Drug metabolism

METABOLISM
Ms. Nita B. Deore
Assistant Professor
Sir Dr MS Gosavi College Of Pharmaceutical
Education, Nasik

OVERVIEW
• Definition
• Introduction
• Types
• Phase I/II in detail
• Factors affecting metabolism
• Bioactivation
• Tissue toxicity

Introduction
Onset of pharmacological response depends on
Drug absorption
Drug distribution
Duration and intensity of action depends on
Tissue redistribution
The rate of elimination

Elimination
• Irreversible loss of drug from the body
• Occurs by two processes
Biotransformation
Excretion

Biotransformation
• Defined as chemical conversion of one form to another form
• Term synonymously used with metabolism
• Need for biotransformation-
• Xenobiotics-
 chemical substances that are not nutrients for the body
 enter the body through, ingestion, inhalation or absorption
are called as xenobiotics
• or exogenous compounds-
• Drugs are xenobiotics

Need for biotransformation-
• For effective absorption-a drug needs to be sufficiently lipid soluble
• But if it remains the same physicochemical property means remains
lipid soluble that enables it to bypass excretion.
• Only water-soluble agents undergo renal excretion (major route for exit
of drugs from the body)
• Whereas lipid soluble substances are passively reabsorbed from the renal
tubules into the blood after glomerular filtration.
• Thus, if such a phenomenon continues, drugs would accumulate in the
body and precipitate toxic reactions.

• However, to prevent such a consequence, the body is armed with the
metabolic system which transforms the water insoluble, lipophilic,
nonpolar drugs in to polar and water-soluble products that
can be easily excreted by the kidneys and are poorly reabsorbed;
• for instance, hippuric acid, the metabolite of benzoic acid, is 2.5 times more
water-soluble. The N-acetyl derivatives of sulphonamides are less water-
soluble than the parent drug and thus have a tendency to cause crystalluria
• Drug biotransformation is thus a detoxification process.
Need for biotransformation-

Consequences of biotransformation:
• Results in pharmacological inactivation of drugs --
metabolites with little or no pharmacological activity; e.g.
conversion of phenytoin to p-hydroxy phenytoin.
• Yields metabolites with equal activity; e.g. conversion of
phenylbutazone to oxyphenbutazone.
• Leads to toxicological activation of drugs, i.e. it results in
formation of metabolites with high tissue reactivity; e.g.
conversion of paracetamol to reactive metabolites that cause
hepatic necrosis.

Metabolites and Relative Activity of Drugs

Drugs Metabolising Organs
• Liver-primary site for metabolism
Because of its relative richness in possessing a large
variety of enzymes in large amounts.
• Extra hepatic metabolism-
Lungs
Kidneys
Intestine
Placenta
Adrenals
Skin

Drug Metabolising Enzymes
The enzymes that biotransform xenobiotics differ from
those that metabolise food materials.
• Microsomal enzymes
• Non-microsomal enzymes

Microsomal enzymes
• Microsomal enzymes are typically found in the
endoplasmic reticulum of hepatocytes.
• Microsomes are fragments of endoplasmic reticulum
and attached ribosomes that are isolated together when
homogenized cells are centrifuged.

Characteristics of microsomal enzymes
1. The intact nature of lipoidal membrane bound enzyme of the
microsomes is essential
for its selectivity towards lipid-soluble substrates.
2. A number of lipid-soluble substrates (xenobiotics in general) can
interact
nonspecifically with the microsomal enzymes. Natural endogenous
substances which
are generally water-soluble do not interact.
3. The lipid soluble substrate is biotransformed into a water-
soluble metabolite by the
microsomal enzymes which can be readily excreted.

Non microsomal enzymes
• those that are present in soluble form in the
cytoplasm and those attached to the mitochondria but not to endoplasmic reticulum
• These
• are also non-specific enzymes that catalyse few oxidative reactions, a number of
reductive
• and hydrolytic reactions and conjugation reactions other than glucuronidation. It is
• interesting to note that, in contrast to microsomal enzymes, the non-microsomal
enzymes,
• especially the soluble enzymes, act on relatively water-soluble xenobiotics (as well as
• endogenous compounds), e.g. oxidases, peroxidases, dehydrogenases, esterases, etc.

