This document discusses drug metabolism and provides details about its evolution as a science. It describes the key pioneers in the field after World War II and their contributions. It then covers the major pathways of drug metabolism, including phase I reactions like oxidation and reduction and phase II conjugation reactions like glucuronidation, sulfation, and glutathione conjugation. The roles of cytochrome P450 enzymes and various conjugating enzymes are explained.

1. DRUG
METABOLISM
Presented by:
Dr. A.S. Dhake
Principal, SMBT College of Pharmacy,
Nashik
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2. 2

3. 3

4. 4

5. Why study drug metabolism?
 Toxic metabolites are possible
 Drug metabolism now required for drug
approval
• Metabolites must be identified
• Metabolites must be shown non toxic
• Very low concentration and unknown
structures require specialized method.
5

6. Evolution of Drug Metabolism As a Science
Post WWII Pioneers
• Richard Tecwyn Williams – Great Britain
– 1942, worked on the metabolism on TNT with regard to toxicity
in munitions workers; due to the war he assembled teams to
work on metabolism of sulfonamides, benzene, aniline,
acetanilide, phenacetin and stilbesterol.
– Developed concept of Phase 1 & Phase 2 Reactions.
• Biotransformation involves metabolic oxidation, reduction,
or hydrolysis; result in changes in biological activity
(increased or decreased)
• Second phase, conjugation, in almost all cases resulted in
detoxification.
6

7. Evolution of Drug Metabolism As a Science
Post WWII Pioneers
• Bernard B. Brodie, U.S.
– NYU and Laboratory of Industrial Hygiene, NYC 1949 –
Metabolic fate of acetanilide and phenacetin in man
(with Julius Axelrod)
– 1950s, NIH – pioneering studies on all aspects of drug
metabolism; esp. reserpine, serotonin;hexobarbital
tolerance
– 1952 – R.T. Williams spent 6 months at NIH;
subsequently many students went between both labs
(Richard Adamson, James Gillette, and Sidney
Udenfriend)
– 1950s, Brodie lab developed the spectro
photofluorimeter (Robert Bowman)
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8. Hepatic microsomal enzymes
(oxidation, conjugation)
Extrahepatic microsomal enzymes
(oxidation, conjugation)
Hepatic non-microsomal enzymes
(acetylation, sulfation,GSH,
alcohol/aldehyde dehydrogenase,
hydrolysis, ox/red)
Drug Metabolism
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9. How will the drug action be stopped?
Metabolism / Biotransformation
• Drugs and other chemicals that are foreign to the
body (xenobiotics) undergo enzymatic
transformations which usually result in loss of
pharmacological activity.
• Modifies lipophilic molecules into polar, water
soluble products, essential for the elimination and
termination of their biological activity.
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10. • Detoxification: Generates biologically inactive
metabolites and non toxic metabolites .
Readily excreted from body.
• Metabolites may still have potent biological
activity (or may have toxic properties)
• Generally applicable to metabolism of all
xenobiotics as well as endogenous compounds
such as steroids, vitamins and fatty acids
10

11. Ying & Yang concept of Metabolism
Metabolism = Anabolism + Catabolism
Photosynthesis requires Respiration
Respiration requires Photosynthesis
Energy Production = Energy Consumption
11

12. Breakdown
Proteins to Amino Acids, Starch to Glucose
Synthesis
Amino Acids to Proteins, Glucose to Starch
12

13. TYPES OF METABOLITES
1) Inactive metabolites: Drugs gets inactivated or detoxified to
give inactive products
Eg. Procaine
2) Metabolites which retain similar activity to the same or
different extent:
Eg. Imipramine, Acetohexamide, Codein
3) Metabolites with altered activity:
Eg. Iproniazid, Retinoic acid
4) Bioactivated metabolites: inactive compound (prodrug) gets
metabolized to an active compound:
Eg. Cyclophosphamide, Enalapril
13

14. SITE OF BIOTRANSFORMATION
• Enzymatic in nature
• Enzyme systems involved are localized in
liver
• Every tissue has some metabolic activity.
Other organs with significant metabolic
capacity are gastrointestinal tract, kidneys
and lung.
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15. Pathways of drug metabolism
1)Phase I Metabolism:
Includes oxidation, reduction, hydrolysis, hydration
and isomerization
2) Phase 2 Metabolism:
Glucuronide conjugation, acylation, sulfate
conjugation, methylation, acetylation, amino acid
conjugation, glutathione conjugation
3)Phase 3 Metabolism: Detoxification process
15

