1. DRUG METABOLISM F. Keyhanfar Ph.D, D.I.C.

2. METABOLISM (BIOTRANSFORMATION) ------------------------------------ Acetaminophen, NSAIDs inhibit cyclo-oxygenase Amitriptylline & Cimetidine Phenytoin Diazepam - Dopa & Carbidopa Succinycholine Isoniazid

3. Terminology  Metabolism: (Gk: metabole: change)  Metabolite: metabolism produce metabolite  Xenobiotics: xeno (Gk: foreign)… biotics  Inhibition (Latin inhibitus, ): to prohibit from doing something  Induction (Latin inducere): to induce: to call forth or bring about by influence or stimulation  Cytochrome P450s (CYPs)

4. METABOLISM (BIOTRANSFORMATION) ------------------------------------ The processes by which foreign molecules (Xenobiotics) are chemically altered by a living organism.

5. METABOLISM (BIOTRANSFORMATION) The body recognizes these molecules and, if it is unable to eliminate them unchanged, is able to increase their water solubility by a variety of enzymic reactions

6. Result ------------ Water soluble metabolites Increased Excertion Reduced Biological Half-life Minimum Toxicity

7. Phase I Phase II DRUG METABOLITE CONJUGATE Expose or introduce a Conjugate the functional functional group that groups exposed or introduced can be conjugated by during Phase I biotransformation Phase II enzymes Small  in water solubility Large  in water solubility Termination of Pharmacological activity or introduce toxicity The rate and extent to which a drug is metabolized determines the dose of the drug and the duration of the effect of the drug Rate limiting/ Affected by genetic and environmental factors Active/Inactive/Toxic/ Mutagenic/Carcinogen OHO Glucuronide

8. Two-phase biotransformation Phase I (functionalization) reactions:  Oxidation, Reduction, and hydrolytic reactions (makes the drug more polar, but not necessarily inactive) Phase II (conjugation) reactions:  Conjugation to polar groups: glucuronidation, sulfation, acetylation (most of these result in drug inactivation) Ultimate effect is to facilitate elimination

9. BIMM118 Phase I Reactions –Oxidation –Reduction –Hydrolytic cleavage –Alkylation (Methylation) –Dealkylation –Ring cyclization –N-carboxylation –Dimerization –Transamidation –Isomerization –Decarboxylation Cytochromes P-450 (CYP) Flavin Monooxygenase (FMO) Monoamine Oxidase (MAO) Aldehyde dehydrogenase Alchohol dehydrogenase Various amidases/esterases

10. BIMM118 Drug Metabolism - Phase II Conjugation reactions –Glucuronidation by UDP-Glucuronosyltransferase: (on -OH, -COOH, -NH 2, -SH groups) –Sulfation by Sulfotransferase: (on -NH2, -SO 2 NH 2, -OH groups) –Acetylation by acetyltransferase: (on -NH 2, -SO 2 NH 2, -OH groups) –Amino acid conjugation (on -COOH groups) –Glutathione conjugation by Glutathione-S-transferase: (to epoxides or organic halides) –Fatty acid conjugation (on -OH groups) –Condensation reactions

11. An Example of Drug Metabolism Non – specific Hydrolytic enzyme

12. Complex Metabolism

13. Metabolism Hepatic microsomal enzymes (oxidation, conjugation) Extrahepatic microsomal enzymes (oxidation, conjugation) (oxidation, conjugation) Hepatic non-microsomal enzymes (acetylation, sulfation,GSH, alcohol/aldehyde dehydrogenase, hydrolysis, ox/red)

14. Important CYP Isoforms (12 ) CYP3A4 & 3A5 & 3A7 CYP2D6 CYP1A1 &1A2 CYP2A6 CYP2B6 CYP2C8 & 2C9 & 2C19 CYP2E1

15. Important CYP Isoforms (12 ) CYP3A4 VERAPAMIL (CA CHANNEL BLOCER) CYP2D6 VERAPAMIL CYP1A1 &1A2 CYP2A6 CYP2B6 CYP2C8 & 2C9 VERAPAMIL CYP2C19 CYP2E1

