Drugs and other foreign substances that enter the body undergo a process of biotransformation or metabolism within the body. This involves chemical alterations through phase I and phase II reactions. Phase I reactions introduce functional groups like OH or NH2 through oxidation, reduction or hydrolysis. Phase II then involves conjugating these groups to molecules like glucuronic acid or sulfate, making the substances more polar and excretable in urine or bile. This overall process of metabolism and conjugation serves to terminate the pharmacological effects of drugs and remove them from the body.

1. AMJAD ANWAR
Department of Pharmacy, UOM

2.  Drugs and other non-food foreign substances or
chemicals (Xenobitics), which enter the body, are
gradually removed from it after a period of time.
While inside the body, it undergoes a series of
chemical changes or alterations, giving rise to
compounds which are;
◦ Less active or inactive
◦ More active or more toxic
◦ Having similar or different activity

3.  Drug metabolism or Drug biotransformation is a
process of chemical transformation or alterations,
which a drug undergoes inside the body or
biological system to some other form, resulting in
alteration of its pharmacological activity, Duration
of action and toxicity.
 Drug metabolism is a process of conversion of a
drug molecule to a relatively more polar compound
so that it is cannot be re-absorbed from the renal
tubules and may be readily excreted from the
body.

4.  It is important to be noted that as a result of drug
metabolism, Termination of the pharmacological
activity of a drug molecule takes place with its
ultimate elimination from the body.
 Following scheme shows the steps of drug
elimination
Drug Elimination from the body
➢Renal Excretion
✓(Unchanged drug + Conjugated Drug)
➢Hepatic Elimination
✓ Drug Biotransformation
✓ Biliary Drug Excretion (Unchanged drug + Conjugated
Drug)

5. Generally following are the net consequences of drug
metabolism
 Drugs undergoes chemical transformation either in
a single step or two step reactions.
1) Single Step Conversion Reaction
 In this case, a biologically active compound is
converted to an inactive compound in a single step
which is then excreted from the body.
 E.g. conjugation of Phenol with Sulfate or
Glucoronic acid

6. Two Step Conversion Reaction
 It involves 3 types of conversion;
i) Active compound is inactivated followed by
conjugation
 E.g. Hydroxylation of phenobarbital followed by
conjugation and ultimate excretion from the body.
ii) Pro-drug is converted to an active compound
followed by conjugation and excretion.
 E.g. Phenacetin -----> Paracetamol
 Sulfasalazine -------> Sulfapyridine + 5-amino salicylic acid
 Enalapril -----------> Enalaprilate
 Levo Dopa ---------> Dopamine

7. iii) A two-step process in which an active compound
is converted to an active metabolite and then
followed by its conjugation.
 E.g. After demethylation, codeine gives morphine
which then undergoes conjugation with sulfate or
glucuronic acid.
 Other examples include;
 Diazepam ------------> Oxazepam
 Phenyl Butazone -------> Oxyphenbutazone
 Propranolol -----------> 4-Hydroxy Propranolol
 Amitriptyline ----------> Nor-triptyline
 Chloral hydrate --------> Trichloro-ethanol

8.  Sometimes an active drug is converted to a reactive
intermediate. So for some compounds such as
Acetaminophen, Benzopyrene and other drugs
containing aromatic rings, reactive intermediates
such as epoxides or oxiranes are formed during
Hydroxylation reactions
 The aromatic epoxides are highly reactive and react
with macromolecules (Cell proteins), possibly
causing liver necrosis (Acetaminophen) or cancer
(Benzopyrene)
Xenobiotics
 The foreign non-food substances or chemicals that
initiate the process of biotransformation are called
xenobiotics.

