1. Drug metabolism
Refers to enzyme-mediated biotransformations (detoxication) that alter the pharmacological activity of both endogenous and exogenous compounds.
Metabolism may result in:
Pharmacologically inactive drug (detoxification).
Pharmacologically active drug (bioactivation…prodrug approach).
Change the pharmacological activity (toxic effect).

2. Drug metabolism
Drugs undergo a variety of chemical changes in the animal organism by enzymes of the liver, intestine, kidney, lung, plasma and other tissues.
The importance of studying drug metabolism:
Understanding the pharmacological and toxicological activity of drugs.
The importance of shortening the drug’s duration of action.
The complications of drug-drug interactions mainly depends on the induction or inhibition of metabolic enzymes

3. Drug metabolism Can be divided into two distinct categories: Phase-I:
Reactions which introduce or unmask hydrophilic groups in the drug structure (functionalisations).
Phase-II:
Reactions which conjugate the drug or its phase-I metabolite with a hydrophilic, endogenous species (conjugation reactions).

4. Phase-I reactions Aliphatic hydroxylation. Oxidation: Reduction.
Oxidative Dealkylation.
Oxidative deamination.
N and S oxidation.
Alcohol/aldehyde dehydrogenase.
Reduction.
Hydrolysis.

5. Phase-I reactions Two enzyme systems take part in these reactions:
Cytochrome P-450 dependent mono-oxygenase: catalyze the oxidation at carbon, nitrogen and sulfur.
Flavin mono-oxygenase: catalyze oxidation at nitrogen and sulfur.

6. Phase-I reactions Cytochrome P-450 dependent mono-oxygenase:
Membrane–bound mixed function oxidase found in the smooth endoplasmic reticulum of the liver.
has the ability to activate molecular oxygen permitting the incorporation of one oxygen atom into the organic (drug) molecule and the reduction of the other atom into water molecule.

7. Oxidative Phase-I involving cytochrome P-450 enzymes:
Aromatic hydroxylation:

8. Oxidative Phase-I involving cytochrome P-450 enzymes:
Aliphatic hydroxylation:

9. Oxidative Phase-I involving cytochrome P-450 enzymes:
Aliphatic hydroxylation:
Mainly occur on the ultimate (ω) or penultimate (ω-1) carbon atom in the structure.
Also it occurs at an activated carbon atom, that is next to sp , sp2 carbons:

10. Oxidative Phase-I involving cytochrome P-450 enzymes:
Aromatic epoxidation:
Polycyclic aromatic
Hydrocarbons: highly toxic ,
Obtained from burning,
industrial process and
Cigarette smoking

11. Oxidative Phase-I involving cytochrome P-450 enzymes:
Alkene epoxidation:

12. Glutathione conjugation
For electrophilic drugs and metabolites:

13. Detoxication by glutathione adduct formation

14. Glutathione conjugation
Toxicity of aromatic compounds came from the formation of arene oxide during the metabolism that will be attacked by endogenous nucleophile such as proteins, DNA or RNA.

15. Oxidative Phase-I involving cytochrome P-450 enzymes:
O-dealkylation:

16. Oxidative Phase-I involving cytochrome P-450 enzymes:
N-dealkylation:

17. Oxidative Phase-I involving cytochrome P-450 enzymes:
Oxidative deamination:

18. Oxidative Phase-I involving cytochrome P-450 enzymes:
N-oxidation:
Mostly for primary and secondary amines as well as aromatic amines:
This gives N-oxide that will be rapidly converted to hydroxylamines.

20. Reductive phase-I metabolism
Mainly for carbonyl and nitro groups.
Carbonyl reduction by aldo-keto reductase:

21. Reductive phase-I metabolism
Nitro reduction by cytochrome P-450 or the bacterial nitroreductase:

22. Hydrolytic phase-I metabolism
By non-specific esterase and amidase enzymes that present in plasma, gut, liver and kidney.
It has a beneficial role in most of prodrugs that after hydrolysis inside the body release the active form of the drug.
Procaine Procainamide

23. Hydrolytic phase-I metabolism
Examples of prodrugs activated by hydrolytic enzymes:
Dipivefrine: is a di-tertbutylcarboxy ester of adrenaline…. More lipophilic… better penetration through the corneal membrane….then will be hydrolyzed to give the active form (adrenaline)

24. Hydrolytic phase-I metabolism
Ibuprofen as most of the non-steroidal anti-inflammatory has a local inhibition of protective prostaglandin synthesis in stomach which results in gastric irritation

25. Hydrolytic phase-I metabolism
-methydopa is an anti-hypertensive agent which have poor oral bioavailability because of the zwitter-ionic form.

