Drug Metabolism

METABOLISM OR BIOTRANSFORMATION
 The conversion from one chemical form of a substance to another.

 The term metabolism is commonly used probably because products of drug

transformation are called metabolites.

 Metabolism is an essential pharmacokinetic process, which renders lipid soluble and

non-polar compounds to water soluble and polar compounds so that they are
excreted by various processes.
 This is because only water-soluble substances undergo excretion, whereas lipid
soluble substances are passively reabsorbed from renal or extra renal excretory sites
into the blood by virtue of their lipophilicity.

 Metabolism is a necessary biological process that limits the life of a substance in the
body.

 Biotransformation: It is a specific term used for chemical transformation of

xenobiotics in the body/living organism.
• a series of enzyme-catalyzed processes—that alters the physiochemical properties of
foreign chemicals (drug/xenobiotics) from those that favor absorption across biological
membranes (lipophilicity) to those favoring elimination in urine or bile (hydrophilicity )
 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.

 Xenobiotics : These are all chemical substances that

are not nutrient for body (foreign to body) and which
enter the body through ingestion, inhalation or dermal
exposure.

 They include :
drugs, industrial chemicals, pesticides, pollutants,
plant and animal toxins, etc.
 Functions of Biotransformation

It causes conversion of an

active drug to inactive or less
active metabolite(s) called as
pharmacological
inactivation.

It causes conversion of an

active to more active
metabolite(s) called as
bioactivation or toxicological
activation.

• It causes conversion of an
inactive to more active toxic
metabolite(s) called as lethal
synthesis
 Functions of Biotransformation….contd

• It causes conversion of an
inactive drug (pro-drug) to
active metabolite(s) called as
pharmacological activation

• It causes conversion of an
active drug to equally active
metabolite(s) (no change in
pharmacological activity)

• It causes conversion of an
active drug to active
metabolite(s) having entirely
different pharmacological
activity (change in
pharmacological activity) 
 Site/Organs of drug metabolism
The major site of drug metabolism is the liver
(microsomal enzyme systems of hepatocytes)
Secondary organs of biotransformation
• kidney (proximal tubule)
• lungs (type II cells)
• testes (Sertoli cells)
• skin (epithelial cells); plasma. nervous tissue
(brain); intestines
Sites of Biotransformation…contd
Liver
 The primary site for metabolism of almost all drugs because it is relatively
rich in a large variety of metabolising enzymes.

 Metabolism by organs other than liver (called as extra-hepatic metabolism)

is of lesser importance because lower level of metabolising enzymes is
present in such tissues.

 Within a given cell, most drug metabolising activity is found in the smooth
endoplasmic reticulum and the cytosol.
 Drug metabolism can also occur in mitochondria, nuclear envelope and
plasma membrane.

 A few drugs are also metabolised by non-enzymatic means called as 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.
Subcellular Locations of Metabolizing Enzymes

• ENDOPLASMIC RETICULUM (microsomes): the primary location for the

metabolizingenzymes.
• (a) Phase I: cytochrome P450, flavin-containing monooxygenase, aldehydeoxidase,
carboxylesterase, epoxide hydrolase, prostaglandin synthase, esterase.
• (b) Phase II uridine diphosphate-glucuronosyltransferase, glutathione S-
transferase, amino acid conjugating enzymes.
• CYTOSOL (the soluble fraction of the cytoplasm): many water-soluble enzymes.
• (a) Phase I: alcohol dehydrogenase, aldehyde reductase, aldehyde dehydrogenase,
epoxide hydrolase, esterase.
• (b) Phase 11: sulfotransferase, glutathione S-transferase, N-acetyl transferase,
catechol 0-methyl transferase, amino acid conjugating enzymes.

• MITOCHONDRIA.
• (a) Phase I: monoamine oxidase, aldehyde dehydrogenase, cytochrome P450.
• (b) Phase II: N-acetyl transferase, amino acid conjugating enzymes.
• LYSOSOMES. Phase I: peptidase.
• NUCLEUS.
• Phase II: uridine diphosphate-glucuronosyltransferase (nuclear membrane of
enterocytes).
Drug Metabolising Enzymes

A number of enzymes in animals are capable of metabolising

drugs. These enzymes are located mainly in the liver, but may
also be present in other organs like lungs, kidneys, intestine,
brain, plasma, etc.