9.  Although biotransformation reactions may occur in many tissues,
but liver is the most important organ. It is particularly rich in almost
all of the drugs metabolizing enzymes. Liver is a well perfused organ
and plays an important role in the detoxification and metabolism of
endogenous and exogenous compounds in the blood.
 Orally administered drugs that are absorbed from the GIT must first
pass through the liver. Therefore, they are much susceptible to
hepatic metabolism (First Pass Effect).
 In humans, several drugs are metabolized extensively by the first
pass effect.
 Following are the examples of some drugs;
 Isoproterenol
 Lidocaine
 Meperidine
 Nitroglycerine
 Morphine
 Pentazocin
 Propoxyphene
 Propranolol
 Salicylamide etc.
Some drugs, e.g. Lidociane, are removed so effectively by the first pass
metabolism that they are in-effective when given orally.

10. Intestines
 As most of the drugs are administered orally so intestines
also plays an important role in the extra-hepatic metabolism
of xenobiotics.
 For example, orally administered Isoproterenol undergoes
considerable sulfate conjugation in the intestinal wall.
 Several other drugs like Levodopa, Chlorpromazine and
Diethyl stilbestrol also have been reported to be metabolized
in the GIT.
 Esterases and Lipases present in the intestines may carry out
hydrolysis of many ester pro-drugs.
 Bacterial Flora present in the intestine and colon play an
important role in the reduction of many aromatic, AZO and
Nitro Compounds e.g. Sulfasalazine.
 Intestinal B-Glucuronidase enzymes are capable of
hydrolyzing glucuronide conjugates excreted in the bile,
thereby liberating the free drug or its metabolites for
possible re-absorption (Enterohepatic circulation)
Other tissues such as kidneys, Lungs, adrenal gland, placenta,
brain and skin also have some degree of metabolizing capacity.

11.  The group of enzymes or enzyme system carrying out
the oxidative biotransformation of drugs and other
foreign compounds or chemicals and certain natural
metabolites is referred to as Mixed Function Oxidases
(MAOs) or Hepatic Cytochrome P450 Mixed function
Oxidase system or Cytochrome P450 mono-oxygenase
enzyme system.
 The abbreviation “CYP450” will be used for this
enzyme system.
 These enzymes are located in the lipophilic
membrane of the smooth Endoplasmic Reticulum of
the liver and other extra-hepatic tissues.
 When these lamellar membranes are isolated by
Homogenization and Fractionation of the cell, they
reform into vesicles called “microsomes”

12.  Microsomes retain most of morphological and functional
characteristics of the intact membrane including both the rough
and smooth surface features of the rough and smooth ER.
 ER is a network of Lipo-proteins membranes within the
cytoplasm.
 Rough Microsme (Derived from rough ER) containing
ribosomes, are responsible for protein biosynthesis whereas
smooth Microsomes are rich in drug metabolizing enzymes.
 Now it is obvious to say that most of the detoxification
reactions and metabolic changes of drugs which occur in the
liver or other Extra-hepatic tissues are carried out in smooth ER
Why it is called cytochrome P450?
 The name Cytochrome P450 is derived from the spectral properties of
this Heme protein. This component of the enzyme system in its
reduced form (Ferrous Fe2+) binds with carbon Monoxide to give a
complex which absorbs UV light at a maximum wavelength of 450 nm.

13.  Neibert classified the CYP450 super gene family on the basis of
their structural relationships.
 They have been sub-divided into families with greater than
40% amino acids homology and sub-families with greater
than 55% homology.
 CYP450 are named using the root symbol “CYP” which denotes
Cytochrome P450
 It is followed by a number that specifies the cytochrome
family (CYP1, CYP2, CYP3) etc.
 Next is the capital letter that denotes the sub-family (CYP1A,
CYP1B, CYP2A, CYP2C etc.)
 Finally the name ends with a number that represents the
specific enzyme (Individual gene)
 (CYP 1A1, CYP 1A2, CYP 2C9, CYP 2C19, CYP 3A4) etc.