16. OXIDATION
• First studied in vitro in 1940’s using rat liver
homogenates
• Require NADP+, O2, microsomal fraction and
NADPH
•Active towards broad spectrum of compounds.
• Incorporates only one O atom into the substrate.
Involves a heme protein, which absorbs visible light
of 450nm after reduction and absorption of CO
• Named cytochrome P450
16

17. 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 for different
individual isoforms; eg. CYP1A2
Italics indicate gene (CYP1A2); regular font for enzyme
17

18. Cytochrome P450 Isoforms (CYPs) - An Overview
•NADPH + H+ + O2 + Drug NADP+ + H2O + Oxidized metabolite
•Carbon monoxide binds to the reduced Fe(II) heme and absorbs
at 450 nm (origin of enzyme family name)
•CYP-450 enzymes are heme proteins. Heme part is
Protoporphyrin IX ie an iron containing porphyrin. Protein part in
enzyme is apoprotein.
•CYP -450 enzyme family found in endoplasmic reticulum in liver,
kidney, G.I. tract, skin, lungs and adrenal cortex.
• Oxidative reactions require the CYP heme protein, the
reductase, NADPH, phosphatidylcholine and molecular oxygen.
18

19. OTHER
36%
CYP2D6
2%
CYP2E1
7%
CYP 2C
17%
CYP 1A2
12%
CYP 3A4-5
26%
RELATIVE HEPATIC CONTENT
OF CYP ENZYMES
% DRUGS METABOLIZED
BY CYP ENZYMES
ROLE OF CYP ENZYMES IN HEPATIC DRUG
METABOLISM
CYP 1A2
14%
CYP 2C9
14%
CYP 2C19
11%
CYP2D6
23%
CYP2E1
5%
CYP 3A4-5
33%
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20. Humans CYP450 -18 families, 43 subfamilies
• CYP1 drug metabolism (3 subfamilies, 3 genes, 1 pseudogene)
• CYP2 drug and steroid metabolism (13 subfamilies, 16 genes, 16 pseudogenes)
• CYP3 drug metabolism (1 subfamily, 4 genes, 2 pseudogenes)
• CYP4 arachidonic acid or fatty acid metabolism (5 subfamilies, 11 genes, 10 pseudogenes)
• CYP5 Thromboxane A2 synthase (1 subfamily, 1 gene)
• CYP7A bile acid biosynthesis 7-alpha hydroxylase of steroid nucleus (1 subfamily member)
• CYP7B brain specific form of 7-alpha hydroxylase (1 subfamily member)
• CYP8A prostacyclin synthase (1 subfamily member)
• CYP8B bile acid biosynthesis (1 subfamily member)
• CYP11 steroid biosynthesis (2 subfamilies, 3 genes)
• CYP17 steroid biosynthesis (1 subfamily, 1 gene) 17-alpha hydroxylase
• CYP19 steroid biosynthesis (1 subfamily, 1 gene) aromatase forms estrogen
• CYP20 Unknown function (1 subfamily, 1 gene)
• CYP21 steroid biosynthesis (1 subfamily, 1 gene, 1 pseudogene)
• CYP24 vitamin D degradation (1 subfamily, 1 gene)
• CYP26A retinoic acid hydroxylase important in development (1 subfamily member)
• CYP26B probable retinoic acid hydroxylase (1 subfamily member)
• CYP26C probabvle retinoic acid hydroxylase (1 subfamily member)
• CYP27A bile acid biosynthesis (1 subfamily member)
• CYP27B Vitamin D3 1-alpha hydroxylase activates vitamin D3 (1 subfamily member)
• CYP27C Unknown function (1 subfamily member)
• CYP39 unknown function (1 subfamily member)
• CYP46 cholesterol 24-hydroxylase (1 subfamily member)
• CYP51 cholesterol biosynthesis (1 subfamily, 1 gene, 3 pseudogenes) lanosterol 14-alpha demethylase
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21. CYP
Enzyme
Examples of substrates
1A1 Caffeine, Testosterone, R-Warfarin
1A2 Acetaminophen, Caffeine, Phenacetin, R-Warfarin, Imipramine, estradiol
2A6 17-Estradiol, Testosterone
2B6 Cyclophosphamide, Erythromycin, Testosterone
2C8 S-Warfarin, Acetaminophen, Tolbutamide,
Hexobarbital, Phenytoin, Testosterone, R- Warfarin,
2C9 S- Warfarin, Tolbutamide, Ibuprofen, Naproxen, Phenytoin.
2C19 Omeprazole, Mephenytoin, Diazepam, Propranolol
2D6 Quinidine, Lignocaine, Propranolol, Metaprolol, Fluoxetine,
Acetaminophen, Codeine
2E1 Acetaminophen, Caffeine, Halothane, Theophylline
3A4 Acetaminophen, Caffeine, Codeine, Cortisol, Erythromycin, S- and R-
Warfarin, Phenytoin, Testosterone, Halothane, Zidovudine, Diazepam,
Lignocaine, Steroids. 21

