1. By Dr. fatmah alomary Falomary@ksu.edu.sa Drug Metabolism

2. Drug metabolism is the transformation of foreign compounds ( xenobiotics) into a water soluble derivatives which can be easily eliminated in the urine.

3. Example

4. In General, the metabolism of xenobiotics takes place in two steps known as phase I & phase II reactions

5. Phase I ( functionalization reaction ) Is the process of increasing of the hydrophilicity of lipophilic drug by introducing a polar functional group eg; OH,COOH,NH2,SH to the molecule through oxidative, reductive & hydrolytic biotransformations.

6. Phase II ( conjugation reactions )  Is Linking of an endogenous solubilizing moiety either to the original drug (if polar function is already present) or to the phase I metabolite.  Common solubilizing groups are glucuronic acid, various amino acids or sulphate groups.  The conjugate molecule, being more polar and water-soluble, is usually excreted via the renal route

7. Effect of metabolism on the therapeutic activity of drugs

9. Factors affecting drug metabolism  Genetic factors  Physiological factors  Pharmaceutical factors  Pharmacodynamic factors.  Enviromental factors.

10. Genetic factors Biological half –life (t 1/2 ) of various drug Genetic Polymorphism: Different expression of metabolizing enzymes according to the Race (ethnicity)

11. Physiological factors Age,Gender,maternity status,liver function & Nutritional status. eg: Age which is the ability of the body to metabolize the drug lower in v. young & elderly.

12. Pharmacodynamic factors  The dose, the route and the frequency of administration of drugs & Drug interaction can affect their metabolic profiles.  Drugs given too frequently may overload the metabolic system available to it, leading to elevated blood and tissue levels of the drugs. The effect of protein binding also influences the metabolism.  Drug interactions for example:- Phenobarbital stimulate the metabolism of Diphenylhydantoin. Plasma Concentration of anticoagulants such as Warfarin are reduced by simultaneous application of barbiturate

13. Enviromental Factors  Inhaled gases,toxins eg:Nicotine ( cigarette – 8 to 10 mg )  -Acute nicotine exposure  (From – insecticide sprays or tobacco)  Nausea, vomiting, salivation, diarrhea, dizziness, mental confusion, weakness  -Fatal exposure (60 mg fatal for adult)  Decreased blood pressure, irregular pulse, convulsions, respiratory failure and death  -Cotinine - Major metabolite  -Lung – First site of metabolism  -Liver – Major site  -Half-life – about 2 hours

14. (Phase I (Functionalization reactions  Oxidations (electron removal, dehydrogenation and hydroxylation)  Reduction ( electron donation, hydrogenation and removal of oxygen )  Hydrolytic reactions of amides & esters. -Two general types of enzyme systems take part in these reactions: -a) Microsomal Mixed Function Oxidases (MFOs) Flavoprotein, NADPH-monooxygenase Cytochrome P450 -b) Non-cytochrome oxidizing enzymes. Xanthine oxidase Alcohol/aldehyde dehydrogenase

15. I) Oxidation Reactions  The main enzymes involved in the oxidation of xenobiotics called mixed – function oxidases (MFO) or monooxygenases, found mainly in the liver but also occur to less extent in other tissues. Cytochrom P450 ( CYP450 ) catalyze the majority of Drug metabolism oxidation reactions.  MFO is an old terminology,the enzyme are most frequently known as CYP450 Superfamily

16. - The enzyme systems carrying out this biotransformation are referred to as monooxygenases or microsomal (non specific enzymes in liver). -The reaction requires both molecular oxygen and the reducing agent (Activation of O 2  1 atom goes to organic molecule, the other reduced to H 2 O0. -NADPH (nicotinamide adenosine dinucleotide phosphate). - Monooxygenases are made up of several components :- 1) Cytochrome P-450 which is the most important component and is responsible for transferring an oxygen atom to the substrate R-H. 2) Cofactors supply the reducing equivalents (electrons) needed in the overall metabolic oxidation

17. a) NADPH. Dependent cytochrome P-450 reductase. b) NADH. Linked cytochrome P-450. *Cytochrome P-450 is found in high concentration in the liver, also present in other tissues like lung, kidney, intestine, skin, placenta and adrenal cortex. C) FMO is also a member of the mono-oxygenase system

