Metabolism II

Phase II Reactions in
Biotransformation
Mr. K. K. Mali
Assistant Professor,
YSPM’s Yashoda Technical Campus,
Faculty of Pharmacy, Satara

Content
• Introduction of biotransformation
• Phase II Reactions
• Conjugation with glucuronic acid
• Conjugation with sulphate moieties
• Conjugation with alpha amino acids
• Conjugation with glutathione and mercapturic acid
formation
• Acetylation reaction
• Methylation reaction
• Miscellaneous conjugation reactions
April 13, 2018 2Phase II Reactions In Biotransformation

Content
• Factors affecting biotransformation of drugs
• Physicochemical properties of drug
• Chemical factors
• Induction of drug metabolising enzymes
• Inhibition of drug metabolising enzymes
• Environmental factors chemicals
• Biological factors
• Species differences
• Strain differences
• Sex differences
• Age
• Diet

Content
• Factors affecting biotransformation of drugs
• Altered physiological factors
• Pregnancy
• Hormonal imbalance
• Diseased states
• Temporal factors
• Circadian rhythm

Introduction of biotransformation

Introduction of biotransformation
• True detoxification reaction
• Phase II metabolites are
• Highly polar
• Free from pharmacological activity
• Non-carcinogenic due to conjugation

Phase II reactions
• Phase II metabolites are
• Attachment of small polar endogenous molecules such as glucuronic acid,
sulfate and amino acids to Phase I metabolites or parent drugs
• Products are more water-soluble and easily excretable
• Trapping highly electrophilic molecules with endogenous nucleophiles
such as glutathione prevent damage to important macromolecules (DNA,
RNA, proteins)
• Regarded as true detoxifying pathway (with few exceptions)
• In general, appropriate transferase enzymes activate the transferring group
(glucuronate, sulphate, methyl, acetyl) in a coenzyme form

Phase II reactions
Characteristics of conjugation reactions
• Initial activation step
Drug is activated
or
Conjugating reagent activated
• Capacity limited reaction
a. Limited amount of conjugating reagent
b. Limited amount of enzyme
Order of capacity limited reaction
Glucuronidation > Amino acid conjugation > Sulphation and Glutathione conjugation

Phase II reactions
Phase II reactions and their characteristics
Conjugation
reaction
Conjugating
Agent
Enzyme
involved
Activated
intermediate
Functional
groups
Glucuronidation Glucuronic Acid UDP-glucuronyl
transferase
UDPGA -OH, -COOH,
-NH2, -SH
Sulphation Sulphate Sulpho
transferase
PAPS -OH, -NH2
Amino Acid Glycine Acyl
transferase
Acyl CoA -COOH, NH2
Glutathione Glutathione Glutathione-S
transferase
- Alkyl halide, alkyl
nitrate, alkyl
epoxide, etc
Acetylation Acetyl CoA N-Acetyl
Transferase
Acetyl CoA Hydrazines, -
NH2, -SO2NH2
Methylation L-Methionine Methyl
Transferase
S-adenosyl
Methionine
-OH,
-NH2, -SH

• Glucuronidation is the most common conjugation pathway
• The coenzyme, UDP glucuronic acid is synthesized from the corresponding
phosphate
• UDP-glucuronic acid contains D-glucuronic acid in the a-configuration at the
anomeric center, but glucuronate conjugates are b-glycoside, meaning inversion
of stereochemistry is involved in the glucuronidation
• Glucuronides are highly hydrophilic and water soluble
• UDP glucuronosyl transferase is closely associated with Cyp450 so that Phase I
products of drugs are efficiently conjugated
• Four general classes of glucuronides: O-, N-, S-, and C-
• Neonates have undeveloped liver UDP-glucuronosyl transferase activity, and
may exhibit metabolic problem. For example, chloramphenicol (Chloroptic)
leads neonates to “gray baby syndrome”
• Neonatal jaundice may be attributable to their inability to conjugate bilirubin
with glucuronic acid
Conjugation with glucuronic acid

