• 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.

• The term metabolism
is commonly used
probably because products
of drug transformation are called
metabolites.

• 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.

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

• 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)

• skin (epithelial cells)

• Intestines

• Plasma

• Nervous tissue (brain)

• Testes (Sertoli cells)

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 pseudocholine esterase enzyme.

Subcellular
locations of metabolising enzymes

ENDOPLASMIC RETICULUM
(microsomes): the primary location for the metabolizing enzymes.

(a) Phase I: cytochrome P450, flavin-containing monooxygenase,
aldehyde oxidase, 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 II:
sulfotransferase, glutathione S-transferase, N-acetyl transferase, catechol 0-methyl
transferase, amino acid conjugating enzymes.

MITOCHONDRIA

Phase I: monoamine oxidase, aldehyde dehydrogenase,
cytochrome P450.

Phase II: N-acetyl transferase, amino acid conjugating
enzymes.

LYSOSOMES

Phase I: peptidase.

NUCLEUS

Phase II: uridine diphosphate-glucuronosyl transferase
(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.

• 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, glucronyl 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

Factors
Affecting Drug Metabolism

1. Species differences:
eg in phenyl butazone, procaine and barbiturates.

2. Genetic
differences – variation exist.

3. Age of animal –
feeble in fetus, aged, newborn.

4. Sex: under the
influence of sex hormones.

5. Nutrition:
starvation and malnutrition

6. Pathological
conditions: Liver/Kidney dysfunction

Phase 1 reaction. (Non
synthetic phase)

• Change in drug molecule generally results in the introduction
of a functional group into molecules or the exposure of new functional groups
of molecules.

• Phase I (non-synthetic or non-conjugative phase) includes
reactions which catalyse oxidation,
reduction and hydrolysis of drugs.

• In phase I reactions, small polar functional groups like
-OH, -NH2. -SH, -COOH, etc. are either added or unmasked (if already present)
on the lipid soluble drugs so that the resulting products may undergo phase II
reactions.

• It results in activation,
change or inactivation of drug.

PHASE 1
REACTION

Phase I metabolism is
sometimes called a “functionalization reaction,”

Results in the introduction of new hydrophilic functional
groups to compounds.

Function:
introduction (or unveiling) of functional group(s) such as –OH, –NH2, –SH, –COOH
into the compounds.

Reaction types:
oxidation, reduction, and hydrolysis

Enzymes:

Oxygenases and oxidases: Cytochrome P450 (P450 or CYP),
Flavin containing monooxygenase (FMO), peroxidase, monoamine oxidase (MAO),
alcohol dehydrogenase, aldehyde dehydrogenase, and xanthine oxidase.

Reductase:
Aldo-keto reductase and quinone reductase.

Hydrolytic enzymes:
esterase, amidase, aldehyde oxidase and alkyl hydrazine oxidase.

Enzymes that scavenge
reduced oxygen: Superoxide dismutases, catalase,glutathione peroxidase,
epoxide hydrolase, y-glutamyl
transferase, dipeptidase, and cysteine conjugate β-lyase

Oxidation

Oxidation by cytochrome P450 iso enzymes (microsomal
mixed-function oxidases). Oxidation by enzymes other than cytochrome P450s.

Most of these are:

(a) Oxidation of alcohol by alcohol dehydrogenase

(b) Oxidation of aldehyde by aldehyde dehydrogenase

• Oxidative reactions are most important metabolic reactions,
as energy in animals is derived by oxidative combustion of organic molecules
containing carbon and hydrogen atoms.

• The oxidative reactions are important for drugs because
they increase 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 oxidation
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 monooxygenases or mixed function
oxidases (MFO).

• These enzymes are located mainly in the hepatic
endoplasmic reticulum and require both molecular oxygen (O2) 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 monooxygenases 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+O2+NADPH+H+ —————-► ROH+H2O+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

Cytochrome P450 Monooxygenase (Cytochrome P450, P450, or
CYP)

• Flavin-Containing Monooxygenase (FMO)

• Esterase

• Alcohol Dehydrogenase (ADH)

• Aldehyde Dehydrogenase (ALDH)

• Monoamine Oxidase (MAO))

PHASE II ENZYMES

• Uridine Diphosphate- Glucuronosyltransferase (UDPGT)

• Sulfotransferase (ST)

• N-Acetyltransferase (NAT)

• Glutathione S-Transferase (GST)

• Methyl Transferase

• Amino Acid Conjugation

The
cytochrome P-450 ENZYMES

• Super family of haem-thiolate proteins that are widely
distributed across all living creatures.

