drug metabolism

1. Drug Metabolism

2. 2
• Xenobiotics (including drugs) are taken up into the
body
• Some are excreted from the human body
unchanged
• Most are modified structurally to facilitate
excretion
• Such modification processes are called drug
metabolism
• Drug metabolism is a detoxification mechanism for
the human body
• Each drug metabolite must be considered as a new
drug entity to the body with potential actions and
toxicity

3. Metabolism (Biotransformation)
 Drugs undergo biotransformation by many of
the same reactions as endogenous
compounds.
 Drugs are usually metabolized to less active
and more ionized (water-soluble) forms, but
equally or more active metabolites can also be
created.
 An inactive parent drug that forms active
metabolites is called a prodrug.

4. Drug metabolism Can Lead To Inactivation of Drug
Procaine p-aminobenzoic acid
(active) (inactive)
The hydrolysis of procaine to p-aminobenzoic acid
results in a loss of anesthetic activity
CONSEQUENCES OF DRUG METABOLISM.

5. 5
Drug metabolite Can Retain Activity of Drug
Imipramine is demethylated to the essentially equiactive
antidepressant desipramine.

6. 6
In the case that the metabolite has the desired
pharmacological activity although the parent is inactive,
the metabolism is called bioactivation. The parent
compound devoid of pharmacological activity before
metabolism is called a prodrug.
Examples:
 Proton-pump inhibitors – omeprazole, lansoprazole are
prodrugs. (Used to reduce stomach acid secretion)
 Levo-dopa is a prodrug. It is converted into the active
compound dopamine (used for Parkinson’s disease)
Drug Metabolism Can Also Activate Drugs -
Prodrugs

7. Bioactivation of a drug can also result in a toxic
metabolite. The parent activated is called a protoxicant.
Bioactivation of acetaminophen; under certain conditions, the electrophile N-
acetylbenzoquinoneimine reacts with tissue macromolecules, causing liver
necrosis.

8. Organ Sites of Drug Metabolism
 Liver
 Small and large intestine
 Kidney
 Skin
 Lungs
 Plasma
 All organs of the body
Within the cell, xenobiotic-metabolizing enzymes are found in
the intracellular membranes and in the cytosol. The phase 1
CYPs, FMOs, and EHs, and some phase 2 conjugating
enzymes, notably the UGTs, are all located in the
endoplasmic reticulum of the cell.

9. 9
Drug metabolism is performed by a large number of different
enzymes and even by some nonenzymatic processes. A
general classification of enzymatic processes based on the
type of reactions involved includes phase I and phase II.
General Pathways of Drug Metabolism

10. 10
Phase I characteristics:
 Parent drug is altered by introducing or
exposing a functional group (-OH,-NH2, -SH)
 Drugs transformed by phase I reactions usually
lose pharmacological activity.
 Inactive prodrugs are converted by phase I
reactions to biologically-active metabolites.
 Phase I reaction products may be directly
excreted in the urine or react with endogenous
compounds to form water soluble conjugates.

11. 11
Phase II characteristics:
 Parent drug participates in conjugation reactions
that form covalent linkage between a parent
compound functional group and glucuronic acid,
sulfate, glutathione, amino acids, acetate
 Conjugates are highly polar and rapidly excreted
in the urine
 Generally inactive: exception to the rule:
morphine glucuronide metabolite-- more potent
analgesic then parent compound
 High molecular weight conjugates: Excreted in
the bile

12. Example of Phase I & II - Phenytoin
Phase I
•Introduction of polar
functional group (OH,
COOH, SH, O, or NH2)
“Functionalization”
•Slightly soluble in Water
Phase II
•Attachment of highly polar
group (Glucuronide,
sulfate, glutathione)
“Conjugation”
•Much more soluble in
Water

13. 13
Comparing Phase I &
Phase II
Enzyme Phase I Phase II
Types of reactions Hydrolysis
Oxidation
Reduction
Conjugations
Increase in
hydrophilicity
Small Large
General mechanism Exposes functional
group
Polar compound
added to functional
group
Consquences May result in
metabolic
activation
Facilitates excretion