26. Other phase-I metabolic enzymes
Monoamine oxidase: involved in deamination of catecholamines.
Alcohol dehydrogenase and aldehyde dehydrogenase:

27. Other phase-I metabolism
Heterocyclic ring oxidation:
S-dealkylation:

28. Other phase-I metabolism
Sulfoxidation: by flavin monooxygenase
Azoreduction:

29. Other phase-I metabolism
Miscellaneous reductions:
Reduction of disulfide and sulfoxide into sulfides:
Dehydroxylation of aromatic and aliphatic hydroxyl derivatives.
Ketone reduction by ketone reductase rather than the alcohol dehydrogenase (using NADPH as the cofactor) for naltexone, naltrexone, and hydromorphone)

30. Other phase-I metabolism
β-oxidation :
This occurs for alkyl carboxylic acids .
Involves the oxidative cleavage of two carbon units at a time (as acetate), starting from the carboxyl terminus until no more acetate can be removed:

31. General notes regarding phase-I metabolism
Hydrolysis normally catalyzes by carboxylesterases:
Cholinesterase…. Hydrolyzes choline-like esters (such as succinylcholine), procaine and acetylsalicylic acid.
Arylcarboxyesterase.
Liver carboxyesterase

32. General notes regarding phase-I metabolism
Esters that are sterically hindered are hydrolyzed more slowly and may be appeared unchanged in urine:
Amides are more stable to hydrolysis than esters….large fraction of amide containing drugs are normally excreted unchanged.

33. Phase-II metabolism
Involves the following conjugation reactions that are catalyzed by transferase enzymes:
Glucuronidation.
Sulfation.
Amino acid conjugation.

34. Glucuronidation
Involves conjugation of phenols, alcohols, hydroxylamines, carboxylic acids (O-glucuronidation), amines, sulfonamides, amides (N-glucuronidation) and thiols (S-glucuronidation) with glucuronic acid:

35. Glucuronidation
Estradiol

36. Sulfation (sulfate conjugation)
Mainly for phenols, alcohols, arylamines, and N-hydroxy compounds, catalyzed by sulfotransferases.
Salbutamol

37. General notes regarding sulfation
Sulfation and glucuronidation occur side by side, even competing for the same substrate….phenols in many cases.
Sulfation mainly occur in intestine for orally administered drugs…leads to pre-systemic first pass elimination….decreasing the oral bioavailability.
Co-administration of acetaminophen with oral contraceptive ethynylestradiol may affect oral availability of both drugs.
The rate of sulfation is age dependent, decreasing with age

38. Glycine conjugation
Is the conjugation of carboxylic acid group with glycine and glutamine (mainly for phenylacetic acids) in animals) whereas in primates the conjugation take place with glutamate or ornithine.
The carboxylic acid should be activated by forming CoA thioester which serve as a good leaving group.

39. Glycine conjugation
AMP
Coenzyme-A

40. General notes regarding drug carboxylic acids
The activation of carboxylic acids by forming the CoA ester is a stereo-selective reaction:

41. Acetylation
Is a reaction of amino groups involving the transfer of acetyl CoA to an aromatic primary or aliphatic amine, amino acids, hydrazine and sulfonamides.
Secondary amines are not acetylated.
Acetylation normally ends with pharmacologically inactive metabolites with some exceptions:

42. Acetylation
The rate of acetylation is mainly affected by the existence of genetic polymorphism.
Two acetylator phenotypes:
Slow acetylators: tend to accumulate higher blood concentrations of un-acetylated drugs...toxicity (isoniazid… peripheral nerve damage, procainamide…lupus erythematous).
Fast acetylators: eliminate drugs more rapidly, at the same time can form toxic metabolite very fast.
80-90% of orientals are fast acetylator
100% of Eskimos are fast acetylators

43. Methylation Mainly O- and N-methylation.
It is more important for endogenous compounds than for drugs.
The methylated metabolite in general is more lipophilic than the parent compound, sometimes this lipophilic metabolite is more active than the parent:

44. Methylation
The methyl source here is the activated intermediate S-adenosylmethionine (SAM), the enzyme catalyze this reaction is the methyl transferase enzyme.
Results in the formation of O-methylated, N-methylated and S-methylated products.

45. Methylation
O-methylation is catalyzed by Catechol-O-methyl transferase (COMT):
Regioselective….methylates m-hydroxyl group much more than the O-hydroxyl group.
Substrate selective… only methylates catechol-containing compounds, does not work on monohydric or other dihydric phenols.
The main importance is the biological inactivation of adrenergic neurotransmitter, norepinephrine and other Catechol-like structures.
Found in liver, kidney, and nervous system.

46. Methylation N-methyltransferase: S-methyltransferase:
Catalyzes the methylation of phenylethanolamines such as
histamine, norepinephrine and norephedrine.
Requires SAM as a methyl source.
S-methyltransferase:
One of the detoxication pathways for thiol containing compounds.
Needs SAM for methyl transfer.