1. Catalysed by microsomal enzymes.
2. Dominates at high substrate concentration
3. Most common phase II reaction. Why?

Steps in glucuronidation formation
1. Synthesis of activated coenzyme UDPGA
2. Transfer of glucuronyl moiety from UDPGA to substrate RXH
pyrophosphorylase
-D-Glucose-1-phosphate + UTP UDPG + PPi
UDPG-dehydrogenase
UDPG + 2NAD+ + H2O UDPGA + 2NADH +2H+
UDP-glucuronyl transferase
UDPGA + RXH RX-glucuronic acid + UDP
Where, X = O, COO, NH or S

April 13, 2018 Phase II Reactions In Biotransformation 13

1. O-glucuronide conjugation
a. OH compounds
Ex. Alcohol:- Chloramphenicol
Phenol:- Morphine, paracetamol
b. COOH compounds
Ex. Salicylic acid
2. N-glucuronide conjugation
Ex. Desimipramine, Meprobamate
3. S-glucuronide conjugation
Ex. Thiophenol
4. C-glucuronide conjugation
Ex. Phenylbutazone

• O-Glucuronide: Phenols
Acetaminophen Morphine
OH
CH3
O
H
N
HO OH
N
O
CH3

• O-Glucuronide: Alcoholes
Chloramphenicol Propranolol
O2N
Cl
Cl
O
H
N
OH
OH
H
CH3
CH3
OH
NO

• O-Glucuronide: Enols
Hydroxycoumarine
O O
OH

• O-Glucuronide: N-hydroxyamines/amides
N-hydroxydapsone N-Hydroxy-2-
acetylaminoflourene
S
O2
H2N NHOH
N
CH3
OH

• O-Glucuronide:
Aryl acids
Salicylic Acid
Arylalkyl Acids
Fenoprofen
O
OH
O
CH3
OH
COOH

• N-Glucuronide:
Arylamine
7-Amino-5-nitroindazole
N
H
N
NH2
O2N
Alkylamines
Desipramine
N
H
CH3
N

• N-Glucuronide:
Sulphonamides
Sulfisoxazole
OO
H2N
N
H
S
CH3
CH3
N
O
H3C
O
NH2
NH2
H3C O
O
O
Amides
Meprobamate

• N-Glucuronide:
Amitryptiline
3o Amines
Cyproheptadine
CH3N
3o Amines

• N-Glucuronide:
Cotinine
Aromatic Amine

• S-Glucuronide:
Sulfhydryl
Methimazole
Carbodithioic acid
Disulfirum
(reduced form)
CH3
HS
N
N
H3C
H3C
S
SH
N

• C-Glucuronide:
Phenylbutazone
CH3
O
O
N
N

Example: Benzoic acid
Water Soluble, Excreted

Conjugation with sulphate moiety
• Occurs less frequently than does glucuronidation presumably due to
fewer number of inorganic sulfates in mammals and fewer number
of functional groups (phenols, alcohols, arylamines and N-hydroxy
compounds)
1. Catalysed by nonmicrosomal enzymes
2. Less common
3. Dominates at low substrate concentration

Steps in Sulphate Conjugation
1. Synthesis of activated coenzyme PAPS
2. Transfer of sulphate moiety from PAPS to substrate RXH
ATP-sulfurylase/Mg++
ATP + SO4
2- APS + PPi
APS-phosphokinase/Mg++
APS + ATP PAPS + ADP
Sulfotransferase
PAPS + RXH RX-SO3 + PAP
Where, X = O, NH

Three enzyme-catalyzed reactions are involved in sulfate
conjugation
S
O
O
-
O O-
ATP PPi
Mg+2
ATP sulfurylase
Sulfate
O
OP
O
O-
OS
O
O
-
O
Ad
HO OH
Adenosine-5'-
phosphosulfate (APS)
Mg
+2
APS phosphokinase
O
OP
O
O-
OS
O
O
-
O
Ad
-2
O3PO OH
ATP ADP
3'-phosphoadenosine-5'-
phosphosulfate (PAPS)
PAP
Sulfotransferase
RXH
S
O
O
-
O XR
Sulfate
conjugate
(soluble)