14.  Oxidation is probably the most common and
important in drug metabolism and the
phenomenon of oxidative biotransformation is
termed as “Microsomal Drug Oxidation”
 CYP450 function as multi-component electron
transport system, responsible for the oxidative
metabolism of a variety of endogenous
substances (Such as Steroids, Fatty acids,
Prostaglandins and Bile acids etc.) and
Exogenous substances (Xenobiotics) including
drugs, Carcinogens, Insecticides, Plants Toxins,
Environmental Pollutants and other foreign
chemicals.

15.  Following is the general equation which describes
the oxidation of many xenobiotics (R-H) to their
corresponding oxidized metabolites (R-OH)
RH + NADPH + O2 + H+ ----> R-OH + NADP+ + H2O
 The reaction requires both molecular oxygen (O2)
and NADPH (Reduced form of Nicotinamide adenine
dinucleotide Phosphate)
 During the process of Oxidation, one atom of the
molecular oxygen is introduced into the substrate
(R-H) to form R-OH and the other oxygen atom is
incorporated into water.

16.  There are two major protein components of CYP450
which play a key role in oxidative
biotransformation of xenobiotics
 A Heme-Protein, called Cytochrome P450.
◦ The Heme portion is an Iron containing Porphyrin, Called
Protoporphyrin IX and the protein portion is called
apoprotein.
◦ The cytochrome P450 is a super family of enzymes
(Currently at 57 genes). It is responsible for transferring an
oxygen atom to the substrate (R-H) molecule.
 A Flavoprotein, called NADPH Cytochrome P450
reductase
◦ It contains both Flavin-Mono-Nucleotide (FMN) and Flavin
Adenine Di-Nucleotide (FAD)
◦ This component serves as an electron carrier i.e. shuttling
electrons from NADPH to Cytochrome P450

17.  A third component is Phospholipid called
Phosphatidyl Choline.
◦ The phospholipid is involved in the binding of the
drug molecule to the cytochrome P450 and
coupling the NADPH Cytochrome P450 reductase
to the cytochrome P450.
◦ The phospholipids make about 1/3 of the Hepatic
ER.

18.  Important Features
◦ An important feature of the CYP450 is its ability to
metabolize an unlimited number of substrate by a
variety of oxidative transformation
 This shows the substrate non-specificity of CYP450
as well as the presence of multiple forms of the
enzymes
 The multiple forms of the enzymes are called
Isoenzymes (Isoforms) which are closely related to
each other and differ somewhat in amino acid
sequence.
◦ Because of its membrane bound nature the
cytochrome P450 mono-oxygenase system has a
lipidoidal environment.
 This explains, why lipophilic compounds are
generally good substrates for these enzymes. The
catalytic role that CYP450 plays in the oxidation of
xenobiotics can be summarized in the following.

19. Following steps are involved in this reaction cycle
 The ferric Cytochrome P450 binds with a molecule of
substrate (R-H) to form CYT-P450-Substrate complex
 Transfer of one electron from NADPH dependent
Cytochrome P450 Reductase to the CYT-P450-Substrate
complex. This one electron transfer reduces Fe3+ to Fe2+
 The reduced CYT-P450-Substrate complex readily bind
di-Oxygen (O2) to form oxy-complex.
 The oxy- CYT-P450-Substrate complex undergoes auto-
oxidation to form a ferric superoxide anion CYT-P450-
Substrate complex

20.  The ferric superoxide anion then undergoes further
reduction by accepting one electron from NADPH
dependent CYT-P450-Reductase to give Ferric Per
Oxide di-anion CYT-P450-Substrate complex
 Water is released from the complex to form an
activated oxygen CYT-P450-Substrate complex
 The activated oxygen (FeO)3+ is highly electron
deficient and a potent oxidizing agent. The
activated oxygen is transferred to the substrate (R-
H) and the oxidized product (R-OH) is released
from the enzyme complex and the oxidized form
of CYT P450 is regenerated.