Chemical pathways of drug biotransformation
• R.T.Williams, the leading pioneer in drug biotransformation
research
• Drug metabolism reactions are divided into two general
categories
Phase-I Reactions
Phase-II Reactions

Phase-I reactions:
• These reactions generally precede phase II reactions and
include oxidative, reductive and hydrolytic reactions. By
way of these reactions, a polar functional group is either
introduced or unmasked if already present on the otherwise
lipid soluble substrate, e.g. -OH, -COOH, -NH2 and -SH.
Thus, phase I reactions are also called as functionalisation
reactions.
• These transformations are also called as asynthetic
reactions, opposite to the synthetic phase II reactions.
The resulting product of phase I reaction is susceptible to
phase II reaction

Phase-I reaction
• Oxidative reactions
• Reductive reactions
• Hydrolytic reactions

• Oxidative reactions increase hydrophilicity of
xenobiotics by introducing polar functional groups
such as—OH. Such a polar metabolite can thus
rapidly undergo phase II reaction or is excretable by
the kidneys.

Oxidation of Aromatic Carbon
Atoms (Aromatic Hydroxylation)
• This reaction proceeds via formation of a reactive
intermediate arene oxide (epoxide), which in most
cases undergoes rearrangement to yield arenols, and
in some cases catechols and glutathione conjugates.

Oxidation of Aromatic Carbon
Atoms (Aromatic Hydroxylation)

Oxidation of Benzylic Carbon
Atoms

Oxidation of Allylic Carbon
Atoms

Oxidation of Carbon Atoms
Alpha to Carbonyls and Imines

Oxidation of Aliphatic Carbon
Atoms (Aliphatic Hydroxylation

Oxidation of Alicyclic Carbon
Atoms (Alicyclic Hydroxylation)

Oxidation of Alcohol, Carbonyl and
Carboxylic Acid

Oxidation of Carbon-Heteroatom
Systems
• 1. Hydroxylation of carbon atom attached to the
heteroatom and subsequent cleavage at carbon-
heteroatom bond, e.g. N-, O- and S- dealkylation,
oxidative deamination and desulphuration.
• 2. Oxidation of the heteroatom itself, e.g. N- and S-
oxidation.

REDUCTIVE REACTIONS
• Bioreductions are also capable of generating polar functional groups such as hydroxy and
• amino which can undergo further biotransformation or conjugation. A number of reductive
• reactions are exact opposite of oxidation. For example:
• Alcohol dehydrogenation Carbonyl reduction
• N-Oxidation Amine oxide reduction
• Thus, in this sense, bioreduction comprises one-half of reversible reactions. Such
• reactions may be catalysed by –
• Same enzyme (true reversible reaction), or
• Different enzymes (apparent reversible reaction).
• Since reversible reactions usually lead to conversion of inactive metabolites into active
• drug, they may result in delay of drug removal from the body and hence prolongation of
• action.

Reduction of Carbonyls
(Aldehydes and Ketones)
• Depending on their reactivity towards bioreduction,
carbonyls can be divided into 3 categories –
1. The aliphatic aldehydes and ketones.
2. The aromatic aldehydes and ketones.
3. The esters, acids and amides
The order of reactivity of these categories of drugs in
undergoing reduction is –
1 > 2 > 3

Reduction of Alcohols and
Carbon-Carbon Double Bonds
• These two reductions are considered together
because the groups are interconvertible by
• simple addition or loss of a water molecule. Before
an alcohol is reduced, it is dehydrated to
• C=C bond, e.g. bencyclane (antispasmodic).

Reduction of N-compounds
(Nitro, Azo and N-Oxide)
• The N-containing functional groups that commonly
undergo bioreduction are nitro, azo and
• N-oxide. It is important to note that such a reaction
is reverse of oxidation.
• Reduction of nitro group proceeds via formation of
nitroso and hydroxylamine
• intermediates to yield amines.