• Phenolic sulfation predominates
• Phenolic O-glucuonidation competes favorably with sulfation due to
limited sulfate availability
• Sulfate conjugates can be hydrolyzed back to the parent compound by
various sulfatases
• Sulfoconjugation plays an important role in the hepatotoxicity and
carcinogenecity of N-hydroxyarylamides
• In infants and young children where glucuronyltransferase activity is
not well developed, have predominating O-sulfate conjugation
• Examples include: a-methyldopa, albuterol, terbutaline,
acetaminophen, phenacetin, phenol (salbutamol, paracetamol),
amines (aniline)

• Examples
COOHH3C
H
H
N
HO
HO HO CH3
HO
CH3
CH3
HOH
N
-Methyldopa
CH3
CH3
CH3
OH
HOH
NHO
Albuterol Terbutaline

Conversion of Minoxidil to Minoxidil Sulphate
Minoxidil Monoxidil Sulphate

Conjugation with alpha amino acids
• The first mammalian drug metabolite isolated, hippuric acid, was the
product of glycine conjugation of benzoic acid
• Amino acid conjugation of a variety of caroxylic acids, such as
aromatic, arylacetic, and heterocyclic carboxylic acids leads to amide
bond formation
• Glycine conjugates are the most common
• Taurine, arginine, asparagine, histidine, lysine, glutamate, aspartate,
alanine, and serine conjugates have also been found
COH
R O
Benzoic Acid, R = H
Salicylic Acid, R = OH
CONHCH2COH
R O O
Hippuric Acid, R = H
Salicyluric Acid, R = OH

Less common due to limited number of amino acids
Steps:
1. Activation of carboxylic acid drug substrate
2. Acetylation of amino acid
Acyl synthetase
RCOOH + ATP RCOAMP + H2O + PPi
Acyl CoA transferase
RCOAMP + CoASH RCOSCoA + AMP
N-acyl transferase
RCOSCoA + H2N-R’ -COOH RXH RCONH-R’-COOH + CoASH
Where, R’ = -CH2 If glycine, CH-CH2-CH2-NH2 if glutamine

Mechanism of Amino Acid conjugation
Examples:
1. Aryl acid: Salicylic acid
2. Heterocyclic aryl acid: Nicotinic acid
An Acyl-CoA Intermediate
Glycine Conjugate R = H
Glutamine Conjugate R = CH2CH2CONH2
Drug-COOHDrug-COOH

Brompheniramine Metabolism
Br
N
N
CH3
CH3
P450
Br
N
NH
CH3
P450
Br
N
NH2
P450
Br
N CHO
Br
N
N
CH3
CH3
Br
N
O
H
N COOH
Br
N COOH
Brompheniramine
Aldehyde
dehydrogenase
Glycine
N-acyltransferase
Carboxylic Acid metabolite
Brompheniramine N-oxide Glycine conjugate

Conjugation with glutathione
Structure of glutathione
• Glutathione is a tripeptide (Glu-Cys-Gly) – found virtually in all
mammalian tissues
• Its thiol functions as scavenger of harmful electrophilic parent drugs
or their metabolites
• Examples include SN2 reaction, SNAr reaction, and Michael addition

Mechanisms of GSH conjugation
1. Nucleophilic substitution
RX + GSH R-S-G + H+ + x-
Ex. Sulphates, Organophosphates
2. Nucleophilic addition
R-CH=CH-R’ + GSH RCH-S-G-CH2-R’
Ex. Paracetamol

SN2 : Example

SNAr : Example

Michael Addition: Example

Mercapturic acid Conjugate
N
H
H
N
NH2
O
S
O
O
HO
O
OH
Drug Amino Acid
(AA)
-Glutamyl
transpeptidase
-Glutamyl-AA
NH2
H
N
S
O
O
HO
Drug
Glutathione Conjugate
Glycine
Cysteinyl
Glycinase
NH2
HO
S
O
Drug
S-substituted
Cysteine
Derivative
Acetyl
CoA CoASH
N
H
H2N
S
O
Drug
CH3
O
Mercapturic
acid conjugate