18. *It is characterized by the substrate nonspecificity, this versatility may be attributed to the multiple forms of the enzyme. - Consequently, the biotransformation of a parent xenobiotic to several oxidized metabolites is carried out not just by one form of P-450 by several different forms. -It is now actually proven that the metabolism of drug is carried out by different isoforms,members of the CYP450 superfamily,eg:CYP2A1,CYP2D6,CYP3A4…etc

19. -A large number of families (at least 18 in mammals) of cytochrome P-450 (abbreviated “CYP”) enzymes exists as well as many subfamilies. each member catalyzes the biotransformation of a unique group of drugs -CYP450 SUPERFAMILY: classified according to sequence homology. -High homology: > 90%, intermediate: ~ > 60%; Low: ~ > 40% -FAMILY: members have > 40% homology (low). E.g.: CYP1 vs. CYP2 -SUBFAMILY: members have > 60% homology (intermediate). E.g.: CYP2A vs. CYP2B ISOFORM: CYP2A1, CYP2A2. (High) CLASSIFICATION

21. Major reactions of oxygenation catalyzed by CYP450: 1-Carbone oxidation reaction 2-N-Oxygenation reactions. 3-S-oxidation.

22. 2-N-Oxygenation reactions:1-Carbone oxidation reactions a)Hydroxylation of Saturated aliphatic C atom. ring b)Hydroxylation of aromatic aliphatic c)Oxidation of unsaturated

23. Major reactions of oxygenation catalyzed by CYP450 3-S-oxidation.

24. Oxidation reactions 1. Carbon oxidation reaction A) –Aliphatic hydroxylation B) -Aromatic hydroxylation 2. N-Dealkylation 3. N-oxide formation 4. Oxidative Deamination 5. O-Dealkylation reactions 6. S-Dealkylation. 7. S-oxidation reactions

25. A) –Aliphatic hydroxylation i)saturated aliphatic carbon atoms  Saturated aliphatic C-H bonds are metabolised by hydroxylation on the penultimate carbon atom ( ω -1 )and on the ultimate carbon( ω )to lesser extent.

26. ii)Enzymatic introduction of a hydroxyl group into cyclohexane ring generally occurs at C-3 or C-4 -In humans the trans-4-hydroxycyclohexyl product has been reported as a major metabolite of acetohexamide ( hypoglycemic agent )

27. iii(Terodiline Aromatic p-hydroxylation predominate with R- isomer where as benzylic hydroxylation is preferred with S-isomer.

28. iv)Tolbutamide CYP450 Tolbutamide

29. Pentobarbital CYP450 Ibuprofen CYP450

30. Phenmetrazine Valproic Acid CYP450

31. (v(v

32. VI)Oxidation at Benzylic Carbon Atoms Benzylic carbon atoms are susceptible to oxidation forming the corresponding alcohol or carbinol which is further oxidized to or conjugated with glucuronic acid.

33. )Oxidation at Carbon Atoms Alpha to Carbonyl and Imines An important class of drugs undergoing this type of oxidation is the benzodiazepines e.g. diazepam and flurazepam. The C-3 carbon atom is  to both a lactam carbonyl and an immino functionality. Hydroxylation of the carbon atom  to carbonyl group generally occurs only to a limited extent e.g. glutethimide

34. vi) Aliphatic hydroxylation (alkene epoxidation).

35. B ) Aromatic Hydroxylation (Oxidation of aromatic rings) : Aromatic epoxidation: It involves oxidation of aromatic compounds (arenes) to their phenolic metabolites (arenols). It is a major route of metabolism for many drug containing phenyl groups.

36. :Rules for Aromatic Oxidation -In most of drugs containing aromatic moieties, microsomal aromatic hdroxylation occurs at the para-position. -Microsomal aromatic hydroxylation reactions proceed most readily in activated (electron-rich) rings e.g. rings containing electron donating group as NH 2 group. -Deactivated aromatic rings (e.g., those containing electron-withdrawing groups as Cl, N + R 3, COOH, SO 2 NHR are generally slow or resistant to hydroxylation.

37. For compounds in which two aromatic rings are present, hydroxylation occurs preferentially in the more electron-rich ring.