14. 14
In phase 1 reactions, enzymes carry out oxidation,
reduction, or hydrolytic reactions. The phase 1 enzymes
lead to the introduction of what are called functional
groups, resulting in a modification of the drug, such that
it now carries an -OH, -COOH, -SH, -O- or NH2 group.
Phase 1 metabolism is classified as the functionalization
phase of drug metabolism. Many drug-metabolizing
enzymes are located in the lipophilic endoplasmic
reticulum membranes of the liver and other tissues.
Phase 1 reactions

15. 15
Cytochrome P450 (CYP450) is a superfamily of mixed
function oxidases that are responsible for the majority of
oxidation reactions. CYP450 is classified as a microsomal
enzyme and in the cell is bound to the ER. CYPs have
molecular weights of 45-60 kDa.
It is a hemoprotein containing an
iron atom which can alternate
between the ferrous (Fe++) and ferric
(Fe+++) states.
Cytochrome P450 enzyme complexes
are located close to their cofactors
(eg. NADPH is required as an
electron donor). These enzymes
catalyze an oxidation-reduction
process that requires CYP450,
CYP450 reductase, NADPH
(reducing agent), and O2.

16. Cytochrome P450 Enzymes (CYP)

17. 17
There are several isoforms of CYP450 responsible for drug
metabolism, which gives wide substrate specificity to the family
of CYP450 enzymes. Cloning and sequencing of CYP
complementary DNAs have revealed 57 putatively functional
genes and 58 pseudogenes in humans. These genes are grouped,
based on amino acid sequence similarity, into a large number of
families and subfamilies. A limited number of CYPs (12 in
humans) that fall into families 1 to 3 are primarily involved in
drug metabolism. In humans, CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8,
2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 are known to be important
for metabolism of xenobiotics. CYP3A4 is very common to the
metabolism of many drugs; its presence in the GI tract is
responsible for poor oral availabilty of many drugs
CYP Families

18. Nomenclature of cytochrome P450 enzymes
CYP
CYP1 CYP2 CYP3
CYP1A CYP1B
CYP1A1 CYP1A2 CYP1B1 CYP1B2
Family
Sub-family
Gene number
cytochrome P450
The nomenclature for the CYP450 enzymes is based on amino acid
homology and divides the CYP450’s into families designated by an
Arabic number, subfamilies designated by a letter, and individual
genes designated by an Arabic number preceded by “CYP.”

19. Cytochrome P450 Enzymes (CYP)
The importance of a particular CYP450 isoform in drug
metabolism is not necessarily a function of its relative
abundance in the liver. . .

20. Cytochrome P450 Enzymes (CYP)
Oxidation of organic molecules by P450s is quite complex, but
the overall reaction can be represented simply by:
RH + O2 + NADPH + H+ → ROH + H2O + NADP+
An electron from NADPH is transferred via the flavin domain
of NADPH-P450 Reductase to the heme domain of the CYP450
where the activation of molecular oxygen occurs. Substrates
react with one of the oxygen atoms and the other is reduced to
water.

21. 21
Step 1 Oxidized (Fe3+)
P450 combines with a drug
substrate to form a binary
complex.
Step 3 A second electron is introduced from NADPH via
the same P450 reductase, which serves to reduce
molecular oxygen and to form an "activated oxygen"-P450-
substrate complex.
Step 4 This complex in turn transfers activated oxygen to
the drug substrate to form the oxidized product.
Step 2 NADPH donates an
electron to the
flavoprotein P450
reductase, which in turn
reduces the oxidized
P450-drug complex.
Cytochrome P450 cycle in drug
oxidations. RH, parent drug;
ROH, oxidized metabolite; e–,
electron.