Miscellaneous Reductive Reactions
• 1. Reductive Dehalogenation: This reaction
involves replacement of halogen attached to the
carbon with the H-atom, e.g. halothane.

• 2. Reduction of Sulphur Containing Functional
Groups-An example of S-S reductive cleavage is
disulphiram.

HYDROLYTIC REACTIONS
These reactions differ from oxidative and reductive reactions in 3
respects:
1. The reaction does not involve change in the state of oxidation
of the substrate.
2. The reaction results in a large chemical change in the substrate
brought about by loss of relatively large fragments of the
molecule.
3. The hydrolytic enzymes that metabolise xenobiotics are the ones
that also act on endogenous substrates. Moreover, their activity is
not confined to liver as they are found in many other organs like
kidney, intestine, etc.

Hydrolysis of Esters and Ethers
• Esters on hydrolysis yield alcohol and carboxylic acid.
The reaction is catalysed by esterases

Organic acid (carboxylic acid)
esters
Esters with a large acidic (and small alcohol) group e.g. clofibrate

• Esters with large alcoholic (and small acidic) group e.g.
aspirin

• Esters with large acidic and alcoholic groups (generally amine
alcohols) e.g. succinylcholine.

Inorganic Acid Esters
Phosphates e.g. stilbestrol diphosphate

Sulphates
• Sulphates e.g. isopropyl methanesulphonate

Nitrates
• Nitrates e.g. pentaerythritol tetranitrate

Hydrolysis of Amides (C-N bond
cleavage)
• Amides are hydrolysed slowly in comparison to
esters. The reaction, catalysed by amidases, involves C-
N cleavage to yield carboxylic acid and amine.
Primary amides are rare. Secondary amides form the largest group of amide drugs.

• Secondary amides with aromatic substituent on N-atom
(anilides) e.g. lidocaine

• Tertiary amides (N-atom contained in a ring) e.g.
carbamazepine

• Hydrazides are also a class of amides e.g.
isocarboxazide

Hydrolytic Cleavage of Non-
aromatic Heterocycles
• Nonaromatic heterocycles also contain amide
functions, e.g. lactams (cyclic amides).
• Several lactams that undergo hydrolysis are:

Hydrolytic Dehalogenation
• Chlorine atoms attached to aliphatic carbons are
dehalogenated easily, e.g. DDT

Miscellaneous Hydrolytic
Reactions
• These reactions include hydration of epoxides and
arene oxides, hydrolysis of sulphonyl ureas,
carbamates, hydroxamates and of glucuronide and
sulphate conjugates.

Phase-II Reaction
• Phase II reactions involve transfer of a suitable
endogenous moiety such as glucuronic acid, sulphate,
glycine, etc. in presence of enzyme transferase to drugs or
metabolites of phase I reactions having suitable functional
groups to form highly polar, readily excretable and
pharmacologically inert conjugates

Phase II Reactions
• Involve covalent attachment of small polar endogenous molecules such as glucuronic acid,
sulphate, glycine, etc. to either unchanged drugs or phase I products having suitable
functional groups viz. -OH, -COOH, -NH2 and -SH and form highly water soluble
conjugates which are readily excretable by the kidneys (or bile).
• Thus, these reactions are called as conjugation reactions.
• Since the outcome of such processes are generally products with increased molecular size
(and altered physicochemical properties), they are also called as synthetic reactions.
• Quite often, a phase I reaction may not yield a metabolite that is sufficiently hydrophilic or
pharmacologically inert but conjugation reactions generally result in products with total
loss of pharmacological activity and high polarity. Hence, phase II reactions are better
known as true detoxification reactions.
• Since these reactions generally involve transfer of moieties to the substrate to be
conjugated, the enzymes responsible are called as transferases.
• The biotransformation of drug metabolites, particularly the glutathione conjugates which
are excreted via bile in the gut, by the intestinal microflora, is considered by few
researchers as phase III reactions.