47. General notes regarding drug carboxylic acids
Small, unbranched carboxylic acids normally undergo β-oxidation.
Branched aliphatic and aromatic carboxylic acid are resistant to β-oxidation and form either glycine or glucuronide conjugates.]
Substitution at the α-carbon favors glucuronidation rather than glycine conjugation.
Benzoic and heterocyclic carboxylic acids favor glycine conjugation.

48. General notes regarding phase-II metabolism
Phase-II metabolism (especially the glucuronidation and sulfation) thought to terminate pharmacological activity of drugs (why?):
By transforming the drug into readily excreted ionizable metabolite.
This metabolite will have poor affinity for the active site of drug’s receptors.
This metabolite will have poor cellular diffusion.

49. General notes regarding phase-II metabolism
Phase-II metabolism sometimes give more active metabolites:
Morphine-6-glucuronide has more analgesic activity than morphine.
Minoxidil sulfate is the active form for the anti-hypertensive agent minoxidil.
Sometimes, Phase-II reactions result in toxic derivatives , especially glucuronidation and sulfation:

51. Enzyme induction
Many drugs, and environmental chemicals enhances the metabolism of themselves or other co-ingested compounds ……this will alter the their pharmacologic and toxicologic effects.
It is a dose-dependent phenomenon.
This mainly occur by inducing transcription of CYP450mRNA which leads to overproduction of these enzymes in the liver and other extra-hepatic tissues.
Enzyme induction: is the process by which the rate of synthesis of an enzyme is increased relative to the un-induced organism

52. Enzyme induction Enzyme inducers:
Many drugs (table attached) have the ability to stimulate the activity of CYP450 isoforms.
Many environmental chemicals also alter the activity of CYP450 isoforms:
Cigarette smoking.
Polycyclic aromatic hydrocarbons.
Xanthines and flavones in food.
Halogenated hydrocarbons in insecticides.
Food additives
These chemicals do not have something in common except they are all metabolised by one or more CYP450 isoforms

53. Enzyme induction The importance of studying enzyme induction:
Evaluating the pharmacologic, toxicologic and explaining certain unexpected drug interaction in patients.
Studying the drug-drug interactions
As a result of induction:
The drug may be metabolized more rapidly to more potent or more toxic metabolite.
Or enhance the activation of procarcinogens
Or metabolized to less active metabolite.

54. Enzyme induction and drug-drug interactions
Many drugs are CYP450 inducers of CYP450 subfamilies, at the same time may be also substrates for the same isoforms:
Examples:
Phenobarbital
Phenytoin
Rifampicin
Cigarette smoking

55. Enzyme induction and drug-drug interactions
Examples of drug-drug interactions result from enzyme induction:
Rifamipicn induce metabolism of contraceptives by CYP3A Reducing their serum levels..... Increase the risk for pregnancy.
Phenobarbital, cigarette smoking and dexamethasone induce the metabolism of estrogens, vitamin D and bilirubin in women …….decrease their biological activity……. So cigarette smoking in premenopausal women increases the risk of osteoporosis and early menopause.

56. Enzyme induction and drug-drug interactions
Examples of drug-drug interactions result from enzyme induction:
Cigarette smoking lowers the serum levels of theophylline, imipramine, estradiol, decrease the urinary excretion of nicotine and decrease the drowsiness from diazepam, chlordiazepoxide
Administration of some drugs for a long time may stimulate their own metabolism….. Apparent tolerance:
The sedative action of Phenobarbital becomes shorter with repeated doses due to the increase in self metabolism.

57. Enzyme induction and drug-drug interactions
Examples of drug-drug interactions result from enzyme induction:
Heavy alcoholics metabolize phenobarbital, tolbutamide, and phentoin more rapidly than non-alcoholics due to the enzyme induction of CYP2E1.
The usefulness of enzyme induction:
Certain inducers have been used therapeutically for hyperbilirubinemia in children.
Some inducers stimulate the conversion of drugs into more polar metabolites.

58. Enzyme induction and food-drug interactions
Examples:
Cabbage and cauliflower stimulate monooxygenase activity in rat intestine.
Flavones, safrole, eucalyptol, xanthines and volatile oils present in food and plants also have enzyme induction properties.

59. Enzyme inhibition Can be divided into three major categories:
Reversible inhibition.
Metabolite intermediate complexation of CYP450.
Mechanism-based inactivation of CYP450.
Reversible inhibition:
is a result of reversible interaction at the heme-iron active centre of CYP450, the lipophilic site or both.
This action will be abolished once the enzyme inhibitor is discontinued.
Examples: fluoroquinolones, cimetidine, azoles antifungal agents.