• The name given to this group of proteins because their
reduced form binds with carbon monoxide to form a complex, which has maximum
absorbance at 450 nm.

• Depending upon the extent of amino acid sequence homology,
the cytochrome P- 450 (CYP) enzymes superfamily contains a number of isoenzymes,
the relative amount of which differs among species and among individuals of the
same species.

• These isoenzymes are grouped into various families
designated by Arabic numbers 1, 2, 3 (sequence that are greater than 40%
identical belong to the same family), each having several
subfamilies designated by
Capital letter A,
B, C, while
individual isoenzymes are again allotted Arabic numbers 1.2,3 (e.g.,
CYP1A1, CYP1A2, etc.).

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.

Participation
of the CYP Enzymes in Metabolism of Some Clinically Important Drugs

CYP
Enzyme

Examples
of substrates

1A1

Caffeine, Testosterone, R-Warfarin

1A2

Acetaminophen, Caffeine, Phenacetin, R-Warfarin

2A6

17 β -Estradiol, Testosterone

2B6

Cyclophosphamide, Erythromycin, Testosterone

2C-family

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

2E1

Acetaminophen, Caffeine, Chlorzoxazone, Halothane

2D6

Acetaminophen, Codeine, Debrisoquine

3A4

Acetaminophen, Caffeine, Carbamazepine, Codeine, Cortisol,
Erythromycin, Cyclophosphamide, S- and RWarfarin, Phenytoin, Testosterone,
Halothane, Zidovudine

2.
Reduction

Enzymes responsible for reduction of xenobiotics require
NADPH as a cofactor. Substrates for reductive reactions include azo- or nitro
compounds, epoxides, heterocyclic compounds, and halogenated hydrocarbons:

(a) Azo or nitro reduction by cytochrome P450;

(b) Carbonyl (aldehyde or ketone) reduction by aldehyde
reductase, aldose reductase, carbonyl reductase, quinone reductase

• The acceptance of one or more electron(s) 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.

• Many reductive reactions are exact opposite of the
oxidative reactions (reversible reactions) catalysed cither by the same enzyme
(true reversible reaction) or by different enzymes (apparent reversible
reactions).

• Such reversible reactions usually lead to conversion of
inactive metabolite into active

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 reaction or Conjugation Reaction (Synthetic
phase)

• Last step in detoxification reactions and almost always results in loss of biological activity
of a compound.

• May be preceded by one or more of phase one reaction.

• It involves conjugation of functional groups of molecules
with hydrophilic endogenous substrates such as carbohydrates and amino acids (formation of conjugates) with drug or
its metabolites formed in phase 1 reaction.

• Involve attachment of small polar endogenous molecules
like glucuronic acid, sulphate group, methyl group, amino acids etc., to either
unchanged drugs or phase I products.

• Products called as
‘conjugates’ are water-soluble metabolites, which are readily excreted from
the body.

Phase II
metabolism (conjugation reactions)

• Generally, the conjugation reaction with endogenous
substrates occurs on the metabolite(s) of the parent compound after phase I
metabolism; however, in some cases, the parent compound itself can be subject
to phase II metabolism.

Function:
conjugation (or derivatization) of functional groups of a compound or its
metabolite(s) with endogenous substrates.

Reaction types:
glucuronidation, sulfation, glutathione-conjugation, N-acetylation, methylation
and conjugation with amino acids (e.g., glycine, taurine, glutamic acid).

Enzymes:

• Glucuronidation by uridine
diphosphate-glucuronosyltransferase

• Sulfation by sulfotransferase

• Acetylation by N-acetyltransferase

• Glutathione conjugation by glutathione S-transferase

• Methylation by methyl transferase

• Amino acid conjugation

• 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.

1.
Conjugation with glucuronic acid/ 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.

• The biochemical donor (cofactor) of glucuronic acid is
uridine diphosphate-D- glucuronic acid (UDPGA) and the reaction is carried out
by enzyme uridine diphosphate-glucuronyl transferase (UDP-glucuronyl
transferase; glucuronyl transferase).