Majority of drugs are acted upon by relatively non-specific

enzymes, which are directed to types of molecules rather than
to specific drugs.

The drug metabolising enzymes can be broadly divided into two

groups: microsomal and non-microsomal enzymes.
 Microsomal enzymes: The endoplasmic reticulum (especially
smooth endoplasmic reticulum) of liver and other tissues
contain a large variety of enzymes, together called microsomal
enzymes

 (microsomes are minute spherical vesicles derived from

endoplasmic reticulum after disruption of cells by
centrifugation, enzymes present in microsomes are called
microsomal enzymes).

 They catalyse glucuronide conjugation, most oxidative

reactions, and some reductive and hydrolytic reactions.

 The monooxygenases, glucuronyl transferase, etc are

important microsomal enzymes.
Non-microsomal enzymes: Enzymes occurring in
organelles/sites other than endoplasmic reticulum
(microsomes) are called non-microsomal enzymes.

These are usually present in the cytoplasm, mitochondria, etc.

and occur mainly in the liver, Gl tract, plasma and other tissues.

They are usually non-specific enzymes that catalyse few

oxidative reactions, a number of 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.
 Drug Metabolism

Extrahepatic microsomal enzymes

(oxidation, conjugation)

Hepatic microsomal enzymes

(oxidation, conjugation)

Hepatic non-microsomal enzymes

(acetylation, sulfation,GSH,
alcohol/aldehyde dehydrogenase,
hydrolysis, ox/red)
Factors Affecting Drug Metabolism

1. Species differences : eg in phenylbutazone, procaine and

barbiturates.
2. Genetic differences – variation exist with species
3. Age of animal –feeble in fetus,aged, newborn.
4.sex: under the influence of sex hormones.
5. Nutrition: starvation and malnutrition
6. Patholigical conditions: Liver/Kidney dysfunction
 TYPES OF BIOTRANSFORMATION

Phase 1 reaction. (Non synthetic phase). Phase II reaction. (Synthetic phase)

• a change in drug molecule. generally • Last step in detoxification reactions
results in the introduction of a and almost always results in loss of
functional group into molecules or the biological activity of a compound.
exposure of new functional groups of • May be preceded by one or more of
molecules phase one reaction
 : Phase I (non-synthetic or non- • Involves conjugation of functional
conjugative phase) includes reactions groups of molecules with hydrophilic
which catalyse oxidation, reduction and endogenous substrates- formation
hydrolysis of drugs. of conjugates - is formed with (an
endogenous substance such as
 In phase I reactions, small polar carbohydrates and amino acids. )with
drug or its metabolites formed in
functional groups like-OH, -NH2. -SH,
phase 1 reaction.
-COOH, etc. are either added or
 Involve attachment of small polar
unmasked (if already present) on the
endogenous molecules like glucuronic
lipid soluble drugs so that the resulting
acid, sulphate, methyl, amino acids,
products may undergo phase II
etc., to either unchanged drugs or
reactions.
phase I products.
• result in activation, change or
 Products called as 'conjugates' are
inactivation of drug.
water-soluble metabolites, which are
readily excreted from the body.
• Phase I metabolism is sometimes called a •
Phase II metabolism includes what are known
“functionalization reaction,”
as conjugation reactions.
• Results in the introduction of new
hydrophilic functional groups to compounds. • Generally, the conjugation reaction with
• Function: introduction (or unveiling) of endogenous substrates occurs on the
functional group(s) such as –OH, –NH2, –SH, metabolite( s) of the parent compound after
–COOH into the compounds. phase I metabolism; however, in some cases,
• Reaction types: oxidation, reduction, and the parent compound itself can be subject to
hydrolysis phase II metabolism.
• Function: conjugation (or derivatization) of
• Enzymes: functional groups of a compound or its
• Oxygenases and oxidases: Cytochrome P450 (P450 metabolite(s) with endogenous substrates.
or CYP), flavincontaining
• monooxygenase (FMO), peroxidase, monoamine
• Reaction types: glucuronidation, sulfation,
oxidase(MAO), alcohol dehydrogenase, aldehyde glutathione-conjugation, Nacetylation,
dehydrogenase, and xanthine 0xidase. Reductase: methylation and conjugation with amino acids
Aldo-keto reductase and quinone reductase. (e.g., glycine, taurine, glutamic acid).
• Hydrolytic enzymes: esterase, amidase, aldehyde
oxidase, and alkylhydrazine • Enzymes: Uridine diphosphate-Glucuronosyltransferase
• oxidase. (UDPGT): sulfotransferase (ST), N-acetyltransferase,
glutathione S-transferase (GST),methyl transferase, and
• Enzymes that scavenge reduced oxygen: Superoxide
amino acid conjugating enzymes.
dismutases, catalase,
• Glucuronidation by uridine diphosphate-
• glutathione peroxidase, epoxide hydrolase, y-
glucuronosyltransferase; Sulfation by sulfotransferase
glutamyl transferase,
• 3. Acetylation by N-acetyltransferase; Glutathione
• dipeptidase, and cysteine conjugate β-lyase
conjugation by glutathione S-transferase;. Methylation by
methyl transferase; Amino acid conjugation
 PHASE I BIOTRANSFORMATION

