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Drug metabolism
Topics covered
Introduction
Site of drug metabolis
Metabolic enzymes
Phases of metabolism
Phase I metabolism
Cytochrome P450 enzymes
Oxidation Reactions
Reduction Reactions
Hydrolysis
Phase II metabolism
Glucuronide conjugates
Sulfate conjugates

Glycine and glutamate conjugates

Glutathione conjugates
Acetylation
Methylation
Factors affecting drug metabolism
Internal factors
External factors
Administration factors

Drug metabolism;
Generates more polar (water soluble), inactive 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.
Most metabolic products are less pharmacologically active.
Important exceptions:
Where the metabolite is more active (Prodrugs, e.g. Erythromycin-succinateErythromycin).
Where the metabolite is toxic, carcinogenic (acetaminophen).
There is close relationship between the biotransformation of drugs and normal
biochemical processes occurring in the body. Many of the enzymes involved in drug
metabolism are principally designed for the metabolism of endogenous compounds.
These enzymes metabolize drugs only because the drugs resemble the natural
compound.

Sites of action
Drug metabolism can occur in all tissues and most biological fluids. However, the
widest range of metabolic reactions occurs in the liver. A more substrate selective
range of metabolic processes takes place in the kidney, lungs, brain, placenta and
other tissues. Orally administered drugs may be metabolized as soon as they are
ingested. Figure 2.1
.

Figure 2.1 Sites of drug metabolism

Metabolic enzymes
Microsomal:
CYP450 monooxygenases (CYP=Cytochrome)
Flavin monooxygenase
Non-microsomal
Alcohol dehydrogenase
Aldehyde dehydrogenase
Monoamine and diamine oxidases
Both
Esterases and Amidases
Prostaglandin synthase Peroxidases

Phases of drug metabolism

PHASE I or
FUNCTIONALIZATION REACTIONS
Introduction of functional group
Hydrophilicity increases slightly.
May inactivate or activate original compound.
Major player is CYP or mixed function oxygenase (MFO) system in conjunction
with NAD(P)H.
Location of reactions is smooth endoplasmic reticulum.

Oxidation Reactions
Oxidation of Aromatic Moieties
Oxidation of Olefins
Oxidation of Benzyclic, Allylic carbon atoms, Carbon atoms to carbonyl and imines
Oxidation of Aliphatic and Alicyclic carbon atoms.
Oxidation of Carbon-heteroatom systems:

* Caron-nitrogen system
*Carbon-Sulfur system
* Caron-Oxygen system
Oxidation of Alcohols and Aldehydes.
Other miscellaneous oxidative reactions

Reduction Reactions
Reduction of aldehydes and ketones
Reduction of Nitro and Azo compounds
Miscellaneous Reductive reactions
Hydrolytic Reactions
Hydrolysis of Esters and Amides
Hydration of Epoxides and arene oxide by epoxide hydrase
Other Phase I reactions.
Alkylation (Methylation)
Dealkylation

Ring cyclization
N-carboxylation
Dimerization
Transamidation
Isomerization
Decarboxylation

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PHASE II or CONJUGATION REACTIONS

Conjugation with endogenous molecules (GSH, glycine, cystein, glucuronic acid)
Hydrophilicity increases substantially
Neutralization of active metabolic intermediates
Facilitation of elimination
Location of reactions is cytoplasm
Glucuronidation by UDP-Glucuronosyltransferase: (on -OH, -COOH, -NH2 , -SH
groups)
Sulfation by Sulfotransferase: (on NH2, -SO2 NH2 , -OH groups)
Acetylation by acetyltransferase: (on -NH2 , -SO2 NH2 , -OH groups)
Amino acid conjugation: (on -COOH groups)
Glutathione conjugation by Glutathione-S-transferase: (to epoxides or organic
halides)
Fatty acid conjugation: (on -OH groups)
Condensation reactions
Phase I usually precede phase II reactions

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Drug metabolism pathways

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Phase I reactions
Drug Metabolism Oxidation
Two types of oxidation reactions:
Oxygen is incorporated into the drug molecule (e.g. hydroxylation)
Oxidation causes the loss of part of the drug molecule (e.g. oxidative deimination,
dealkylation)
Microsomal Mixed Function Oxidases (MFOs)
Microsomes
form in vitro after cell homogenization and fractionation of endoplasmic
reticulum
Mixed Function Oxidases or Monooxygenases
These enzymes require a reducing agent (NADPH) and molecular oxygen
(one oxygen atom appearing in the product and the other in the form of water)
MFO consists of two enzymes:
1. Flavoprotein, NADPH-cytochrome c reductase

