METABOLISM/BIOTRANS

FORMATION

 Metabolism or biotransformation may be

defined as the conversion from one chemical
form of a substance to another.
Metabolism is an essential pharmacokinetic
process that renders the lipid soluble and
non-polar compounds to the water
soluble and polar compounds so that they
are easily excreted by various processes.
If a lipid soluble substance is not converted
into a water-soluble form, it may remain in
body for very long period -precipitate toxic
reaction.

 Biotransformation: It is a specific term used

for chemical transformation of xenobiotics in
the body/living organism.
Metabolism: It is a general term used for
chemical transformation of xenobiotics and
endogenous
nutrients
(e.g.
proteins,
carbohydrates and fats) within or outside the
body.

Functions of
Metabolism

It causes conversion of an active drug to

inactive or less active metabolite(s) called
inactivation or pharmacological inactivation
e.g. Phenobarbital, Amfetamine, Morphine,
Phenytoin and Propranolol.
It causes conversion of an active drug to more
active metabolite(s) called bioactivation or
toxicologicai activation e.g. codeine, isoniazid
and malathion.

 It causes conversion of an inactive drug (prodrug)

to
active
metabolite(s)
called
pharmacological activation e.g. enalapril,
phenacetin, pivampicillin and levodopa.
It causes conversion of an active drug to
equally active metabolite(s) (no change in
pharmacological
activity)
e.g.
digitoxin,
diazepam, amitriptyline and phenylbutazone.
It causes conversion of an active drug to
active
metabolite(s)
having
entirely
different pharmacological activity (change
in pharmacological activity) e.g. iproniazid

Sites of Metabolism
Liver is the primary site for metabolism
(rich in a large variety of metabolising
enzymes).
However, all tissues in body carry some
degree of metabolic activities. Metabolism
by organs other than liver (called extrahepatic
metabolism)
is
of
lesser
importance.
Within a given cell, most drug metabolising
activity is found in the smooth endoplasmic
reticulum and cytosol.

 Drug metabolism can

mitochondria, nuclear
plasma membrane.

also occur in
envelope and

A few drugs are also metabolised by nonenzymatic means called non-enzymatic

metabolism. For example, atracurium, a
neuromuscular
blocking
drug,
is
inactivated in plasma by spontaneous
non-enzymatic degradation (Hoffman
elimination) in addition to that by
pseudocholinesterase enzyme.

Drug Metabolising Enzymes

A large variety of enzymes -are located
mainly in the liver, but may also be present
in other organs like lungs, kidneys, intestine,
brain, plasma, etc.
The drug metabolising enzymes can be
broadly divided into two classes: microsomal
and non-microsomal enzymes.

Microsomal enzymes
located within the lipophilic membranes of the
smooth endoplasmic reticulum (SER) of liver and
other tissues.
The endoplasmic reticular enzymes are called
microsomal enzymes because when the SER is
isolated in laboratory by tissue homogenation and
differential centrifugation, the cells are disrupted
and the SER membrane re-forms into minute
spherical vesicles called microsomes; all enzymes
present in vesicles thus becomes microsomal
enzymes.
Microsomal enzymes are the most important group
of enzymes which catalyze glucuronide conjugation,
most oxidative reactions, and some reductive and
hydrolytic reactions.

Non-microsomal enzymes
Enzymes occurring in organelles/ sites other than
endoplasmic reticulum (microsomes) are called nonmicrosomal enzymes.
These are mostly present in the cytoplasm,
mitochondria, body fluids etc. and occur mainly in the
liver, GI tract, plasma and other tissues.
They are usually non-specific enzymes which catalyse
few oxidative reactions, some reductive and hydrolytic
reactions and all conjugative reactions other than
glucuronidation.
None of the non-microsomal enzymes involved in drug
biotransformation is known to be inducible.

PATHWAYS OF
BIOTRANSFORMATION
Biotransformation of drugs has been divided into two
major pathways or phases: Phase I and phase II.
Phase I
Phase I (Non-synthetic or non-conjugative phase)
includes reactions which catalyse oxidation,
reduction and hydrolysis of drugs.
In phase 1 reactions, small polar functional
groups like -OH, -NH2, -SH, -COOH, etc. are either
added or unmasked (if already present) on the
otherwise lipid soluble drugs so that the resulting
products may either undergo phase II reactions or
are directly excreted.
Phase I reactions generally precede phase II

 Phase II
Phase II (Synthetic or conjugative phase)
includes reactions those involve attachment of
polar endogenous molecules such as glucuronic
acid, sulphate, methyl, amino acids, etc., to
either parent drugs or their phase I products.
Phase II products, generally called conjugates,
are
generally
water-soluble
and
polar
metabolites those are readily excreted from the
body.
The products of phase II biotransformation may
be further metabolized in body (sometimes
termed phase III reactions) before final
excretion.

Oxidation
Oxidative reactions
metabolic reactions

are

the

most

important

The oxidative reactions are important for drugs

because they enhance hydrophilicity of drugs by
introducing polar functional groups such as -OH.
Oxidation of drugs is non-specifically catalysed by
a number of enzymes located primarily in the
microsomes. Some of the oxidative reactions are
also catalysed by non-microsomal enzymes (e.g.
aldehyde dehydrogenase, xanthine oxidase and
monoamine oxidase).