Acetylation reaction
• Metabolism for drugs containing a primary amino group,
(aliphatic and aromatic amines), amino acids, sulfonamides,
hydrazines, and hydrazides
• The function of acetylation is to deactivate the drug, although
N-acetylprocainamide is as potent as the parent antiarrhythmic
drug procainamide (Procanbid) or more toxic than the parent
drug, e.g., N-acetylisoniazid
• Examples:
Aliphatic amines: Histamine
Aromatic amines: PAS, PABA, Dapsone
Sulphonamide: Sulphanilamide, Sulphapyridine
Hydrazides: Isoniazide

• Acetylation is two-step, covalent catalytic process involving N-
acetyl transferase
H3C SCoA
O CoASH
H3C X
O
H2N R
X-
H3C
O
NHR
X-
N-Acetylation of amines

• Acetylation of P amino salicylic acid

• Acetylation of Procainamide
H2N
O
N
H
N N
O
N
H
N
O
H
Procainamide NAPA
Urine 85%

Methyl conjugation
Characteristics:
1. Metabolites formed are not polar
2. Drug and metabolite having equal pharmacological activity
3. Less important for xenobiotics
4. Important in biosynthesis of endogenous amines, ex.
Adrenaline, while as inactivation of endogenous amines, ex.
NA, 5-HT, Histamine

Methyl conjugation
• Minor conjugation pathway, important in biosynthesis of
epinephrine and melatonin; in the catabolism of norepinephrine,
dopamine, serotonin, and histamine; and in modulating the
activities of macromolecules (proteins and nucleic acids)
• Except for the formation of quarternary ammonium salts,
methylation of an amine reduces the polarity and hydrophilicity of
the substrates
• A variety of methyl transferase, such as COMT (catechol O-methyl
transferase), phenol-O-methyltransferase, N-methyl transferase, S-
methyltransferase etc are responsible for catalyzing the transfer of
methyl group from SAM to RXH

Methyl conjugation
Steps in methyl conjugation
1. Synthesis of an activated coenzyme SAM
2. Transfer of methyl group from SAM to substrate

Methyl conjugation
Examples
1. O-methylation: Morphine, L-DOPA
2. N-methylation: Normorphine, Nicotine
3. S-methylation: Propylthiouracil, 6-mercaptopurine

Fatty Acid and Cholesterol Conjugation
• Hydroxyl-containing drugs can undergo conjugation with a wide range of
endogenous fatty acids such as saturated acids from C10 to C16 and
unsaturated acids such as oleic and linoleic acids
• Cholesterol ester metabolites have been detected for drugs containing
either an ester or a carboxylic acid
HO
O
O
O
O
N
Cl
Cl
OH
Cl O (CH2)10 COOH
Prednimustine
Fatty Acid
Cholesterol

Miscellaneous conjugation reactions
1. Conjugation of cyanide
2. Conjugation with ribose
Purine/Pyrimidine + Ribose Nucleotides
3. Conjugation with Taurine
Bile acid + Taurine Bile

Factors affecting biotransformation of drug
1. Physicochemical properties
of drug
2. Chemical factors
a. Enzyme Induction
b. Enzyme Inhibition
c. Environmental factors
3. Biological factors

Factor Reaction type
Age (newborns,
children,
elderly)
Decreasing of metabolism speed
Pregnancy Increasing of metabolism speed
Genetic factor Various reactions
Liver pathology Decreasing of excreation speed of drugs, depending on their kinetics,
type and stage of liver disease, increasing of bioavailability and
decreasing of excretion speed of orally administered drugs with high
hepatic clearence
GI pathology Changes in metabolism in GI epithelium
Nutrition
character
Increasing of metabolism speed of certain drugs in case of diet with
dominance of proteins and carbohydrates
Decreasing of metabolism speed in case of heavy digestive disorders
linked with starvation (total or protein)

Environment Increasing of metabolism speed if in contact with chlorine
insecticides
Alcohol
— one time
consumption
Depressing of enzymes that metabolise drugs
— chronic
consumption
Induction of enzyme system
Smoking Increasing of metabolism of certain drugs (i.e. theophyllin)
Way of excretion Metabolism in liver before entering system circulation (first
going-through effect) after peroral administration of drugs
Time of introduction
of drugs
Circade changes in drugs metabolism
Interaction of drugs Stimulation and depression of enzyme reaction

• Molecular size and shape
• pKa
• Acidity/ Basicity
• Liphophilicity
• Steric and electronic characteristics
• The chemical structure (the absence or presence of certain functional
groups) of the drug determines its metabolic pathways.