22. 22
The diverse reactions carried out by mammalian CYPs include:

23. 23
The diverse reactions carried out by mammalian CYPs include:

24. 24
The diverse reactions carried out by mammalian CYPs include:

25. Non-CYP drug oxidations
 Monoamine Oxidase (MAO), Diamine Oxidase (DAO) -
MAO (mitochondrial) oxidatively deaminates endogenous
substrates including neurotransmitters (dopamine,
serotonin, norepinephrine, epinephrine); drugs designed to
inhibit MAO used to effect balance of CNS
neurotransmitters (L-DOPA). DAO substrates include
histamine and polyamines.

26. Non-CYP drug oxidations
Flavin Monooxygenases
– The FMOs are another superfamily of phase 1 enzymes involved
in drug metabolism .
– the FMOs are expressed at high levels in the liver and are bound
to the endoplasmic reticulum,
– There are six families of FMOs, with FMO3 being the most
abundant in liver.
– Require molecular oxygen, NADPH, flavin adenosine
dinucleotide (FAD)
– FMOs are heat labile and metal-free, unlike CYPs
– FMOs are considered minor contributors to drug metabolism
cimetidine cimeditine S-oxide
HN N
S
N N
N
H H
CN HN N
S
N N
N
H H
CNO
FMO3

27. 27
Alcohol & Aldehyde Dehydrogenase - non-specific
enzymes found in soluble fraction of liver; ethanol metabolism
Xanthine Oxidase - converts hypoxanthine to xanthine, and
then to uric acid. Drug substrates include theophylline, 6-
mercaptopurine. Allopurinol is substrate and inhibitor of
xanthine oxidase; delays metabolism of other substrates;
effective for treatment of gout.
CH3CH2OH + NAD+  CH3CHO + NADH + H+
CH3CHO + NAD+  CH3COOH + NADH + H+

28. Hydrolysis
 Nonspecific esterases, amidases
 Amides hydrolyze more slowly than esters
 Occurs in cytosol of cells (liver, kidney, intestine) and in
plasma
Hydrolysis reactions add water across a bond to
produce a more water-soluble metabolite. Common
hydrolysis reactions include ester hydrolysis, amide
hydrolysis, and epoxide hydrolysis.

29. Reductions
Reduction reactions are less common than oxidation but
are still phase I reactions that add or reveal a functional
group to increase the water solubility of the molecule to
facilitate elimination.
There are several reductase enzymes and even CYP450 may
have reductase activity. Common reduction reactions
include the reduction of disulfi de bonds, carbonyls, and
nitro or azo functional groups.

30. 30
Phase II reactions are commonly called conjugation reactions
because they use a functional group on the xenobiotic (either
from phase I metabolism or part of the xenobiotic itself) to add or
conjugate a biomolecule that usually increases the polarity of the
xenobiotic and facilitates elimination from the body. These
conjugation reactions require an enzyme generally termed as
transferase that transfers a high-energy molecule called the
cofactor or cosubstrate to the xenobiotic. The transferred cofactor
is usually large and very polar-forming inactive metabolites.
Conjugation substrates =
– glucuronic acid, glutathione, sulfate, amino acid
(glycine) , acetic acid,
Functional groups which serve as linkers (“handle”) include:
alcohols, amines, phenols, carboxylic acids, and "free radical
intermediates"
Phase II Drug Metabolism

31. PHASE II REACTIONS
Glucuronidation
Sulfate Conjugation
Acetylation
Glycine Conjugation
Methylation
Transulfuration
Glutathione Conjugation
Mercapturic Acid Synthesis

32. Phase II Metabolism
Conjugation: Enzyme
• UDP-Glucuronosyl
transferases (UGT)
• Sulfotransferases (ST)
• Methyltransferases (MT)
• Acetyltransferases (NAT)
• Amino Acid Transferases
• Glutathione Transferases
(GST)
• Fatty acid conjugation
Contribution of Phase II
Enzymes to drug metabolism