Oxidation

• Oxidation by cytochrome P450 isozymes (microsomal mixed-

functionoxidases).
• Oxidation by enzymes other than cytochrome P450s—most of these
• (a) oxidation of alcohol by alcohol dehydrogenase,
• (b) oxidation of aldehyde by aldehyde dehydrogenase,
• (c) N-dealkylation by monoamineoxidase.
The most important group of oxidative enzymes are microsomal
monooxygcnases or mixed function oxidases (MFO).

These enzymes are located mainly in the hepatic endoplasmic

reticulum and require both molecular oxygen (02) and reducing
NADPH to effect the chemical reaction.

Mixed function oxidase name was proposed in order to

characterise the mixed function of the oxygen molecule, which is
essentially required by a number of enzymes located in the
microsomes.
The term monooxygenses for the enzymes was proposed as they
incorporate only one atom of molecular oxygen into the organic substrate
with concomitant reduction of the second oxygen atom to water.

The overall stoichiometry of the reaction involving the substrate RH which

yields the product ROH, is given by the following reaction:
MFO
RH+02+NADPH+H+ ----------------► R0H+H20+NADP+

The most important component of mixed function oxidases is the

cytochrome P-450 because it binds to the substrate and activates oxygen.

The wide distribution of cytochrome P-450 containing MFOs in varying

organs makes it the most important group of enzymes involved in the
biotransformation of drugs.
 PHASE I ENZYMES PHASE II ENZYMES
Cytochrome P450 • Uridine Diphosphate-
Monooxygenase Glucuronosyltransferase
(Cytochrome P450, P450, (UDPGT)
or CYP) • Sulfotransferase (ST)
• Flavin-Containing • N-Acetyltransferase (NAT)
Monooxygenase (FMO)
• Glutathione S-Transferase
• Esterase (GST)
• Alcohol Dehydrogenase • Methyl Transferase
(ADH)
• Amino Acid Conjugation
• Aldehyde
Dehydrogenase (ALDH)
• Monoamine Oxidase
(MAO)
ROLE OF CYP ENZYMES IN HEPATIC DRUG METABOLISM
In human beings, of the 1000 currently known cytochrome P-450s, about 50 are
functionally active. These are categorised into 17 families, out of which the isoenzymes
CYP3A4 and CYP2D6 carry out biotransformation of largest number of drugs.

