1. Phase II or Conjugation Reactions – Conjugation reactions link an endogenous moiety (an endocon) to the original drug (if polar functions are already present) or to the Phase I metabolite.  They are catalyzed by enzymes known as transeferases.  They involve a cofactor which binds to the enzyme in the close proximity of the substrate and carries the endogenous molecule or moiety to be transferred.  Except for methylations and acetylations, this endocon is highly polar and its size is comparable to that of the substrate.

2. Phase II Reactions Include: 1. Glucuronidation 2. Sulfate Conjugation 3. Acetylation 4. Amino Acids (GLYCINE) Conjugation 5. Methylation 6. Glutathione Conjugation

3. 1. Glucuronidation  The mechanism of glucuronidation is one of nucleophilic substitution with inversion of configuration,  -D-glucuronic acid in UDPGA forming  -D-glucuronides.

5. Glucuronidation Pathway and Enterohepatic Recirculation

6.  The functional groups able to undergo glucuronidation are classified as O-, N-, S- and C-glucuronides. O-Glucuronidation includes phenolic xenobiotics or metabolites generally at high doses (O-sulfation is predominating at low doses). – Alcohols are another major substrates, may primary, secondary or tertiary ones.

7.  An interesting example is that of morphine, which is conjugated on its phenolic and secondary alcohol groups to form the 3-O-glucuronide and the 6-O- glucuronide respectively.

8. The second important after O-Glucuronides are the N-glucuronides formed from carboxamides, sulfonamides, and various amines. N-Glucuronidation of aromatic and aliphatic amines in addition to pyridine-type nitrogens has been observed in only a few cases.

9. S-Glucuronides are formed from aliphatic thiols, aromatic thiols and dithiocarboxylic acids. C-Glucuronidation is seen in humans for 1,3-dicarbonyl drugs such as sulfinpyrazone.

10.  Beside xenobiotics, a number of endogenous substrates, notably bilirubin and steroids are eliminated as glucuronide conjugates.  In neonates and children, glucuronidation processes often are not fully developed. In such subjects, drugs and endogenous compounds (e.g. bilirubin) that are normally metabolized by glucuronidation may accumulate and cause serious toxicity.  For example, neonatal hyperbilirubinemia and  gray baby syndrome, result from accumulation of toxic levels of the free chloramphenicol.

11. 2. Sulfate conjugation In reactions of sulfoconjugation, a sulfate molecule is transferred from the cofactor (3’- phosphoadenosine 5’-phosphosulfate, PAPS) to the substrate by cytosolic enzymes known as sulfotransferases.

12.  Phenols compose the main group of substrates undergoing sulfate conjugation. Thus, drugs containing phenolic moieties often susceptible to sulfate formation.

13. The sulfate conjugation of N-hydroxylamines and N-hydroxyamides takes place occasionally as well. O-Sulfate conjugates of N-hydroxy compounds are of considerable toxicologic concern because they can lead to reactive intermediates that are responsible for cellular toxicity.

14. 3. Amino acid conjugation This is a major route for many xenobiotic acids, involving the formation of an amide bond between the xenobiotic acyl-CoA and the amino acid.

15. Glycine is the amino acid most frequently used for conjugation. The xenobiotic acids undergoing amino acid conjugation are mainly benzoic acids such as benzoic acid itself and salicylic acid, forming hippuric acid and salicyluric acid respectively.

16. 4. Conjugation with glutathione (GSH) – Glutathione (GSH,  -glutamylcysteinylglycine) is a thiol-containing tripeptide. – GSH conjugation is an important pathway by which chemical reactive electrophilic compounds are detoxified. – Many serious drug toxicities may be explainable also in terms of covalent interaction of metabolically generated electrophilic intermediates with cellular nucleophiles. – GSH protects vital cellular constituents against chemically reactive species by virtue of its nucleophilic sulfhydryl (SH) group.

18.  Unlike other conjugative phase II reactions, GSH conjugation does not require the initial formation of an activated enzyme or substrate. –Two general mechanisms are known for the reaction of compounds with GSH: 1. Nucleophilic displacement at an electron-deficient carbon or heteroatom.

19. 2- Nucleophilic addition to an electron-double bond. – The major reactions of glutathione are summarized in the following scheme:

20. – In a few cases, GSH conjugation has been implicated in causing toxicity e.g. 1,2-dichloroethane. – In most instances, GSH conjugation is regarded as a detoxifying pathway

21. 5- Acetylation Reactions –The most common reactions are acetylations of xenobiotics containing a primary amino group. –The major reactions of N-acetylation are summarized in the following scheme.