Phase II reactions are the real drug detoxication
pathways because
1. The conjugates/products of phase II reactions are
absolutely free of pharmacological activity.
2. The conjugates/products of phase II reactions are
highly polar and thus easily excretable either in bile or
urine.
3. Tissue-reactive and carcinogenic metabolites formed
as a result of phase I reaction are rendered harmless by
conjugation with moieties such as glutathione.

Phase II reaction possess characteristics
• They are simple endogenous molecules such as
carbohydrates, proteins and fats.
• They are of large molecular size.
• They are strongly polar or ionic in nature in order to
render the substrate water soluble

Two outstanding characteristics of conjugation reactions are –
1. The reaction involves an initial activation step – either
(a) The drug is activated e.g. conjugation with amino acids and acetylation reaction;
or
(b) The conjugating reagent is activated e.g. glucuronidation, sulphation and
methylation.
2. The reaction is capacity-limited – the limited capacity of conjugation reactions is
attributed to –
(a) Limited amount of conjugating agent, for example, glycine.
(b) Limited ability to synthesise the active nucleotide intermediate.
(c) Limited amount of enzyme conjugate transferase.
Thus, when doses of drugs are higher than normal levels of conjugating molecules,
saturation of metabolism occurs and the unconjugated drug/metabolite precipitates
toxicity.

• The increase in the molecular weight of the drug
following conjugation with glucuronic acid, sulphate and
glutathione is 176, 80 and 300 Daltons respectively.
• The molecular weight of the conjugate is important in
dictating its route of excretion –
• High molecular weight conjugates (>350) are excreted
predominantly in bile
• Low molecular weight conjugates (<250) are excreted in
urine.
• Thus, glutathione conjugates are always excreted in bile.

Phase-II reactions
• Conjugation with glucuronic acid
• Conjugation with sulphate moieties
• Conjugation with alpha amino acids
• Conjugation with glutathione and mercapturic acid formation
• Acetylation reactions
• Methylation reactions
• Miscellaneous conjugation reactions
The order of capacities of important conjugation reactions is –
• Glucuronidation > Amino Acid Conjugation > Sulphation and Glutathione
Conjugation

CONJUGATION WITH GLUCURONIC ACID
• Also called as glucuronidation,
• it is the most common and most important phase II reaction
1. Readily available source of conjugating moiety, D-glucuronic acid which is derived from
D-glucose.
2. Several functional groups viz. alcohols, acids, amines, etc. can combine easily with
Dglucuronic acid.
3. Quantitatively, conjugation with D-glucuronic acid occurs to a high degree.
4. All mammals have the common ability to produce glucuronides,
5. The free carboxyl function of glucuronic acid has a pKa in the range 3.5 to 4.0 and
hence ionisable at both plasma and urine pH thereby greatly increasing the water solubility
of the conjugated substrate.
6. The glucuronidation enzymes are in close association with the microsomal mixed
function oxidases, the major phase I drug metabolising enzyme system; thus, a rapid
conjugation of phase I metabolites is possible.
7. Lastly, glucuronidation can take place in most body tissues since the glucuronic acid
donor, UDPGA is produced in processes related to glycogen synthesis and thus, will never
be deficient unlike those involved in other phase II reactions.

The steps involved in glucuronide
synthesis are depicted below:
where X = O, COO, NH or S.

Glucuronide formation occurs in 2 steps
1. Synthesis of an activated coenzyme uridine-5'-diphospho- -D-
glucuronic acid (UDPGA) from UDP-glucose (UDPG). The
coenzyme UDPGA acts as the donor of glucuronic acid.
UDPG is synthesized by interaction of -D-glucose-1-
phosphate with uridine triphosphate (UTP).
2. Transfer of the glucuronyl moiety from UDPGA to the
substrate RXH in presence of enzyme UDP-glucuronyl
transferase to form the conjugate. In this step, the -
configuration of glucuronic acid undergoes inversion and thus,
the resulting product is-D-glucuronide (also called as
glucosiduronic acid or glucopyranosiduronic acid
conjugate).

glucuronidation of benzoic acid

Oxygen or O-Glucuronides
• 1. Hydroxyl Compounds: These form ether
glucuronides
• Carboxyl Compounds These form ester glucuronides

Nitrogen or N-Glucuronides
• Xenobiotics with amine, amide and sulphonamide
functions form N-glucuronides
• Sulphur or S-Glucuronides
Thiols (SH) form thioether glucuronides e.g. thiophenol.
• Carbon or C-Glucuronides
Xenobiotics with nucleophilic carbon atoms such as
phenylbutazone form C-glucuronides.