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One mole of this enzyme contains one mole each of flavin mononucleotide
(FMN) and flavin adenine dinucleotide (FAD)
Enzyme is also called NADPH-cytochrome P450 reductase
2. Cytochrome P450 enzymes
Cytochrome P450 enzymes
CYP enzymes have been identified in all domains of life, i.e., in animals, plants,
fungi, bacteria, archaea, and even viruses.
More than 11500 distinct CYP proteins are known.
Named based on its light absorption at 450 nm when complexes with carbon
monoxide
Human CYPs are primarily membrane-associated proteins, located either in the
inner membrane of mitochondria or in the endoplasmic reticulum of cells.
CYP is a hemoprotein containing an iron atom which can alternate between the
ferrous (Fe++) and ferric (Fe+++) states

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Electron acceptor
Serves as terminal oxidase
Its relative abundance compared to NADPH-cytochrome P450 reductase makes it
the rate-limiting step in the oxidation reactions
Involved in around ~75% of the total metabolism.

Structure of Cytochrome P450

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CYP Nomenclature
Families - CYP plus arabic numeral (>40% homology of amino acid sequence, eg.
CYP1)
Subfamily Alphabet ( 40-55% homology of amino acid sequence; eg. CYP1A)
Isoform (gene) - additional arabic numeral when more than 1 subfamily has been
identified; (eg. CYP1A2)
Format of nomenclature:
CYPFamily/Subfamily/Gene
Family = 1, 2, 150 and counting
Subfamily = A, B,H
Gene (Isoform) = 1, 2..10 or above
Families grouped in Clans

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Figure Format of nomenclature

CYP Families
Humans have 18 families of cytochrome P450 genes and 43 subfamilies:
CYPs have molecular weights of 45-60 kDa.
Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families.
Key forms: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4

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CYP3A4 is very common to the metabolism of many drugs; its presence in the GI
tract is responsible for poor oral availability of many drugs

Figure 2.5 Proportion of drugs metabolized by different families of CYPs.

Drug oxidation requires:
Cytochrome P450
Cytochrome P450 reductase
NADPH
Molecular oxygen

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The cycle involves four steps:

1. Oxidized (Fe3+) cytochrome P-450 combines with a drug substrate to form a binary
complex. 2. NADPH donates an electron to the cytochrome P-450 reductase, which in
turn reduces the oxidized cytochrome P-450-drug complex. 3. A second electron is
introduced from NADPH via the same cytochrome P-450 reductase, which serves to
reduce molecular oxygen and form an "activated oxygen"-cytochrome P-450-substrate
complex. 4. This complex in turn transfers "activated" oxygen to the drug substrate to
form the oxidized product. The potent oxidizing properties of this activated oxygen
permit oxidation of a large number of substrates.

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Drug oxidation cycle (cytochrome P450)

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Participation of the CYP Enzymes in Metabolism of Drugs

CYP
Examples of substrates
Enzyme
1A1

Caffeine, Testosterone, R-Warfarin

1A2

Acetaminophen,
Warfarin

2A6

17-Estradiol, Testosterone

2B6

Cyclophosphamide,
Testosterone

2Cfamily

Acetaminophen,
Tolbutamide
(2C9);
Hexobarbital, S- Warfarin (2C9,19); Phenytoin,
Testosterone, R- Warfarin, Zidovudine
(2C8,9,19);

Caffeine,

Phenacetin,

R-

Erythromycin,

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2E1

Acetaminophen,
Halothane

Caffeine,

Chlorzoxazone,

2D6

Acetaminophen, Codeine, Debrisoquine

3A4

Acetaminophen, Caffeine, Carbamazepine,

Codeine,
Cortisol,
Erythromycin,
Cyclophosphamide, S- and R-Warfarin,
Phenytoin,
Testosterone,
Halothane,
Zidovudine

Oxidation reactions NOT catalyzed by Cytochrome P450:

Flavin containing monoxygenase system
Present mainly in liver but some is expressed in gut and lung
Located in smooth endoplasmic reticulum
Oxidizes compounds containing sulfur and nitrogen
Uses NADH and NADPH as cofactors
Alcohol dehydrogenase (cytosol)
Aldehyde oxidation (cytosol)

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Xanthine oxidase
Amine oxidases
Monoamine oxidase (nerve terminals, mitochondria)
Diamine oxidase found in liver microsomes
Primarily endogenous metabolism
Monoamine Oxidases (MAO):
Catalyze oxidative deamination of endogenous catecholamines (epinephrine)
Located in nerve terminals and peripheral tissues
Substrates for catecholamine metabolism found in foods (tyramine) can cause a
drug/food interaction
Inhibited by class of antidepressants called MAO inhibitors

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Metabolic Oxidation reactions:

Aromatic Hydroxylation
Many drugs possess aromatic rings
Hydroxylation of these rings is a major route of drug metabolism
Hydroxylation occurs most readily on rings that are electron-rich, i.e. those that have
electron-donating groups directly attached to the ring (OH, OCH3, NH2, alkyl groups)
The presence of electron-withdrawing groups like (F, Cl, Br, I), nitro, nitriles,
carbonyls, sulfoxides and sulfones. on the ring usually inhibits hydroxylation on that
ring.
MFOs metabolize aromatic rings by epoxidation of electron-rich p-bonds in the rings.
The resulting epoxides(3-membered ring ethers) are called arene oxides

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Metabolic Oxidation of Alkenes

The oxidative metabolism of olefins (alkenes) is identical to that of aromatics except
that the epoxides that are formed have little tendency to undergo rearrangement.
Most typically, they are attacked by epoxide hydrolaseto give trans-dihydrodiols.

Metabolic Oxidation of Alkyl Groups

Aliphatic carbons are also subject to metabolic oxidation.
Oxidation of aliphatic carbons that are adjacent to (at an a-position) a functional
group
Oxidation of aliphatic carbons at or near the end of a chain of aliphatic carbons.

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The product of these oxidation reactions is an alcohol.

The carbon undergoing metabolism must have at least one attached hydrogen.

-Oxidations
The carbon adjacent to a functional group is the -carbon. The carbons shown in
bold-face type below are all -carbons:
The result of these oxidations is that an oxygen atom is inserted into a C-H bond to
give a C-OH group.

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Metabolic Oxidation of Alcohols

The alcohol functional group is present in many drugs and is also formed by
metabolic hydroxylation of aliphatic carbons.
The enzyme alcohol dehydrogenase catalyzes the oxidation of alcohols to carbonyl
compounds.
Primary alcohols are oxidized to aldehydes and secondary alcohols are oxidized to
ketones.
Aldehydes are highly reactive and undergo rapid metabolic oxidation to carboxylic
acids.
Tertiary alcohols and ketones cannot be further oxidized.

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Metabolic Oxidation of Sulfides and Sulfoxides

Sulfides can be metabolically oxidized by MFO to sulfoxides and to sulfones.
Sulfoxides can be oxidized to sulfones.
Sulfones, in which sulfur is already in its highest oxidation state cannot be oxidized
any further.

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Metabolic Oxidation at sp2 Nitrogen

The MFO system is also capable of oxidizing sp2 nitrogen as found in imines and
certain aromatic heterocycles such as pyridine and quinoline.
The products of these oxidations are called imine oxides.

Metabolic Dealkylation
Many drugs contain a heteroatom such as nitrogen, oxygen, or sulfur that is attached
to an alkyl group.
When such drugs undergo metabolism these alkyl groups may be removed in a
process called N-(or O-or S-)-dealkylation.
The enzymes that are responsible for this are called monoamine oxidases (MAOs).
MAOs contain flavin, which is a compound that oxidizes other compounds by
removing a single electron from the nitrogen via free-radical mechanism.
For tertiary amines (R-NR2) N-dealkylation can remove either one alkyl group to
give a secondary amine (R-NHR), or two alkyl groups to give a primary amine

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If a drug contains a cyclic amine such as a piperidine unit, it is also subject to

metabolism by MAO.

Metabolic Reductions
Reduction is a lowering of the oxidation state of a group by adding electrons (usually
as a hydride (H-) ion).

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Functional groups that most typically undergo metabolic reduction include:

Ketones
Nitro groups
Azo groups
Less commonly, aldehydes and sulfoxides can be reduced.
Thus a group that has one double bond gets reduced to a species containing only a
single bond (C=O .CH-OH).
Metabolic Reduction of Carbonyl Groups

only ketones and aldehydes usually undergo metabolic reduction to alcohols.

Ketones however, are resistant to further oxidation. They are readily reduced
metabolically to secondary alcohols.

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Nitro groups readily undergoes metabolic reduction.

Metabolic Reduction of Azo Groups

An azo compound is one which has a N=N bond within its structure.
Bacteria within the intestine have nitro-reductases that can reduce azo compounds to
amines.

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Metabolic Reduction of Sulfoxides

As with aldehydes, sulfoxides are most commonly oxidized to sulfones by
metabolism.
Occasionally they may undergo metabolic reduction to sulfides.

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2.3.1.3 Hydrolysis
Hydrolysis is the process of breaking bonds by the addition of water.
Esterases and amidases are present in plasma and other tissues in the body
Functional groups that are most often metabolized by hydrolysis include esters (and
lactones) and amides (and lactams).
Hydrolysis of esters ALWAYS results in two products a carboxylic acid and an
alcohol.
Hydrolysis of amides ALWAYS results in two products a carboxylic acid and an
amine.
Hydrolysis of cyclic esters and amides (lactones and lactams) results in ring opening,
giving a compound having a carboxylic acid at one end of the chain and an alcohol or
amine at the other.