 The most important group of oxidative enzymes are

microsomal mixed function oxidases (MFO) or
monooxygenases.
These enzymes are located mainly in the hepatic
endoplasmic reticulum (microsomes) and require
both molecular oxygen (02) and reducing NADPH to
effect the chemical reaction.
The most important component of mixed function
oxidases is the cytochrome P-450 (CYP-450), a
microsomal superfamily of isoenzymes that catalyze
the phase I oxidation of many xenobiotics.
In humans up to 18 families, 43 subfamilies and 57
genes of CYPs have been described. Out of these,
CYP I, 2 and 3 account for 70% of total hepatic CYPs
content and are responsible for more than 90% of
drugs metabolism in liver; the isoenzymes

Reduction
Reduction is the acceptance of one or more
electrons or their equivalent from another
substrate.
Reductive reactions, which usually involve
addition of hydrogen to the drug molecule, occur
less frequently than the oxidative reactions.
Biotransformation by reduction is also capable of
generating polar functional groups such as
hydroxy and amino groups, which can undergo
further biotransformation.

Hydrolysis
The hydrolytic reactions, contrary to
oxidative or reductive reactions, do not
involve change in the state of oxidation of
substrate, but involve the cleavage of drug
molecules by taking up a molecule of water.
A number of drugs with ester, ether, amide
and hydrazide linkages undergo nydrolysis.
Important
examples
are
cholinesters,
procaine, procainamide and pethidine.

Phase II Reactions
Phase II or Conjugation (Latin word conjugatus
=
yoked
together)
reactions
involve
combination of the drug or its phase I
metabolite with an endogenous substance to
form a highly polar product, which can be
efficiently excreted from the body.
In the biotransformation of drugs, such products
or metabolites carry two parts: exocon, the
portion derived from exogenous compound or
xenobiotic, and endocon, the portion derived
from endogenous substance.
The molecular weight of the conjugate
(metabolite) determines mainly its route of
excretion. High molecular weight conjugates are
excreted predominantly in bile (e.g. glutathione

Glucuronidation
Conjugation with glucuronic acid (glucuronide
conjugation or glucuronidation) is the most common
and most important phase II reaction in vertebrates,
except cats and fish.
It is the only conjugation reaction that occurs in the
liver microsomal enzyme system.
The source of glucuronide is Uridine diphosphate a-Dglucuronic
acid
(UDPGA)
through
glucuronyl
transferase.
The product is highly soluble and is excreted by urine
(if MW<300 500) or via bile ( if >500).
Beta -glucuronidase in intestine may liberate the
active drug which is again reabsorbed and undergoes
enterohepatic recirculation which leads to longer
duration of action of the drug.
In cats, there is reduced glucuronyl transferase
activity, while in fish there is deficiency of
endogenous glucuronic acid donor.

CONJUGATION WITH SULPHATE/SULPHATION

Conjugation with sulphate is similar to glucuronidation but
is catalyzed by non-microsomal enzymes and occurs
less commonly.
The endogenous donor of the sulphate group is 3'phosphoadenosine-5-phosphosulphate (PAPS) and
enzyme catalysing the reaction is sulphotransferase.
The conjugates of sulphate are referred to as sulphate
ester conjugates or ethereal sulphates. Sulphate esters
are polar and readily excreted in urine.
Unlike glucuronide conjugation, sulphoconjugation in
mammals is less important because the PAPS donor that
transfers sulphate to the substrate is easily depleted.
In cats, where glucuronidation is minimum, sulphate
conjugation is important.
Functional groups capable of forming sulphate conjugates
include phenols, alcohols, arylamines, N-hydroxylamines

CONJUGATION WITH METHYL

GROUP/METHYLATION

Conjugation with methyl group (Methyl conjugation

or methylation) involves transfer of a methyl group
(-CH3) from the cofactor S-adenosyl methionine
(SAM) to the acceptor substrate by various methyl
transferase enzymes.
Methylation reaction is of lesser importance for
drugs, but is more important for biosynthesis (e.g.
epinephrine and melatonin) and inactivation (e.g.
histamine) of endogenous amines.
Occasionally the metabolites formed by methylation
are not polar or water-soluble and may possess
equal or greater activity than the parent compound

CONJUGATION WITH GLUTATHIONE AND

MERCAPTURIC ACID FORMATION
Conjugation

with

glutathione

(Glutathione

conjugation) and mercapturic acid formation is a

minor but important metabolic pathway in animals.
Glutathione (GSH, G=glutathione and SH = active -SH
group) is a tripeptide having glutamic acid, cysteine
and glycine.
It has a strong nucleophilic character due to the
presence of a -SH (thiol) group in its structure.

 Thus it conjugates with electrophilic substrates, a

number of which are potentially toxic compounds
(superoxides, epoxides and toxic intermediates) and
protects the tissues from their adverse effects (highly
electrophilic substrates have a tendency to react
with

tissue

toxicity

nucleophilic

from

tissue

groups
necrosis,

and

precipitate

carcinogenesis,

mutagenesis, etc.)
The interaction between the substrate and the GSH
is

catalysed

by

enzyme

glutathione-S-

transferase, which is located in the soluble fraction

of liver homogenates.