CONJUGATION WITH
SULPHATE MOIETIES
• Sulphation is similar to glucuronidation but it is
catalysed by nonmicrosomal enzymes and occurs less
commonly as the moiety that transfers sulphate to
the substrate is easily depleted

• Like glucuronidation, sulphation also occurs in 2 steps:
1. Synthesis of an activated coenzyme 3'-phosphoadenosine-
5'- phosphosulphate (PAPS) which acts as a donor of sulphate
to the substrate. This also occurs in two steps —
(a) An initial interaction between the sulphate and the adenosine
triphosphate (ATP) to yield adenosine-5'-phosphosulphate (APS),
followed by
(b) Activation of APS to PAPS.
2. Transfer of sulphate group from PAPS to the substrate
RXH in presence of enzyme sulphotransferase (sulphokinase)
and subsequent liberation of 3'-phosphoadenosine-5'-
phosphate (PAP).

CONJUGATION WITH ALPHA
AMINO ACIDS
• This reaction also occurs to a limited extent because of
limited availability of amino acids.
The reaction occurs in two steps:
1. Activation of carboxylic acid drug substrate with ATP and
coenzyme A (CoA) to form an acyl CoA intermediate. Thus,
the reaction is a contrast of glucuronidation and sulphation
where the donor coenzyme is activated and not the substrate.
2. Acylation of the -amino acid by the acyl CoA in presence
of enzyme N-acyl transferase.

where R’ = -CH2- (if glycine) or >CH-CH2-
CH2-CONH2 (if glutamine)

CONJUGATION WITH GLUTATHIONE AND
MERCAPTURIC ACID FORMATION
• Glutathione ( -glutamyl cysteinyl glycine or GSH) is a
tripeptide with a strongly nucleophilic character due
to the presence of a -SH (thiol) group in its structure

• Thus, it has great affinity for electrophilic substrates, a
number of which are potentially toxic compounds.
• It is important to note that a highly electrophilic
metabolite has a tendency to react with tissue nucleophilic
groups such as -OH, -NH2 and -SH and precipitate
toxicities such as tissue necrosis, carcinogenesis,
mutagenesis, teratogenesis, etc.
• Conjugation with glutathione protects the tissue from
such reactive moieties and thus, the reaction is an
important detoxication route.

ACETYLATION
• This reaction is basically an acylation reaction and
thus similar to conjugation with –amino acids. The
analogy also lies in the fact that both reactions yield
amide products

METHYLATION
• This reaction differs from general characteristics of phase II reactions in several ways:
1. The metabolites formed are not polar or water-soluble.
2. The metabolites, in a number of instances, have equal or greater pharmacological activity
than the parent drug, e.g. morphine formed from normorphine.
3. The reaction is of lesser importance in metabolism of xenobiotics.
It is more important in the biosynthesis (e.g. adrenaline, melatonin) and inactivation of
endogenous amines (e.g. noradrenaline, serotonin, histamine).
• Methylation can be considered as intermediate of phase I and phase II reactions. It can be
called as a phase I reaction as it is reverse of demethylation reaction and can be classed as
a phase II reaction because of its mechanism.

BIOACTIVATION AND
TISSUE TOXICITY
• Formation of highly reactive metabolites (from relatively inert
chemical compounds) which interact with the tissues to precipitate one
or more of the several forms of toxicities such as carcinogenesis and
teratogenesis is called as bioactivation or toxicological
activation.
• The reactive, chemically unstable species, capable of
toxication, are broadly divided into two
• Electrophiles
• Free radicals

BIOACTIVATION AND
TISSUE TOXICITY

Drug metabolism

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