RELATIVE HEPATIC CONTENT % DRUGS METABOLIZED

OF CYP ENZYMES BY CYP ENZYMES
CYP2E1
CYP2D6 7%
2%

CYP 2C19
CYP 2C 11%
17% CYP 2C9
OTHER 14%
CYP2D6
36% 23%
CYP 1A2
12% CYP 1A2
14%
CYP 3A4-5
26% CYP2E1
CYP 3A4-5
5%
33%
 2. Reduction :
Reduction
Enzymes responsible for reduction of xenobiotics require NADPH as a cofactor.
Substrates for reductive reactions include azo- or nitrocompounds, epoxides,
heterocyclic compounds, and halogenated hydrocarbons:
(a) Azo or nitroreduction by cytochrome P450;
(b) Carbonyl (aldehyde or ketone) reduction by aldehyde reductase, aldose
reductase, carbonyl reductase, quinone reductase
(c) other reductions including disulfide reduction, sulfoxide reduction, and
reductive dehalogenation.
3. Hydrolysis :
Esters, amides, hydrazides, and carbamates can be hydrolyzed by
various
enzymes.
 The hydrolytic reactions, contrary to oxidative or reductive
reactions, do not involve change in the state of oxidation of the
substrate, but involve the cleavage of drug molecule by taking up
a molecule of water.

The hydrolytic enzymes that metabolise drugs are the ones that
act on endogenous substances, and their activity is not confined
to liver as they are found in many other organs like kidneys,
intestine, plasma, etc.

A number of drugs with ester, ether, amide and hydrazide

linkages undergo hydrolysis. Important examples are
cholinesters, procaine, procainamide, and pethidine.
 PHASE II REACTIONS

 Phase II or conjugation (Latin, 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

have two parts:
 Exocon, the portion derived from exogenous compound or
xenobiotic,
 Endocon, the portion derived from endogenous substance.

 Conjugation reactions have high energy requirement and they

often utilise suitable enzymes for the reactions.
The endogenous substances (endocons) for conjugation
reactions are derived mainly from carbohydrates or amino acids
and usually possess large molecular size.

 They are strongly polar or ionic in nature in order to render the

substrate water-soluble. The molecular weight of the conjugate
(metabolite) is important for determining its route of excretion.

High molecular weight conjugates are excreted predominantly in

bile (e.g., glutathione exclusively, glucuronide mainly),
while low molecular weight conjugates are excreted mainly in the
urine.

As the availability of endogenous conjugating substance is limited,

saturation of this process is possible and the unconjugated
drug/metabolite may precipitate toxicity.
 Induction of Drug Metabolising Enzymes

 Several drugs and chemicals have ability to increase the drug

metabolising activity of enzymes called as enzyme induction.

 These drugs known as enzyme inducers mainly interact with DNA and
increase the synthesis of microsomal enzyme proteins, especially
cytochrome P-450 and glucuronyl transferase.

 As a result, there is enhanced metabolism of endogenous substances

(e.g., sex steroids) and drugs metabolised by microsomal enzymes.
Some drugs (e.g., carbamazepine and rifampicin) may stimulate their
own metabolism, the phenomenon being called as auto-induction or
self induction.
Since different cytochrome P450 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.

However, some drugs like Phenobarbitone may affect metabolism of a

large number of drugs because they induce isoenzymes like CYP3A4 and
CYP2D6 which act on many drugs.

Enzyme inducers are generally lipid-soluble compounds with relatively long

plasma half-lives.

Repeated administration of inducers for a few days (3 to 10 days) is often

required for enzyme induction, and on stoppage of drug administration,
the enzymes return to their original value over 1 to 3 weeks.

Non-microsomal enzymes are not known to be induced by any drug or

chemical.
Clinical importance of enzyme induction

 It reduces efficacy and potency of drugs metabolised by these

enzymes.

 It reduces plasma half-life 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

 Contrary to metabolising enzyme induction, several drugs or chemicals

have the ability to decrease the drug metabolising activity of certain
enzymes called as enzyme inhibition.

 Enzyme inhibition can be either non-specific of microsomal enzymes

or specific of some non-microsomal 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).
 Nutritional deficiency, hormonal imbalance or hepatic
dysfunction, etc.also inhibit microsomal enzymes indirectly.

 Inhibition of non-microsomal enzymes with specific function

usually results when Structurally similar compounds compete for
the active site on the enzymes.

 Such an inhibition is usually rapid (a single dose of inhibitor

may be sufficient) and clinically more important than the non-
specific microsomal enzyme inhibition.

 Enzyme inhibition generally results in depressed metabolism

of drugs.
As a result, the plasma hall-life, 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. Enzyme inhibition may also
produce undesirable drug interactions.

 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
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