33. Uridine diphosphate (UDP)
glucuronic acid
Glucuronidation = Quantitatively most important Phase II reaction.
Glucuronosyltransferase is the microsomal enzyme that uses
uridine diphosphate glucuronic acid (UDP-GA) as the cofactor to
transfer the glucuronic acid to several different functional groups,
including hydroxyl groups, carboxylic acid groups, hydroxylamines, and
sulfonamides. Th e glucuronic acid adds a significant amount of
hydrophilicity to the molecule and facilities its elimination in the urine.
Phase II Pathways: Glucuronidation
UGT
O-Glucuronide

34. UDP-glucuronosyltransferase:
Microsomal enzyme (SER) found in a
liver, lung, kidney, GI tract, heart &
brain
- 19 UGT genes spread between two
main families
UGT1 and UGT2
- Oligomers (1-4) with MW 50-60k
- Gilbert’s Syndrome – Defective
UGT1A1
Hyperbilirubinemia + Imparied glucuronidation
Abbreviations = UDPGT or UGT
Glucuronidation
UDP-glucuronosyltransferase

35. O- or N-
Glucuronidation
Foye’s Medicinal Chemistry, 4th Edition, 1995

36. Enterohepatic Recycling
 High molecular-weight
glucuronides (MW 500 Da) are
secreted into the bile, which
ends up in the intestine.
Drug-glucuronide complexes
excreted into gut can be broken
down by gut β-glucuronidase
(mostly in flora).
 The released drug can then
be reabsorbed into the body –
forming a cycle.
 Can prolong the half life of
drug in the body

37. Biosynthesis
SO4
2- + ATP ATP-sulfurylase adenosine-5’-phosphosulfate (APS) + PPi
APS + ATP
APS-kinase
3’-phosphoadenosine-5’-phosphosulfate (PAPS) + ADP
Critical route of metabolism for catecholamine neurotransmitters,
steroid hormones, thyroxine, and phenolic drugs.
Conducted by the soluble enzyme sulfotransferase located in the
cytosol and conjugate sulfate to the hydroxyl groups of aromatic
and aliphatic compounds.
Energy rich donor: Endogenous donor molecule to conjugation is
3’-phosphoadenosine-5’-phosphosulfate (PAPS)
PAPS
N
N
OP
N
N
O
O
OH
O
-
NH2
O
P OO
O
-
O-SO3
Sulfation
•Cytosolic enzyme
• Liver, kidney lung, gut, brain
• Homodimers with MW 32-34 kD

38. Sulfation of Acetaminophen
O
CH3
OH
H
H
H
O
CH3
HO3SO
H
H
H
Estrone
Sulfotransferas

39. 39
METHYLATION
Drugs and xenobiotics can undergo O-, N-, and S-methylation.
Humans express three N-methyltransferases, one catechol-O-
methyltransferase (COMT) a phenol-O-methyltransferase
(POMT), a thiopurine S-methyltransferase (TPMT), and a
thiol methyltransferase (TMT). MT conducts the donation of
the methyl group from the endogenously synthesized SAM to
various substrates to form methylated conjugates.
N
N
O
N
N
S
+
O
-
O
OH
NH2
OH
NH2
CH3
ATP + Methionine
S-adenosylmethionine (SAM)

40. 40
PMNT

41. Isoniazid
O
N
NH
NH2
O
N
NH
NHCOCH 3
+ CoASH
N-Acetyltransferase (NAT)
Acetyl-CoA
The cytosolic N-acetyltransferases (NATs) are
responsible for the metabolism of drugs and
environmental agents that contain an aromatic amine or
hydrazine group. The addition of the acetyl group from
the cofactor acetyl-coenzyme A often leads to a
metabolite that is less water soluble .

42. 42
Factors Affecting Drug Metabolism
 Age
 Genetic Variation
 State of Health
 Diet and Nutritional status
 Gender
 Degree of Protein Binding
 Species Variation
 Substrate Competition
 Enzyme Induction/Inhibition.
 Route of Drug Administration