16. Important CYP Isoforms CYP3A4 Midazolam, triazolam, cyclosporine, erythromycin, Ca channel blockers THE MOST PREDOMINANT IN HUMAN – AMOUNT IN THE LIVER & VARIETY OF DRUG BEING SUBSTRATES 50% of all CYP-mediated drug oxidations also involved in the greatest number of drug-drug interactions. active site is large, accept substrates up to M.wt = 1200 drugs bind in different regions of the enzyme active sites

17. CYP3A4 Two drugs are metabolized by this enzyme. Will coadministration result in a drug–drug interation ? drugs bind in different regions of the enzyme active sites Two drugs can occupy the active site simultaneously both being available for metabolism by the enzyme. absent interations

18. CYP3A5 Amino acid sequence is similar to that of CYP3A4, It is not present in all individuals. Patients expressing both CYP3A4 & 3A5 Shows increased metabolism of CYP3A substrate

19. CYP3A7 Expressed only in the fetus Disappears following birth and replaced by CYP3A4 & 3A5 Different enzyme expression patterns and thus different drug metabolism capabilities throughout the various stages of life.

20. CYP2D6 Tricyclic antidepressants, codeine, dextromethorphan, antipsychotics antiarrhythmics etc Its relative abundance in the liver is low. Exhibit genetic polymorphism

21. Metabolism Amitriptylline is metabolized by CYP1A2 Cimetidine inhibits CYP1A2 Coadministration results in elevated Amitriptylline levels

22. Cimetidine, Ritonavir, amiodarone, diltiazem, ketoconazole Inhibit CYP3A4 Cimetidine, Fluoxetine, amiodarone Inhibit CYP2D6 Cimetidine, Ketoconazole, Omeprazole Inhibit CYP2C19

23. Potent Inhibitors Fluoroquinolones (ie: ciprofloxacin) H2Blockers (ie: most notably cimetidine) Imidazoles (ie fluconazole) INH Ritonavir Mnemonic: “cip, cim, con, INH, and rit”

24. Barbiturates, Carbamazepine, Phenytoin, pioglitazone, glucocorticoids, … Induce CYP3A4 & 3A5 Phenobarbital, dexamethasone Induce CYP2A6 & 2B6 & 2C9 Smoking, Omeprazole Induce CYP1A1 &1A2

25. Potent Inducers Neuroleptics: Carbamazepine Phenobarbital Phenytoin AND: Rifampin Mnemonic: “carb, barb, pheny, and rif”

26. Hydrolysis Esters & Amides Hydrolyzed by : ESTERASES & AMIDASES Found in : Cytosol of cells in tissues Plasma & microsomes

28. Metabolic Overloading Chemical being metabolised by an alternate pathway, not necessarily comparable to metabolic stress in human exposure situations The chemical may be unmetabolised and accumulate or stored in the body or be excreted unchanged.

29. First order kinetics A constant fraction of drug is eliminated per unit of time. When drug concentration is high, rate of disappearance is high.

30. Zero order kinetics Rate of elimination is constant. Rate of elimination is independent of drug concentration. Constant amount eliminated per unit of time. Example: Alcohol

31. Aspirin, Ethanol Phenytoin Metabolism rate is constant

32. Phase II Metabolism (conjugation) Glutathione Glucuronidation Sulfation Glycine Acetylation Methylation Coenzymes needed: various

33. Sulfation may produce active metabolite

34. Paracetamol Overdose Most common drug taken in overdose Few symptoms or early signs As little as 12g can be fatal Hepatic and renal toxin –Centrolobular necrosis More toxic if liver enzymes induced or reduced ability to conjugate toxin

35. Acetaminophen and p-Aminophenols Acetanilide, 1886 (accidental discovery of antipyretic activity; high toxicity) Phenacetin or acetophenetidin, 1887 (nephrotoxic, methemoglobinemia) Acetaminophen, 1893 Recognized as active metabolite of acetanilide and phenacetin in 1948 (Brodie &Axelrod); popular in US since 1955 70-90% 75-80%

36. Acetominophen Metabolism ~60% ~35% CYP2E1* CYP1A2 CYP3A4 NAPQI N-acetyl-p-benzoquinone imine *induced by ethanol, isoniazid Protein adducts, Oxidative stress Toxicity

