Drug metabolism

1. Drug metabolism Dr.S.O. Ogundele

2. Introduction • Drug metabolism is a process transforming drugs to a more water soluble metabolite within the body • Most drugs are hydrophobic • Drug metabolism results in the following • Prevention of accumulation of drug within the body • Conversion of most drugs to a less active metabolite

3. Introduction • Prodrugs are converted to a more active form by the process of metabolism e.g. • Prednisone • Primaquine • Proguanil • Methyldopa • Disulphiram • Azathioprin

4. Sites of metabolism Extrahepatic microsomal enzymes (oxidation, conjugation) Hepatic microsomal enzymes (oxidation, conjugation) Hepatic non-microsomal enzymes (acetylation, sulfation,GSH, alcohol/aldehyde dehydrogenase, hydrolysis, ox/red)

5. Sites of metabolism • Liver is the principal organ for drug metabolism • Other tissues with considerable activities include • Lungs • Skin • GIT • kidney

6. Sites of metabolism • Metabolising enzymes are located in • Smooth endoplasmic reticulum • Mitochondria • Cytosol • Plasma membrane

7. First pass effect • First pass effect is the metabolism of orally taken drugs before they get into systemic circulation • Site of first pass metabolism include • Intestinal mucosa • CYP Enzymes • P-Glycoprotein • Liver • CYP Enzymes

8. Oral route of drug intake1st pass metabolism

9. IV route

10. Drugs affected by 1st pass metabolism • Examples of such drugs include • Aldosterone • Morphine • Cyclosporine • Nortriptyline • Isoproterenol • Organic nitrates • Lidocaine • Propranolol

11. Phase I metabolism • Phase I (functionalization reaction phase): • This involves conversion of lipophylic molecules to a more polar molecule by introduction or unmasking of a polar functional group • E.g of Phase I reaction: • Hydroxylation (Aliphatic or aromatic) • Dealkylation (N-,O-, S-) • Oxidation (N- or S-oxidation) • Deamination • Hydrolysis, hydration • Dehalogenation

12. Phase I metabolism • Phase I metabolism results in changes in biological activity of most drugs leading to • Increase in activity • Decreased in activity • Sometimes there is no change

13. Phase II metabolism • Conjugation reaction takes place during phase II of metabolism • An endogenous substrate is added to the established functional group from phase I to form a highly polar metabolite

14. Phase II metabolism • Conjugation reactions include: • Glucuronidation • Sulfation • Methylation • Acetylation • Condesation • Amino acid conjugation • In most cases it results in detoxification

15. Process of Phase I metabolism • The smooth ER of the liver contain an important enzyme known as the mixed function oxidase (MFO) or monooxygenase • Activities of MFO require both a reducing agent (NADPH) and a molecular oxygen (reaction is an oxidative-reductive) • In a typical reaction one mol of oxygen is consumed, one atom of oxygen appears in the product and the other forms water

16. Process of Phase I metabolism • One of the enzymes involved in this process is NADPH-cyp reductase • NADPH-cyp reductase is a flavoprotein • One molecule of this enzyme contain 1 mole each of flavine mononucleotide (FMN) and flavine adenine dinucleotide (FAD) • The cytochrome serves to accept electron

17. Process of Phase I metabolism • The second enzyme is an haemoprotein Cyt P-450, it serves as the terminal oxidase • In its reduce form (ferrous) it binds carbon monoxide and absorbs light max at 450nm (this is why it is so named) • Compared to the reductase this enzyme is abundant and its action is the rate limiting step in hepatic oxidation • It is an isoenzyme

18. Electron flow in microsomal drug oxidizing system Drug NADP+ Fe+3 CYP CYP R-Ase Drug PC OH NADPH Fe+3 CYP CO CO Fe+2 Drug CYP CYP-Fe+2 OH hu Drug Drug O2 Fe+2 CYP H2O O2 Drug 2H+ e- Drug e-

19. Consequences of phase I reaction • Oxidative reaction can results in any of the following • Formation of an inactive polar metabolite • Phenobarbital • Formation of an active metabolite • Methyl dopa, prednisone • Formation of a toxic metabolite • Acetaminophen – NAPQI

20. CYP Families • Multiple CYP gene families have been identified in humans • They are categorised according to their protein sequence homology • Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families • CYPs have molecular weights of 45-60 kDa

21. CYP Families • Frequently two or more enzymes can catalyze the same type of oxidation, indicating redundant and broad substrate specificity • CYP3A4 is very common to metabolism of many drugs • Its presence in the GI tract is responsible for poor oral availability of many drugs