60. Enzyme inhibition Metabolite-intermediate complexation of CYP450:
Happens when the metabolite of certain drugs forms stable covalent bond with the reduced ferrous heme intermediate.
Alkylamines are examples of such drugs due to the formation of the nitroso metabolite:

61. Enzyme inhibition
Examples of metabolite intermediate complexation drugs:
Macrolide antibiotics.
Eryhthromycin.
Clarithromycin.
Orphinadrine (anti-parkinson agent).
The clinical significance of this inhibition is the impairment of metabolism of many coadministerd drugs as well as the associated changes in pharmacokinetics for these drugs

62. Enzyme inhibition Mechanism-based inhibition (suicide inhibition):
Some drugs contain functional groups that when oxidized by CYP450 generate metabolites that bind irreversibly to the enzyme.
Examples:
Alkane and alkenes containing drugs can form radical intermediate after oxidation with CYP450…. This will alkylates the heme moiety…. Abnormal porphyrins….. 17-α-acetylenic progestin, norethindrone are examples of such drugs
Cyclophosphamide…forms acrolein and phosphoramide.
Chloramphenicol….(what is the reactive intermediate?).

63. Factors affecting drug metabolism
Species may differ in details of the reaction and enzyme control.
Factors influencing drug metabolism:
Genetic factors: results in differences in the expression of metabolizing enzymes and genetic polymorphism).
Physiologic factors: including age, hormonal changes, sex differences, pregnancy and nutritional status.
Pharmacodynamic factors: including dose, frequency, route of administration and protein binding.
Environmental factors: this depends on the competition with other drugs for the metabolizing enzymes by toxic chemicals such as CO and pesticides.

64. Drug metabolism and age
It is well documented that the metabolism of many drugs and their elimination is impaired in the elderly.
In elderly there are many physiologic changes that affect the plasma concentration and renal clearance….these will decrease the hepatic blood flow, glomerular filtration, hepatic enzymes activity and plasma protein binding.
First pass metabolism of many drugs is reduced in elderly patients: such drugs are diazepam, theophylline, morphine, propranolol and amitriptyline.
All of the common phase-II enzymes are affected by aging

65. Drug metabolism and age
The human fetus has only the cytochrome P450 monooxygenase 3A (CYP 3A)which is capable of metabolizing xenobiotics during the first part of gestation.
Placenta of tobacco smokers has shown increase of CYP1A activity that will form toxic metabolites which will covalently bind to fetus macromolecules….. Teratogenic and hepatotoxic effect.
Phase-II enzymes are found in low to negligible concentration in the fetus….. High risk of toxicity by pregnants metabolites

66. Drug metabolism and age
The ability to carry out metabolic reactions increases after birth and approaches adult levels in about 1 to 2 months.
The inability of infants to conjugate chloramphenicol with glucuronic acid appears to be responsible for the accumulation of toxic levels of this drug….. This will lead to what is called gray-baby syndrome.
Neonatal hyperbilirubinemia results from the newborn baby to glucuronide bilirubin.

67. Species and strain differeneces
There are metabolic differences between species, such as between human and dogs, rabbit, pigs, cats and birds.

68. Species and strain differences
The conjugation with amino acids differs between species as well:
Glycine conjugation is common in most animals.
Birds normally use ornithine amino acid for conjugation.
Strain differences in mice and rabbit have been noted: mainly due to genetic variations that affect the amount of metabolizing enzymes

69. Hereditary or genetic factors
Genetic factors in human is the main cause for the differences in the rate o drug metabolism.
the difference in the rate of acetylation is one example:
Rapid acetylators have more hepatic acetyl N-transferase than the slow acetylators.
90% of Asians and Eskimos are rapid acetylator.
Egyptians and Mediterranean are slow acetylators.
The rate of acetlations is clinically important in terms of therapeutic response and toxicity.
Also, genetic factors affect the arte of oxidation.

70. Sex differences
The rate of metabolism also varies according to sex in some animal species.
Generally it is species dependant:
Rabbit and mice do not show a significant sex differences in drug metabolism.
In humans, few repots of sex differences have been observed:
Nicotine and aspirin seem to be metabolized more rapidly in male compared to female.

71. Genetic Polymorphism
Can be defined as the genetic differences in the natural expression of enzyme isoforms.
Resulted in inter-individual variation in the metabolism of drugs.
Three families of CYP450 exist(CYP1-3).
Two phenotypes:
Extensive metabolizer (EM).
Poor metabolizer (PM).

72. Genetic Polymorphism
Poor metabolizer s(PM) are normally associated with higher risk of serious side effects due to the accumulation of drugs in the body.
Poor metabolizer (PM) also experience loss of activity in some drugs (codeine is an example)

73. Genetic Polymorphism
Poor metabolizer phenotype is inherited as an autosomal recessive gene while the extensive metabolizer is the predominant one.
Extensive metabolizers may be more susceptible than Poor metabolizers to develop cancers because they are better able to activate the pro-carcinogens into the active carcinogen.