38. When para ‑ position of aromatic ring is occupied the oxidation occurs in ortho ‑ position. Estradiol CYP450

39. Metabolic oxid. of C-N & C-S involve hydroxylation of alpha carbone atom attached directly to heteroatom(N,O,S) :General Mechanism a) Hydroxylation of the  -carbon atom attached directly to the heteroatom. 2) N-dealkylation

40. b) Hydroxylation or Oxidation of the Heteroatom (N, S only): Hydroxylol This reaction is catalyzed by cytochrome P-450 and N-oxide amine oxiases or N ‑ oxidases.

41. ؤ cont….. N-dealkylation It involves oxidation of tertiary and secondary amines. oxidative alpha-hydroxylation at alpha-C then dealkylation. i) Oxidation of Tertiary Aliphatic Amines: It is characterized by oxidative removal of alkyl group (particularly –CH 3 group) form tertiary aliphatic and alicylic amines. Removal of the first alkyl group occurs more rapidly than the removal of the second alkyl group. Bisdealkylation may occur but very slowly.

42. i) ii)

43.  -Oxidation of Secondary Amines Amines can undergo deamination. Amphetamine for example is deaminated to phenyl acetone and ammonia iii

44. Nicotine Nornicotine Cotinine Norcotinine CYP450

45. -The biotransformation of amines is the same as the carbon and nitrogen oxidation reactions seen for aliphatic amines but tertiary and secondary aromatic amines are rarely encountered in medicinal agents. 3) N-Oxide formation:

46. MephentermineMephentermine N-Oxide

47. 4) Oxidative Deamination : Amines can undergo deamination. Amphetamine for example is.. deaminated to phenyl acetone and ammonia. Oxidative deamination of most exogenous primary amines is carried out by the mixed oxidases. However, endogenous primary amines, such as dopamine, norepinephrine, tryptamine and serotonin, are metabolized through oxidative deamination by monoamine oxidases (MAO).

48. . This process is similar to N-dealkylation, in that it involves an initial  ‑ carbon hydroxylation reaction to form a carbinolamine intermediate, followed by carbon- nitrogen cleavage to the carbonyl metabolite and ammonia in primary amines Mechanism :

49. Oxygen alkyl groups are removed by liver microsomal preparation by a mechanism involves α-hydroxylation of the alkyl groups 5) Oxidative Dealkylation

50. i) The metabolism of these systems occurs through oxidative O ‑ dealkylation by microsomal enzyme s.

51. . 6) S-dealkylation Methitural S-demethylated metabolite

52. i-Oxidation of Sulfur: Thioethers or sulfides, for example, Chlorpromazine and Cimetidine are oxidized to their sulphoxides

53. ii- Desulfuration It is the conversion of thione (C = S) to the corresponding (C = O).

54. II.Reduction -Play an important role in the metabolism of compunds containing azo,nitro,carbonyl. -Bioreduction of nitro & azo lead to amino derivatives,where as carbonyl compounds reductions lead to alcohol analogs… 1-Azo-reduction

55. 2-Nitro reduction

56. E.g.: The opioid receptor antagonist Naltrexone is reduced in humans to it’s secondary alcohol metabolite 3-Reduction of Carbonyl group

57. Bio reduction of sedative – hypnotic Chloral hydrate yields trichloroethanol. This oxidation is non- microsomal is believed to take place by alcohol dehydrogenase.

58. 4-Reduction of Sulphur containing group. active prodrug inactive

59. III-Hydrolytic Reactions: Metabolism of ester & amide linkage in many drugs catalyzed by hydrolytic enzyme(esterase and amidasea). Procaineamide Procaine

60. Example Short acting local anesthetic Procainamide Long acting antiarrhythmic T 1/2 = 2.5-4.5 hrT 1/2 = 40-84 second

61. Ester vs. Amide bond The duration of actions of ester drugs are less than the amide analogues.why?  Procaine (ester type) injection or topical is usually shorter acting than its amide analogue procainamide administered similarily Ester bond is relatively weaker than amide bond, it will be rapidly hydrolyzed by esterase enzyme

62. Nucleophilic attack of hydroxide anion on ester and amide

63. Esterases and Amidases: Esters are more prone to hydrolysis. Converted to more W.soluble carboxylic acids. E.g.: Meperidine, Succinylcholine. Sterically hindered esters might be excreted unchanged??? Why?

64. Amides: More resistant to hydrolysis than esters why?, Advantage: Procaine vs. Procainamide. Procaine: ester, very short half life, destroyed shortly after entering circulation Procainamide: longer half life than procaine. more than 60% excreted unchanged