37. Paracetamol Metabolism

38. Management General measures <8 hours –Take level after four hours –Start N-aceylcysteine –Patients are usually declared fit for discharge from medical care on completion of its administration. –Patients should be advised to return to hospital if vomiting or abdominal pain develop or recur

39. N-acetylcysteine Supplies glutathione Dosage for NAC infusion - ADULT –(1) 150mg/kg IV infusion in 200ml 5% dextrose over 15 minutes, then –(2) 50mg/kg IV infusion in 500ml 5% dextrose over 4 hours, then –(3) 100mg/kg IV infusion in 1000ml 5% dextrose over 16 hours Side-effects –Flushing, hypotension, wheezing, anaphylactoid reaction Alternative is methionine PO (<12 hours)

40. Acetaminophen overdose results in more calls to poison control centers in the United States than overdose with any other pharmacologic substance. The American Liver Foundation reports that 35% of cases of severe liver failure are caused by acetaminophen poisoning which may require organ transplantation. N-acetyl cysteine is an effective antidote, especially if administered within 10 h of ingestion [NEJM 319:1557- 1562, 1988] Addition of N-acetyl cysteine to acetaminophen tablets proposed to prevent liver toxicity. [British Medical Journal, Vol. 323, Sept. 15, 2001] Acetaminophen Toxicity

41. Metabolism

42. Factors affecting drug metabolism Drug metabolism can be affected by: –First pass effect –Hepatic blood flow –Liver disease –Drugs which alter liver enzymes Main site of drug metabolism = LIVER

43. The phenomenon of “first pass effect” or “first pass metabolism” and its clinical relevance Some drugs are ineffective when given orally – examples: nitroglycerine, nor-adrenaline, insulin

44. Drug Admin: Formulation First Pass Effect Blood from the gastrointestinal tract passes through the liver before entering any other organs. During this first pass through the liver, a fraction of the drug (in some cases nearly all) can be metabolized to an inactive or less active derivative. The inactivation of some drugs is so great that the agents are useless when given orally. (e.g.. lidocaine)

45. Factors affecting drug metabolism Genetic factors –e.g acetylation status Other drugs –hepatic enzyme inducers –hepatic enzyme inhibitors Age –Impaired hepatic enzyme activity Elderly Children < 6 months (especially premature babies)

46. Factors affecting biotransformation age (reduced in aged patients & children) sex (women slower ethanol metabilizers) species (phenylbutazone 3h rabbit, 6h horse, 8h monkey, 18h mouse, 36h man); biotransformation route can change clinical or physiological condition other drug administration (induction (not CYP2D6 ) or inhibition) food (charcoal grill ++CYP1A)(grapefruit juice - -CYP3A) first-pass (pre-systemic) metabolism

47. Factors Influencing Activity and Level of CYP Enzymes Red indicates enzymes important in drug metabolism

48. Enantiomer Drug Shape –Lock and Key phenomenon for the drug and its receptor site –More than half of all useful drugs are chiral molecules and exist as enantiomeric pairs Ex: Carvedilol has a single chiral center and two enantiomers S(-) isomer – potent Beta receptor blocker R(+) isomer – 100-fold weaker at Beta receptor –One drug enantiomer is often more susceptible than the other to drug-metabolizing enzymes One enantiomer could have a longer or shorter duration of action than the other

49. Racemic mixtures Drug Shape –As of now, most clinical studies of drugs have only tested racemic mixtures (two enantiomer pairs) –55% of drugs are only available as racemic mixtures where a patient is receiving 50% inactive or actively toxic drug

50. Pharmacogenetics For pharmacokinetics, change may occur in –drug transporters –drug metabolizing enzymes »more active enzymes »Inactive enzymes »semi-active enzymes

51. Succinylcholine ● Used during anesthesia to induce muscle paralysis ● Paralysis usually lasts minutes, but in some individuals, it may last up to one hour ● Due to altered kinetics of pseudocholinesterase

52. Isoniazid ● Used in the treatment of tuberculosis ● Observed variation in the amount of unchanged isoniazid in the urine ● Differences were due to an individuals ability to convert isoniazid to acetylisoniazid. ● Caused by mutations in the N-acetyltransferase-2 enzyme (NAT2) on chromosome 8 ● Some individuals develop isoniazid toxicity manifested as peripheral neuropathy