• Steric and electronic characteristics

Factors affecting biotransformation of drugs
• Enzyme induction
• Increased drug metabolising ability of the enzymes by drug
• Properties of enzyme inducers
1. Lipophilic
2. Long half life
• Mechanism of enzyme induction
• Increased liver size and blood flow
• Increased total and microsomal protein content
• Increased stability of enzymes
• Increased synthesis of cytochrome P450

• Classification of enzyme inducers
1. Phenobarbital type inducers
Ex. Phenytoin, warfarin etc.
2. Polycyclic hydrocarbon type inducers
Ex. Cigarette smoking can cause increased metabolism and
elimination of theophylline.
• Autoinduction
Ex. Carbamazepine, Meprobamate, Rifampicin

• Consequences of enzyme induction
• Increased rate of metabolism
• Decrease in drug plasma concentration
• Enhanced oral first pass metabolism
• Reduced bioavailability
• If metabolite is active or reactive, increased drug
effects or toxicity

• Enzyme inhibition
• Decrease in the drug metabolising ability
• Classification of enzyme inhibition
• Direct inhibition
• Competitive inhibition
• Non-Competitive inhibition
• Product inhibition
• Indirect Inhibition
• Repression
• Altered physiology

• Direct inhibition
• Competitive inhibition
Ex. Methacholine inhibit metabolism of Ach for choline esterase
• Non-Competitive inhibition
Ex. INH inhibit metabolism of Phenytoin
• Product inhibition
Ex. Xanthine oxidase inhibitor such as allopurionol
• Indirect Inhibition
• Repression
Ex. Disulphiram
• Altered physiology
Due to nutritional deficiency or hormonal imbalance

• Consequences of Enzyme Inhibition
• Increase in the plasma concentration of parent drug
• Reduction in metabolite concentration
• Exaggerated and prolonged pharmacological effects
• Increased liklihood of drug-induced toxicity

“Enzyme inhibition is more important clinically than enzyme induction for drugs
having narrow therapeutic index.”

• Biological Factors
• Sex differences
• Age
• Diet
• Pregnancy
• Disease states
• Temporal factor

• Phenylbutazone half-life is 3 h in rabbit, ~6 h in rat, guinea pig, and
dog and 3 days in humans.
• Isoniazid is known to be acetylated by N-acetyltransferase into
inactive metabolite.
• The rate of acetylation in asian people is higher or faster than
that in eurpoean or north american people. Fast acetylators are
more prone to hepatoxicity than slow acetylator.
• Isoniazide (fast (Whites) and slow (Eskimos) acetylators)

• Sex differences
• Responsiveness to certain drugs is different for men and
women
• Males shows greater metabolism than female
• Hormonal changes during development have a profound effect
on drug metabolism
• Metabolism of Diazepam, caffiene, and paracetamol is faster in
females than in males while oxidative metabolism of lidocaine,
chordiazepoxide are faster in men than in females

• Age
• Neonates: (0-2 months)
• Slow biotransformation
• Drug eliminated as unchanged
• Caffene- Half life 4 d
• Chloramphenicol- Gray baby syndrome or cyanosis
• Sulphonamides- Renal toxicity
• Paracetamol - Hepatotoxicity
• Infants: (2 months to one year)
• Same as neonates with improved enzyme activity.

• Age
• Children : (1-12 years)
• Fast biotransformation
• Require large dose of drug
• Elderly person:
• Liver size reduced
• Blood flow decreased
• Enzyme activity decreased
• Drug conjugation not affected

• Diet
• Low protein diet decreases drug metabolising ability.
• High protein diet increases drug metabolising ability.
• High protein-carbohydrate ratio increases MFO activity.
• Fat free diet decreases CYP 450 levels.
• Vitamin deficiency of A,C,E, and B can result in a
decrease of oxidative pathway in case of vitamin C
deficiency , while vitamin E deficiency decreases
dealkylation and hydroxylation.
• Ca, Mg, Zn deficiencies decreases drug metabolism
capacity whereas Fe deficiency increases it.