• Glucuronyl transferase is present in microsomes of most
tissues but liver is the most active site of glucuronide synthesis.

• Glucuronidation can take place in most body tissues
because the glucuronic acid donor UDPGA is present in abundant quantity in
body, unlike donors involved in other phase II reactions.

• In cats, there is reduced glucuronyl transferase activity,
while in fish there is deficiency of endogenous glucuronic acid donor.

• The limited capacity of this metabolic pathway in cats may
increase the duration of action, pharmacological response and potential of
toxicity of several lipid-soluble drugs (e.g., aspirin) in this species.

• A large number of drugs undergo glucuronidation including morphine,
paracetamol and desipramine. Certain endogenous substances such as steroids,
bilirubin, catechols and thyroxine also form glucuronides.

• Deconjugation
process: Occasionally some glucuronide conjugates that are excreted in bile
undergo deconjugation process in the intestine mainly mediated by β
glucuronidase enzyme.

• This releases free and active drug in the intestine, which
may be reabsorbed and undergo entero-hepatic cycling.

• Deconjugation is an important process because it often
prolongs the pharmacological effects of drugs and/or produces toxic effects.

2.
Conjugation with sulphate/ Sulphation:

• Conjugation with sulphate (sulphate conjugation,
sulphoconjugation or sulphation) is similar to glucuronidation but is catalysed
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. Unlike
glucuronide conjugation, sulphoconjugation in mammals is less important because
the PAPS donor that transfers sulphate to the substrate is easily depleted.

• Capacity for sulphate conjugation is limited in pigs.
However in cats, where glucuronidation is deficient, sulphate conjugation is
important. Functional groups capable of forming sulphate conjugates include
phenols, alcohols, aryl amines, N- hydroxyl amines and N-hydroxyamides.

• Drugs undergoing sulphate conjugation include
chloramphenicol, phenols and steroids.

3.
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., adrenaline, melatonin) and
inactivation (e.g., histamine) of endogenous amines.

• Occasionally, the metabolites formed are not polar or
water-soluble and may possess equal or greater activity than the parent
compound (e.g., adrenaline synthesised from noradrenaline).

4.
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, and protects the tissues from their adverse effects.

• 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.

• The glutathione conjugate either due to its high molecular
weight is excreted as such in the bile or is further metabolised to form
mercapturic acid conjugate that is excreted in the urine.

5. 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 not a true detoxification process because
occasionally it results in decrease in water solubility of an amine and. Thus,
increase in its toxicity (e.g., sulphonamides).

• Acetylation is the primary route of biotransformation of
sulphonamide compounds. Dogs and foxes do not acetylate the aromatic amino
groups due to deficiency of arylamine acetyl transferase enzyme.

6. Conjugation
with amino acids

• Conjugation with amino acids occurs to a limited extent in
animals because of limited availability of amino acids. 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 conjugates
are salicylic acid, nicotinic acid and cholic acid.

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 rhodanse to form inactive thiocyanate.

Thiocyanate formed is much less toxic than the cyanide (true
detoxification) and it is excreted in urine.

SUMMARY

• 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.

• The major site of drug metabolism is the liver.

• 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.

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

• Phase I (non-synthetic or non-conjugative phase) includes
reactions which catalyse oxidation, reduction and hydrolysis of drugs.

• In phase I reactions, small polar functional groups
like-OH, -NH2. -SH, -COOH, etc. are either added or unmasked (if already
present) on the lipid soluble drugs so that the resulting products may undergo
phase II reactions.

• The name given to this group of proteins because their
reduced form binds with carbon monoxide to form a complex, which has maximum
absorbance at 450 nm.

• Depending upon the extent of amino acid sequence homology,
the cytochrome P-450 (CYP) enzymes superfamily contains a number of isoenzymes,
the relative amount of which differs among species and among individuals of the
same species.

• 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.

• Conjugation with glucuronic a./ Glucuronidation

• Conjugation with sulphate/ Sulphation

• Conjugation with methyl group/ Methylation

• Conjugation with glutathione and mercapturic acid
formation

• Conjugation with acetyl group/ Acetylation

• Conjugation with aminoacids

• Conjugation with thiosulphate

M	T	W	T	F	S	S
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30

Drug metabolism, Phase 1 and Phase 2 Reaction – Medicinal Chemistry

DRUG METABOLISM

METABOLISM
OR BIOTRANSFORMATION