22. Examples: Procainamide, isoniazid, sulfanilimide, histamine N-acetyl transferase enzyme is found in many tissues, including liver

23. –Acetylation result in decreased water solubility. – Genetic differences exist in some animal species and in humans, where one distinguishes between slow and fast acetylators. – Examples of drugs exhibiting acetylation polymorphism is the antituberculosis drug isoniazid (INH) –The drugs metabolized by acetylation may become ineffective in fast acetylators and cause toxicity in slow acetylators.This liver toxicity may be attributed to the formation of chemically reactive acylating intermediates as shown in the following scheme:

25. Procainamide Unchanged in Urine, 59% 3% 24% Fast 17% Slow Unchanged in Urine, 85% NAPA 0.3% 1%

26. Procainamide trace metabolite non-enzymatic Lupus?

27. 6- Methylation Reactions Methylation is an important reaction in the biosynthesis of endogenous compounds such as adrenaline, and in the inactivation of biogenic amines such as catecholamines. Reactions of methylation imply the transfer of a methyl group from the onium-type cofactor S- adenosylmethionine (SAM) to the substrate by means of a methyltransferase

28. Methyltransferases having particular importance in the metabolism of foreign compounds include catechol-O- methyltransferase (COMT), phenol-O-methyltransferase and nonspecific N-methyltransferase and S-methyltransferase. Examples of drugs undergoing methylation are given in the following scheme.

29. FACTORS AFFECTING DRUG METABOLISM – Many factors may affect drug metabolism, these include: 1.AGE 2.DIET 3.STATE OF HEALTH 4.GENDER 5.GENETIC VARIATION 6.SPECIES VARIATION 7.SUBSTRATE COMPETITION 8.ENZYME INDUCTION 9.ROUTES OF DRUG ADMINISTRATION

30. AGE  Age – related differences in drug metabolism are generally quite apparent in newborn.  Undeveloped or deficient oxidative and conjugative enzymes are chiefly responsible for the reduced capability. Examples: Undeveloped glucuronidating processes in neonates and children are responsible for neonatal hyperbilirubinemia and gray baby syndrome as dissussed before. The oxidative metabolism of tolbutamide is significantly lower in infants (t ½ more than 40 hrs) compared with adults (t ½ ~ 8 hrs).

31. SPECIES VARIATION – The metabolism of many drugs and xenobiotics is often species dependent. Examples:

32.  Phenylbutazone half-life is 3 h in rabbit, ~6 h in rat, guinea pig and dog and 3 days in humans.

33. Other notable species differences include: – Dogs: deficient in N-acetyl transferase (cannot acetylate aromatic amines) – Guinea-pigs: deficient in sulfotransferase activity; no N- hydroxylation – Cats: poor UDPGlucuronosyltransferase activity ( unable to glucuronidate phenols ) – Rats: often very rapid metabolizers; CYP2C is the major family in the liver with significant gender differences – Cynomolgus monkeys: reported to have low CYP1A2 activity

34. GENETIC VARIATION Marked individual differences in the metabolism of several drugs exists in humans. Many of these genetic or hereditary factors are responsible for the large differences seen in the rate of metabolism of these drugs. Example: Isoniazid - N-acetyltransferase

35. First line drug in the treatment of TB; normally given at a does of 5 mg/kg, max. 300 mg/day for period of 9 months. Rapid and slow acetylators first seen in TB patients; t 1/2 for fast is 70 min; t 1/2 for slow is 180 min. N-acetyltransferase (NAT2 isoform) is in liver, gut Peripheral neuropathy (about 2% patients; higher doses produce effects in 10-20%) seen in slow acetylators. Hepatotoxicity also seen, esp. in older patients.

36. Genetic Polymorphisms in Drug Metabolizing Enzymes

37. GENDER Male Rats metabolize many drugs faster than females through oxidative pathways, glucuronide and GSH conjugation of certain substances, In Humans this difference also existed, though very limited and usually clinically not important. e.g.The clearance of benzodiazepines eliminated by metabolic conjugation is significantly smaller in women than in man.

38. ENZYME INDUCTION Certain drug substrates of CYP, upon repeated administration, “induce” or elevate the amounts of specific forms of the enzyme. Induction results in accelerated metabolism of the drug inducer and any other drugs metabolized by the induced enzyme. Classic Enzyme Inducers Chronic ethanol Phenobarbital Tobacco (benzo[a]pyrene) PCBs (Poly chlorinated biphenyls), dioxin (environmental) Glucocorticoids

39. ENZYME INHIBITION Some drugs are capable of inhibiting metabolizing enzymes. This is usually only important if a patient on a stable drug regimen starts taking a second drug metabolized by the same enzyme. Examples: cimetidine, ethanol

40. Drugs that Inhibit Drug Metabolism by Forming Complexes with CYPs

41. Non-nitrogenous Substances that Effect Drug Metabolism by Forming Complexes with CYPs Grapefruit juice - CYP 3A4 inhibitor; highly variable effects; unknown constituents Isosafrole, safrole - CYP1A1, CYP1A2 inhibitor; found in root pear, perfume Piperonyl butoxide & alcohol -CYP1A1, CYP1A2 inducer; insecticide constituent