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Phase II Metabolism
Conjugation Reactions
During phase II metabolism parent compounds and phase I metabolites are either
detoxified or converted into more water-soluble entities that can then be excreted.
Common classes of phase II metabolisms include:
Glucuronide conjugates
Sulfate conjugates
Glycine and glutamate conjugates
Glutathione conjugates
Acetylation
Methylation

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Glucuronide Conjugates
Among the most common phase II metabolites
Body has large supply of glucuronic acid, which is made from D-glucose.
Functional groups susceptible to glucuronidation include: alcohols and phenols,
carboxylic acids, amines, thiols
Formation of glucuronide conjugates: glucose (UDPG)

The carboxylic acid and three hydroxy groups of the glucuronide conjugate imparts
considerable water solubility to these metabolites.

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Sulfate Conjugates
Sulfate conjugates are formed mainly from phenols, aromatic alcohols and aromatic
amines.
There is less available sulfate in the body than glucuronic acid.
The coenzyme 3-phosphoadenosine-5-phosphosulfate (PAPS) is responsible for
transferring a sulfate to a suitable substrate in the presence of the enzyme
sulfotransferase.

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Glycine and Glutamine Conjugates

Glycine and glutamine form conjugates with carboxylic acids.
The products are carboxyamides, and are more water-soluble than the carboxylic
acids

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Glutathione Conjugates

Glutathione (GSH) conjugation is a means by which the body can detoxify reactive
electrophilic species.
Glutathione possesses a nucleophilic thiol (SH) group that can react with electrophiles
Glutathione with electrophiles reaction is catalyzed by the enzyme Glutathione Stransferase (GST).

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Acetylation
Primary amines undergo acetylation of the amino group to give acetamides.
acetylation does not necessarily lead to more water-soluble metabolites.
Acetylation serves as a means to terminate the activity or detoxify primary amines.
Acetyl-CoA, in the presence of N-acetyl transferases adds an acetyl group onto the
amine.

Methylation
Methylation is a relatively minor phase II metabolic pathway.
Among the groups that undergo this reaction are phenols, catechols (orthodihydroxyaromatic compounds), amines, and thiols.
Methylation does not increase water solubility, but it does usually render the
metabolite biologically inactive.
Monomethylation of catechols is a common metabolic pathway.

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Factors affecting drug metabolism

Internal factors
Age, sex, genetics, diseases, Pregnancy, Body temperature, flora of intestinal
tract, emotional mood and level of enzyme activity.
1. Age. The ability to metabolize drugs is lower in the very young (under 5) and
the elderly (over 60). In the fetus and the very young (neonates), many metabolic
routes are not fully developed. This is because the enzymes required by metabolic
processes are not produced in sufficient quantities until several months after birth.
Children (above 5) and teenagers usually have the same metabolic routes as adults.
2. Sex. Responsiveness to certain drugs is different for men and women
Pregnancy induction of certain drug metabolizing enzymes occurs in second and
third trimester. Hormonal changes during development have a profound effect on drug
metabolism. The metabolic pathway followed by a drug is normally the same for both
males and females. However, some sex related differences in the metabolism of
anxiolytics, hypnotics and a number of other drugs have been observed.
3. Genetic variations. Variations in the genetic codes of individuals can result in
the absence of enzymes, low concentrations of enzymes or the formation of enzymes
with reduced activity. These differences in enzyme concentration and activity result in

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individuals exhibiting different metabolic rates and in some cases different

pharmacological responses for the same drug.
Due to genetic diversity,
Reduction in catalytic ability.
Enhanced activity.
Extensive metabolizers (EMs) have functional enzyme activity.
Intermediate metabolizers (IMs) have diminished enzyme activity.
Poor metabolizers (PMs) have little or no activity.
5-10% of Caucasians and 1-2% of Asians exhibit the PM phenotype
4. Species and metabolism: Different species often respond differently to a drug.
This is believed to be due to differences in metabolism between species. These
metabolic differences may take the form of either different metabolic pathways for the
same compound or different rates of metabolism when the pathway is the same.
5. Disease Factors
Liver Disease Cirrhosis, Alcoholic liver disease, jaundice, carcinoma.
Dysfunction can lead to impaired drug metabolism-decreased enzyme activity.
Cardiac failure causes decreased blood flow to the liver.
Hormonal diseases, infections and inflammation can change drug metabolizing
capacity.

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External factors:
Llifestyle, Poor diet, drinking, smoking, drug abuse, temperature, humidity, light,
other radiations, season, time of day and habitat.
Administration factors
Route of administration, site of administration, volume of administration,
Compostion of excipents, number of doses and frequency of medication