 25 Induction CYP2E1

35. Pharmacological induction - paracetamol cont… Dr. Pavitra Raj Dewda 26

37. Charcoal – barbecued food

38. Plasticizers (polychlorinated biphenyls)

39. High dosing requirements of drugs like ‘Theophylline’ in smokersCarcinogen

40. Enzyme Inhibition What is enzyme inhibition? What are the types? Specific examples Dr. Pavitra Raj Dewda 28

41. What is enzyme inhibition? Certain substrates can block P450 enzyme isoforms (specifically) that metabolize other drugs/substrates A drug may inhibit one P450 isoenzyme while being metabolized its self by another. E.g. Quinidine metabolized by CYP3A4 and inhibits CYP2D6 Also some times a product of drug’s oxidation may inhibit its own metabolism  “Suicide Inhibitor” E.g. Chloramphenicol , Spironolactone etc… Unlike induction (1-3 weeks for onset) , enzyme inhibition usually begins with the first dose of the inhibitor and has a fast time course. Dr. Pavitra Raj Dewda 29

42. The determinant of potency of an inhibitor is the strength of the bond between the prosthetic heme iron and the lone electron pair of inhibitors 30 H2-receptor antagonist has a imidazole ring which binds competitively binding to the H 2 receptor & also selectively to the heme prosthetic group of CYP3A4 and 2D6 imidazole-containing antifungal & CYP3A4 inhibitor Ketoconazole (/Itraconazole>fluconazole )is a more potent inhibitor because of higher lipophilicity resulting in stronger hydrophobic interactions to CYP3A4 Ketoconazoleinhibits metabolism of anti-HIV viral protease inhibitors

43. Dr. Pavitra Raj Dewda 31 e.g. of Irreversible Inhibition due to formation of a MI Inhibitors Erythromycin, Clarithromycin (but not azithromycin) Grape fruit juice (furanocoumarins) Metabolism of drugs Inhibited (increasing Bioavalibility) Astemizole Terfenadine Cisapride Felodipine Cyclosporine Saquinavir Theophylline Triazolam Illustrative Case A 47-year-old man recently diagnosed with HIV infection visited his physician with flushing, dizziness and swelling of the feet and ankles. He had been taking sustained-release nifedipine for treatment of hypertension for about three years. Approximately two weeks earlier, his physician had prescribed a combination of lamivudine, zidovudine and the protease inhibitor ritonavir. Ritonavir (inhibitor CYP3A)increases the Bioavalibility of Nifedipine Erythromycin - oxidised by CYP3A4 Nitroso Metabolite + Haem portion of the CYP enzyme Stable MI complex is inactive CYP3A4 (torsades de pointes / prolonged QT interval) (ventricular arrhythmias ; the new drug ITOPRIDE is metaboilsed by FMO3) nausea, diarrhea, increase in heart rate, arrhythmias, and CNS excitation

44. Suicide inhibitors—inactivators that attack the heme or the protein moiety—includes ; Steroids (ethinyl estradiol, norethindrone, and spironolactone) Fluroxene Allobarbital The analgesic sedatives allylisopropylacetylurea Diethylpentenamide Ethchlorvynol Carbon disulfide Chloramphenicol Grapefruit furanocoumarins Deprenyl Phencyclidine Ticlopidine and clopidogrel Ritonavir Propylthiouracil Dr. Pavitra Raj Dewda 32

45. e.g. of drugs inhibiting CYP2D6 In order of inhibiting potency Paroxetine(is also metabolized)>Fluoxetine>Sertaline Dr. Pavitra Raj Dewda 33 CYP2D6 (substrates) If combined with Trazadone Postural Hypotension & probably Pirpaism If combined with TCAs Cardio toxicity Convulsions Coma

46. Dr. Pavitra Raj Dewda 34

47. ROLE OF DRUG METABOLISM IN THE DRUG DEVELOPMENT PROCESS Efficacy and Safety ; Both depend on drug metabolism It is necessary to determine how and by which enzymes a potential new drug is metabolized drug-drug interactions genetic polymorphisms Ideally, the best drug for a trial would be - metabolized by several CYPs so that variability in expression levels of one CYP or drug-drug interactions would not significantly impact its metabolism and pharmacokinetics Dr. Pavitra Raj Dewda 35

48. Summary Most important CYPs involved in drug metabolism are CYP3A4/5, CYP1A2, CYP2D6, CYP2C9, CYP2C19, CYP2E1 Knowledge about polymorphisms in CYPs (CYP2D6, CYP2C9, CYP2C19), enzyme induction & inhibition helps filtering out drugs for clinical trials and prevents unnecessary patient drug and drug interactions . Dr. Pavitra Raj Dewda 36

49. Dr. Pavitra Raj Dewda 37 Chapter 15 Text 14

50. Dr. Pavitra Raj Dewda 38 Have a great day!

51. Extra Dr. Pavitra Raj Dewda 39

52. How can we demonstrate a role for cytP450 in biotransformation of a XB? cytP450 activity increases after exposure to XB Enzymatic inhibitors of cytP450 (e.g. aminobenzotriazole) have an effect Absorption spectrum of cytP450 is modified in presence of XB Reaction can be reconstituted with components of the catalytically cycle (e.g. NADPH) Dr. Pavitra Raj Dewda 40

53. Hydroxylation of substrates by cytochrome P450-Deactivation (detoxication) Dr. Pavitra Raj Dewda 41

54. Role of cytochrome P450 and peroxidase in activation of benzene in bone marrow Dr. Pavitra Raj Dewda 42 PHS – prostaglandin H synthase

55. Halothane hepatitis and activation by oxidation and reduction Dr. Pavitra Raj Dewda 43

56. Reactions catalyzed by cytochrome P450 Dr. Pavitra Raj Dewda 44

57. Dr. Pavitra Raj Dewda 45 CYP450 ENZYME & SUBSTRATES

58. Some inducers of cytochrome P450 isozymes Dr. Pavitra Raj Dewda 46

59. Some inhibitors of cytochrome P450 isozymes Dr. Pavitra Raj Dewda 47

60. Dr. Pavitra Raj Dewda 48