22. Electron flow in microsomal drug oxidizing system
CO
hu
CYP-Fe+2
Drug
CO
O2
e-
e-
2H+
H2O
Drug
CYP
R-Ase
NADPH
NADP+
OH
Drug
CYP Fe+3
PC
Drug
CYP Fe+2
Drug
CYP Fe+2
Drug
O2
CYP Fe+3
OH
Drug
22

23. Oxidation Reactions
• Mostly occur in liver and some extra hepatic
tissues (intestine, lungs, kidneys)
• Most drugs are oxidised by non specific
enzymes in liver microsomes
• Some oxidation reactions are catalyzed by non
microsomal oxidases located in cytosol and
mitochondria of extra hepatic tissues.
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24. Aromatic Hydroxylation
(Phenytoin, Phenylbutazone)
Aliphatic Hydroxylation
(Pentobarbital, Meprobamate)
Oleifinic Hydroxylation
(Carbamazepine, Secobarbital)
Benzylic Hydroxylation
(tolbutamide, Imipramide)
Allylic Hydroxylation
(Pentazocine, Hexobarbital)
R CH3
R
OH
R
CH3
C
H3
OH
C
H3 CH3
CH3
C
H3
O
H OH
C
H3
CH3
C
H3
CH3
OH
R R
OH
24

25. 25
CH3
NH2
R
S
R
R
O
R
H
O
C
H3
C
H3
OH
O
CH3
O
S
R
S
R
O
R
N
CH3
H
R
N
OH
CH3
R
NH
CH3
NH2
R
Dealkylation (Morphine, Ephedrine)
N-Oxidation (Acetaminophen,
Guanethidine)
S-Oxidation (Chlorpromazine,
Cimetidine)
Oxidative deamination
(Amphetamine, Dopamine)
Desulphuration (Thiopental)
Aldehyde Oxidation (Acetaldehyde)

26. Other Oxidative Enzymes
Primarily protective function
• Flavin monoxygenase
Primarily involved in endogenous metabolism
• Prostaglandin H synthase
• Alcohol dehydrogenase
• Xanthine oxidase
• Monoamine oxidase
• Aromatase
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27. REDUCTION
27

28. HYDROLYTIC REACTION
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29. Phase I Metabolism Summary
Virtually every possible chemical reaction that a compound
can undergo can be catalyzed by the drug metabolizing
enzyme systems
• The final product usually contains a chemical
reactive functional group OH, NH2, SH, COOH.
• This functional group can be acted upon by the
phase II or conjugative enzymes.
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30. PHASE II METABOLISM
• Phase II is usually the true detoxification
of drugs
• Occurs mostly in cytosol
• Gives products that are generally water
soluble and easily excreted
• Includes sugar conjugation, sulfation,
methylation, acetylation, amino acid
conjugation, glutathione conjugation
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31. Phase II transformations
31

32. Conjugate Groups Conjugated Transferases
Glucuronide -OH, -CO2, -NH2, -NR2, -SH,
C-H
UDP-glucuronyl-
transferases
Sulfate -OH, -NH2 Sulfotransferase
Glycine/Gluta
mine
-CO2H Glycine or Glutamine N-
acyltransferase
Glutathione Ar-X, arene oxide,
epoxide, carbocation
Glutathione S-transferase
Acetyl -OH, -NH2 Acetyltransferase
Methyl -OH, -NH2, -SH,
heterocyclic N
Methyltransferase
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33. GLUCURONIDATION
• Most widespread, important of the
conjugation reactions
• Cofactor UDP – glucuronic acid is high in
abundance.
• Closely related to glycogen synthesis.
• Found in all tissues of the body
Other sugars, glucose, xylose or ribose may
be conjugated
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34. Conjugation Reactions
Glucuronidation
Liver has several soluble UDP-Gluc-transferases 34