CONJUGATION WITH ACETYL

GROUP/ACETYLATION

Conjugation with acetyl group (Acetylation) is an

important metabolic pathway for drugs containing
the amino groups.
The cofactor for these reactions is acetyl coenzyme A
and the enzymes are non-microsomal N-acetyl
transferases, located in the soluble fraction of cells of
various tissues.
Acetylation is the primary route of biotransformation
of sulphonamide compounds.
Dogs and foxes do not acetylate the aromatic amino

CONJUGATION WITH
AMINO ACIDS
Conjugation with amino acids occurs to a limited
extent in animals because of limited availability
of amino acids in body.
The

most

important

reaction

involves

conjugation with glycine.

Conjugation

with

other

amino

acids

like

glutamine in man and ornithine in birds is also

seen.
Examples of drugs forming glycine or glutamine

CONJUGATION WITH
THIOSULPHATE
Conjugation with thiosulphate is an important reaction
in the detoxification of cyanide.
Conjugation of cyanide ion involves transfer of sulphur
atom from the thiosulphate to the cyanide ion in
presence of enzyme rhodanese to form inactive
thiocyanate.
Thiocyanate formed is much less toxic than the
cyanide (true detoxification) and it is excreted in urine.

Induction of Drug
Metabolising Enzymes

Several drugs -able to increase the drug

metabolizing activity of certain enzymes referred to
as enzyme induction.
These drugs known as enzyme inducers mainly
interact with DNA and cause an increase in liver
weight, proliferation of the smooth endoplasmic
reticulum and enhance synthesis of microsomal
enzyme proteins, especially cytochrome P-450 and
glucuronyl transferase.
As a result, there is enhanced metabolism of those
endogenous substances (e.g. sex steroids) and drugs
those are metabolized by microsomal enzymes.
Some drugs (e.g. carbamazepine and rifampicin)

 Since
different
cytochrome
P-450
isoenzymes
are
involved
in
the
metabolism of different drugs, enzyme
induction by one drug affects metabolism
of only those drugs which are substrate
for the induced isoenzyme.
Some drugs like phenobarbital may affect
metabolism of a large number of
endogenous
substances
and
drugs
because they induce isoenzymes like
CYP3A and CYP2D6 which act on many

CLINICAL IMPORTANCE OF ENZYME

INDUCTION
It reduces efficacy and potency of drugs
metabolised by these enzymes.
It reduces plasma half-lives and duration
of action of drugs.
It enhances drug tolerance.
It increases drug toxicity by enhancing
concentration of metabolite, if metabolite
is toxic. It increases chances of drug
interactions.
It alters physiological status of animal
due to altered metabolism of endogenous
compounds like sex steroids.

INHIBITION OF DRUG
METABOLISING ENZYMES
Several drugs or chemicals have the ability to
decrease the drug metabolising activity of certain
enzymes known as enzyme inhibition.
Enzyme inhibition can be either non-specific of
microsomal enzymes or specific of some nonmicrosomal enzymes (e.g. monoamine oxidase,
cholinesterase and aldehyde dehydrogenase).
The inhibition of hepatic microsomal enzymes mainly
occurs due to administration of hepatotoxic agents,
which cause either rise in the rate of enzyme
degradation (e.g. carbon tetrachloride and carbon
disulphide) or fall in the rate of enzyme synthesis
(e.g. puromycin and dactinomycin).

 Enzyme inhibition generally results in depressed

metabolism of drugs. As a result, the plasma halflife, duration of action, and efficacy as well toxicity
of the object drug (whose metabolism has been
inhibited) are significantly enhanced.
In case the drug undergoes hepatic first-pass effect,
the bioavailability and toxicity of the drug will be
markedly increased in presence of enzyme
inhibition.
In therapeutics, some specific enzyme inhibitors like
monoamine
oxidase
inhibitors,
cholinesterase
inhibitors and angiotensin converting enzyme (ACE)
inhibitors are purposely used for producing desirable
pharmacological actions.

First-pass metabolism/ First-pass effect

First-pass
effect
(First-pass
metabolism
or
presynthetic metabolism) may be defined as the loss
of drug through biotransformation before it
enters systemic circulation.
Intestinal first-pass effect:
In this type, drugs are metabolized in the GIT by
enzymes present in gut mucosa or gut lumen
before they are absorbed.
Microorganisms present in the GI tract also
inactivate some drugs. Such drugs are not
suitable by oral administration due to poor
bioavailability
Eg: adrenaline, nor-adrenaline
Hepatic first-pass effect:
In this type, drugs are suitably absorbed across

 Enterohepatic recirculation
Some drugs after biotransformation by
glucuronide conjugation, undergo biliary
excretion.
In the large intestine they are detached
from the conjugate, leading to release of
free drug which is absorbed again and
enters liver. Thus the drug recirculates
leading to longer duration of action.
Eg: Phenolphthalein used as purgative.