42. Effect of Grapefruit Juice on Felodipine Plasma Concentration 5mg tablet with juice without

43. STATE OF HEALTH Liver disease Kidney disease Endocrine diseases Reduced blood flow

44. Sample biotransformation problem A……………………………….. B………………………………... C………………………………… D………………………………….

45. Optimising Pharmacokinetic Properties

46. 7. Designing Prodrugs and Bioprecursors Prodrug is any compound that undergoes biotransformation prior to exhibiting its pharmacological effects. Prodrugs can be divided into two classes: 7.1. The Carrier-prodrugs : Result from a temporary linkage of the active molecule with a transport moiety that is frequently of lipophilic nature.

48. Example: Aspirin for salicylic acid Prodrugs to mask toxicity and side effects Mask groups responsible for toxicity/side effects Used when groups are important for activity Salicylic acid Analgesic, but causes stomach ulcers due to phenol group Aspirin Phenol masked by ester Hydrolysed in body

50. Drug Barrier to delivery of drug Drug Barrier to delivery of drug Pro-moiety Drug Biotransformation Efficacy Drug + + Non-toxic & rapidly excreted

51. 7.1.1 Practical applications of carrier-prodrug design 1. Improvement of the lipophilicity

52. Candoxatril for Candoxatrilat (protease inhibitor) Varying the ester varies the rate of hydrolysis Electron withdrawing groups increase rate of hydrolysis (e.g. 5-indanyl) Leaving group (5-indanol) is non toxic

53. 2. Enhancement of water solubility

55. Prodrugs to increase water solubility Often used for i.v. drugs Allows higher concentration and smaller dose volume May decrease pain at site of injection Example: Succinate ester of chloramphenicol (antibiotic) Succinate ester Esterase Chloramphenicol

56. Prodrugs to increase water solubility Example: Phosphate ester of clindamycin (antibacterial) Less painful on injection

57. Prodrugs to increase water solubility Example: Lysine ester of oestrone Lysine ester of oestrone is better absorbed orally than oestrone Increased water solubility prevents formation of fat globules in gut Better interaction with the gut wall Hydrolysis in blood releases oestrone and a non toxic amino acid

58. Increase the duration of Pharmacological effects 3. Prodrugs to prolong activity Add hydrophobic groups

59. Example: Hydrophobic esters of fluphenazine (antipsychotic) Given by intramuscular injection Concentrated in fatty tissue Slowly released into the blood supply Rapidly hydrolysed in the blood supply

60. Example: Azathioprine for 6-mercaptopurine 6-Mercaptopurine (suppresses immune response) Short lifetime - eliminated too quickly Azathioprine Slow conversion to 6-mercaptopurine Longer lifetime Mask polar groups Reduces rate of excretion

61. 4. Improvement of patient acceptance – Used to reduce solubility of foul tasting orally active drugs – Less soluble on tongue – Less revolting taste Example: Palmitate ester of chloramphenicol (antibiotic) Palmitate ester Esterase Chloramphenicol

62. 5. Prodrugs used to target drugs Example: Hexamine Stable and inactive at pH>5 Stable at blood pH Used for urinary infections where pH<5 Degrades at pH<5 to form formaldehyde (antibacterial agent)

63. 6. Increased site-specificity. Two targeting possibilities can be considered: 6.1. Site-directed drug delivery

64. 6.2. Site-specific drug release

65. Chemical Delivery Systems: Pharmacologically inactive molecules that require several steps of chemical and/or enzymatic conversion to the active drug and enhance drug delivery to particular organs or sites. Example: Brain-specific chemical delivery system

67. 7.2. The Bioprecursors : Result from a molecular modification of the active principle itself. This modification generates a new compound, able to be a substrate for the metabolizing enzymes (often, oxidation and reduction). The metabolite being the expected active principle. Example for bioprecursor design based on oxidative bioactivation is the cytotoxic agent cyclophosphamid.

68. Cyclophosphoramide for phosphoramide mustard (anticancer agent) Cyclophosphoramide Non toxic Orally active Phosphoramide mustard Alkylating agent Phosphoramidase (liver)

69. Sulindac (NSAID) is a representative example for bioprecursor bioactivated by reduction.

70. Soft Drugs Biologically active, therapeutically useful chemical molecules (drugs) characterized by a predictable and controllable in vivo deactivation, after achieving the therapeutic objective, to nontoxic, inactive compounds.

71. Soft Drugs Examples:

73. Hard drugs: these can be defined as drugs that are biologically active and non metabolizable in vivo eg: enalaprilat, lisinopril, DDT Insecticide DDT (1,1,1-trichloro-2,2-bis(p- chlorophenyl)ethane)