22. CYP Nomenclature • Families - CYP plus arabic numeral >40% homology of amino acid sequence, eg.CYP1 • Subfamily - 40-55% homology of amino acid sequence; eg. CYP1A • Subfamily - additional arabic numeral when more than 1 subfamily has been identified; eg. CYP1A2 • Italics indicate gene CYP1A2 • Regular font indicate enzyme

23. ROLE OF CYP ENZYMES IN HEPATIC DRUG METABOLISM RELATIVE HEPATIC CONTENT OF CYP ENZYMES % DRUGS METABOLIZED BY CYP ENZYMES A. Atkinson, 2005

24. Participation of the CYP Enzymes in Metabolism of Some Clinically Important Drugs Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002

25. Factors Influencing Activity and Level of CYP Enzymes Red indicates enzymes important in drug metabolism Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002

26. Phase II Metabolism • This is a conjugation reaction • Major Conjugation Reactions are • Glucuronidation (high capacity) • Substrates include phenol, alcohol, sulfonamide • Drugs in this group include, morphine, digoxin, diazepan

27. Phase II Metabolism • Sulfation (low capacity) • e.g. methyl dopa • Acetylation (variable capacity) • Substrates are amines e.g. Procainamide, Isoniazid • Other Conjugation Reactions: • O-Methylation • S-Methylation • Amino Acid Conjugation (glycine, taurine, glutathione) • Many conjugation enzymes exhibit polymorphism

28. Conjugation Reactions Glucuronidation Liver has several soluble UDP-Gluc-transferases

29. Conjugation ReactionsAcetylation E.g.: Procainamide, isoniazid, sulfanilimide, histamine NAT enzyme is found in many tissues, including liver

30. Non-CYP drug oxidations • Monoamine Oxidase (MAO), Diamine Oxidase (DAO) • MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine, serotonin, norepinephrine, epinephrine) • DAO substrates include histamine and polyamines

31. Non-CYP drug oxidations • Alcohol & Aldehyde Dehydrogenase • Non-specific enzymes found in soluble fraction of liver • Responsible for ethanol metabolism • Xanthine Oxidase • Converts hypoxanthine to xanthine, and then to uric acid • Drug substrates include 6-mercaptopurine • Allopurinol is a substrate and inhibitor of xanthine oxidase; delays metabolism of other substrates • Effective for treatment of gout

32. Non-CYP drug oxidations • Flavin Monooxygenases • Family of enzymes that catalyze oxygenation of nitrogen, phosphorus, sulfur – particularly facile formation of N-oxides • Different FMO isoforms have been isolated from liver, lung • Complete structures has been defined • Require molecular oxygen, NADPH, flavin adenosine dinucleotide (FAD)

33. Non-CYP drug oxidations • Single point (loose) enzyme-substrate contact with reactive hydroperoxyflavin monoxoygenating agent • FMOs are heat labile and metal-free, unlike CYPs • Factors affecting FMOs (diet, drugs, sex) not as highly studied as CYPs

34. CYP Inhibition • Inhibition of CYP is usually by competitive binding to the active site. • Onset and offset dependent on the half-life and time to steady-state of the inhibitor. e.g Fluoxetine/CYP2D6Ritonavir/CYP3A4 • Time to maximum interaction effect dependent on time required for substrate drug to reach new steady-state • Mechanism is by enzyme inactivation • Grapefruit juice and intestinal CYP3A content

35. Common CYP inhibitors • Cimetidine (various) • Erythromycin, clarithromycin (3A4) • Ketoconazole, itraconazole (3A4) • HIV protease inhibitors (ritonavir) • Fluoxetine, paroxetine (CYP2D6) • Nefazodone (CYP3A4) • Grapefruit Juice (intestinal CYP3A4 only)

36. CYP Induction • Common suspects (RRCPPNST) • Rifampin • Rifabutin • Carbamazepine • Phenobarbital • Phenytoin • Nevirapine, efavirenz • St. John’s wort • Troglitazone, pioglitazone

37. Effects on induction of drug metabolism • Results in reduction of plasma concentration of substrate drugs • Risk of therapeutic failure • Removal of inducer may lead to toxic concentrations of substrate • Induction may lead to formation of toxic metabolite

38. Note • Sometimes Phase II reaction may precede phase I reaction e.g acetylation of INH • If drug metabolite is sufficiently polar enough, they may be excreted after phase I reaction • Examples of pro-drugs (metabolism results in formation of a more active metabolite) include the following: prednisone, primaquine, proguanil, methyldopa, disulphiram, azathioprin

39. Kinetics of metabolism • 1st order • Rate of metabolism depends on the concentration of the substrate, a constant proportion of drug is metabolised per unit time • Zero order • Constant amount of drug is metabolised irrespective of drug concentration

40. Conclusion Thank you!