22.  Drugs, Plant Toxins, Environmental pollutants,
Insecticides and other chemicals foreign to the body,
undergoes enzymatic transformation that usually
results in the loss of pharmacological activity and
subsequent elimination (Excretion) from the body.
 For most biotransformation reactions, the metabolites
of the drug is more polar than the parent compound.
 The conversion of a drug to a more polar metabolite
enables the drug to be eliminated more quickly as
compared to the less polar lipid soluble compounds.
 The more polar (Hydrophilic) metabolites do not cross
the cell membranes easily so these are not readily
reabsorbed and are more rapidly excreted in urine.
 The pathways of Xenobiotics metabolism are divided
into two major categories
◦ Phase I reactions
◦ Phase II reactions

23.  These are non-synthetic functionalization reactions
 Phase I reactions include Oxidative, Reductive and
Hydrolytic Biotransformation reactions
 The purpose of these reactions is to induce a polar
functional group such as OH, NH2, COOH, SH etc.
into the xenobiotics molecule. This can be achieved
either by;
◦ Direct introduction of the functional group e.g. aliphatic
and aromatic hydroxylation
◦ By modifying or un-masking (exposing) the existing
functional group. E.g.
 Reduction of aldehydes and ketones to alcohols
 Oxidation of alcohols to acids
 Hydrolysis of esters and amides to unmask/expose COOH, NH2
and OH group.
 Reduction of AZO and Nitro compounds to give NH2 moieties.
 Oxidative N, O and S de-alkylation to give NH2, OH and SH group.

24.  Although Phase I reactions may not produce
sufficiently polar metabolites. Generally they tend
to produce a polar functional group or handle in
the molecule that can undergo subsequent phase II
reaction.
 In summary, phase I metabolic transformation
introduce a new polar functional group into the
molecule which may produce one or more of the
following changes.
 Decreased pharmacological activity (De-activation)
 Increased pharmacological activity (activation)
 Increased Toxicity (carcinogen mutagenesis, Cytotoxicity)
 Altered pharmacological activity
 Drugs exhibiting increased activity from the parent
compound/drug generally undergo metabolism +
conjugation, resulting in de-activation and
excretion of inactive conjugates.

25.  Phase II reactions are enzymatic synthesis
 When once a polar constituent or group is revealed
or placed into the drug molecule, then it often
undergoes Phase II or conjugation reactions.
 These reactions involve the coupling or conjugation
of small polar and ionizable compounds such as
Glucuronic acid, Sulfate, acetyl and certain amino
acids like glycine to functional groups of phase I
metabolites to form more polar and water soluble
conjugates.

26.  Most substances undergo both Phase I and
Polar II reactions sequentially while few, for
example isoniazid first undergo acetylation
(Phase II reaction) followed by Hydrolysis
(Phase I reaction).

27.  Parent compounds that already have existing
functional groups undergoes directly Phase II
conjugation reactions. Conjugated metabolites are
readily excreted in urine and are generally devoid
of pharmacological activity and toxicity.
 Generally Phase II pathways terminate or attenuate
the biological activity whereas Glutathione
conjugation protects the body against chemically
reactive compounds or metabolites. Thus phase I &
Phase II reactions complement one another in
detoxifying and facilitating the elimination of drugs
and Xenobiotics.
 Conjugation reactions use conjugating reagents,
which are derived from biochemical compounds
involved in carbohydrates, Fats and Proteins
metabolism.

28.  The conjugate formation involves these high
energy intermediates (Conjugating reagents) and
specific tranferase enzymes. Such enzymes
(Transferases) may be located in microsomes, in
cytosol or in mitochondria.
 These enzymes can catalyze the coupling of high
energy active form of conjugating agents [such as
Uridine-5-Diphospho-Glucuronic acid (UDPGA),
Acetyl CoA, 3-Phosphoadenosine-5-
Phosphosulfate (PAP) or S-adenosyl methionine]
with a drug or endogenous compounds or the drug
may be 1st activated to a high energy compound
(Such as S-CoA derivatives of Benzoic acid) that
then react with the conjugating agent.