35. Glucuronic acid conjugation to
phenols, 3°-amines, aromatic amines
Morphine
O
HO
HO
N CH3
6
3
Amitriptyline
N
N
N
CH3
O
Cotinine
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36. SULFATION
•Major conjugation pathway for phenols, also
alcohols and aromatic amines
• Compounds that can be glucuronidated can also
be sulfated
• In general, sulfate conjugation predominates at
low substrate concentration and glucuronide
conjugation predominates at high substrate
concentration
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37. Conjugation Reactions
Sulfation
Examples: ethanol, p-hydroxyacetanilide, 3-hydroxycoumarin
(PAPS, 3’-phosphoadenosine-
5’-phosphosulfate)
R OH
R O S OH
O
O
H H
NH2
N
N
N
N
OH
O
H H
HO
O P
OH
O
O S
OH
O
O
+
37

38. Sulfation may produce active metabolite
38

39. GLUTATHIONE CONJUGATION
• Glutathione is a protective compound (tripeptide,
Gly-Cys-Glu) within the body for removal of potentially
toxic electrophilic compounds.
• In the presence of glutathione S-transferase,
glutathione reacts with electrophiles such as halides and
epoxides to form harmless inactive products
• Many drugs are metabolized in phase I to strong
electrophiles
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40. • React with glutathione to form non- toxic conjugates
Glutathione conjugates may be excreted directly in
urine or bile, but are usually metabolized further.
• Organophosphate insecticides such as methy
parathion, immuno suppresive drug azathioprine, arene
oxides and aliphatic epoxides, oxiranes like styrene
oxide are highly toxic and if not detoxified by GSH
conjugation produce carcinogenicity.
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41. Phase II Metabolism Summary
Now xenobiotics have been converted into low-
toxicity, higher molecular- weight and high water
solubility metabolites by the combination with
endogenous substances.
Phase 2 metabolites are more water-soluble.
Conjugation reactions: preparing the drug for
excretion by one pathway or another.
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42. PHASE III
Failure to remove the products of Phase II reactions
can lead to:
• Toxicity of conjugates to various cell components
• Hydrolysis of conjugates back to the original reactive
species
• Inhibition of Phase II enzymes.
42

43. The biggest hurdle is the transport of highly
water-soluble entity out of the cell, as the cell
membrane is highly lipophilic
Now the conjugates must be transported against
a concentration gradient out of the cell into the
interstitial space between cells.
They will enter the capillary system and than to
the main blood stream and filtration by the
kidneys.
43

44. Impressive array of multi-purpose membrane
bound transport carrier systems evolved which
can actively remove hydrophilic metabolites and
many other low-molecular-weight drugs and
toxins from cells.
The relatively recent (1990s) term of Phase III
metabolism has been applied to the study of this
essential arm of detoxification process.
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45. Factors affecting Drug Metabolism
Environmental Determinants
•Induction
•Inhibition
Disease Factors
Age and Sex
Genetic Variation
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46. Drug Metabolism Studies
• Radioactive drug administered to an animal
• Urine and feces samples collected
• Extractions used to isolate radioactive
materials from majority of species in mixture
• Chromatographic separations provide further
Separation
• Radioactive compounds identified usually by
mass spectrometry
46

47. References
• Leon Shargel, Alan Mutnick, Paul Souney, Larry Swanson:
Comprehensive Pharmacy Review, 7th edition; Wolter Kluwer,
Lippincott Williams and Wilkins
• G. Gordon Gibson, Paul Skett Fil. Dr: Introduction to Drug
Metabolism, Third edition, (2001 ), Nelson Thornes Publishers
•David Nelson , Michael Cox: Leninger Principles of
biochemistry, fourth edition, W. H. Freeman and Company, New
York
• Michael D. Coleman: Human Drug Metabolism: An
Introduction, 2005 John Wiley & Sons Ltd
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48. • Costas Loannides: Cytochromes P450 Role in the
Metabolism and Toxicity of Drugs and other Xenobiotics,
2008, Royal Society of Chemistry.
• Robert K. Murray, Daryl K. Granner, Lange: Harper’s
Illustrated Biochemistry a LANGE medical book, twenty-sixth
edition, Medical Books/McGraw-Hill
Thomas Lemke, David Williams: Foye’s Principles of
medicinal chemistry, Sixth edition (2008) Lippincott
Williams, Wilkins, Wolters Kluwer business
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49. THANK YOU…!
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