• Diet
• Essential fatty acid (esp. Linoleic acid) deficiency reduce
the metabolism of ethyl morphine and hexobarbital by
decreasing certain drug-metabolizing enzymes.
• Grapefruit juice decrease metabolism of many drugs.
• Starvation- decreases amount of glucuronide formation.
• Malnutrition in women- increases metabolism of sex
hormone.
• Alcohol ingestion results in short term decrease followed
by increase in enzyme activity.

• Pregnancy
• Metabolising ability decreased- high levels of steroid hormons
• Metabolism of promazine and pethidine reduced.
• Metabolism of anticonvulsants increased- induction by circulating
progesterone.
• Enzyme induction or inhibition

• Disease states
• Liver- Primary site for metabolism, Diseases like hepatic
carcinoma, hepatitis, cirrhosis, obstructive jaundice decreases
metabolism.
• Kidney: Glycine conjugation of salicylates, oxidation of vitamin D,
hydroysis of procaine impaired in renal disease.
• CCF and MI: decrease blood flow to liver- decrease metabolism of
high extraction ratio drugs- Propranolol, lidocaine
• Diabetes: decreased glucuronidation

• Temporal factor
• Diurnal variations or variations in enzyme activity with light cycle
• Enzyme activity maximum in early morning.
• Enzyme activity minimum in late afternoon.
• Ex: aminopyrine, hexobarbital, imipamine

• Genetic Variation
• wide variability in the response to drugs between individuals
• consequences of such variation may be therapeutic failure or an
adverse drug reaction
• genetic diversity is the rule rather than the exception with all
proteins, including drug metabolizing enzymes
• allelic variants with different catalytic activities from that of the
wild-type form have been identified
• inheritance leads to subpopulations (genetic polymorphisms) with
different drug metabolizing abilities
lack of activity
reduction in catalytic ability
enhanced activity

• frequency of the polymorphism often varies according to the
ethnic ancestry of the individual
• CYP2D6 is extensively studied, the gene for CYP2D6 is highly
polymorphic
• It’s expression leads to 3 phenotypes (phenotype is the expression
of genetic make-up)
• Extensive metabolizers (EMs) have functional enzyme activity
• Intermediate metabolizers (IMs) have diminished enzyme activity
• Poor metabolizers (PMs) have little or no activity
• 5-10% of Caucasians and 1-2% of Asians exhibit the PM phenotype

• Debrisoquine, formerly used in the treatment of
hypertension, is metabolized by CYP2D6 to 4-
hydroxydebrisoquine
• Remarkable interindividual variation in pharmacological
effect of the drug
• Urine of volunteers given debrisoquine was examined for
presence of 4-hydroxydebrisoquine
• One subject had a very low conversion of parent drug to
metabolite
• was very sensitive to the antihypertensive effects of
debrisoquine

• Drug dosing
• 1- An increase in drug dosage would increase drug concentration and
may saturate certain metabolic enzymes.
• 2- when metabolic pathway becomes saturated, an alternative
pathway may be pursued.

• Route of administration
• Orally administered drugs are absorbed from the GIT and
transported to the liver before entering the systemic circulation.
Thus the drug is subjected to hepatic metabolism (first pass effect)
before reaching the site of action.
• Sublingually and rectally administered drugs take longer time to
be metabolized than orally taken drugs. Nitroglycerine is
ineffective when taken orally due to hepatic metabolism.
• IV administration avoid first pass effect because the drug is
delivered directly to the blood stream.

References
• Brahmankar DM , Jaiswal SB. Biopharmaceutics and Pharmacokinetics A
Treatise. 2nd edition. Vallabh Prakashan;New Delhi, 2009 Page No. 139-
169.
• Gibson G. Gordon, Skett Paul. Introduction to drug Metabolism. 3rd
edition, Nelson Thorne Publishers; Chetenham, 2001 Page No. 1-13, 37-
62.
• Wilson and Gisvold’s Textbook of Organic Medicinal and Pharmaceutical
Chemistry 11th ed. Lippincott, Williams & Wilkins ed.
• Foye’s Principles of Medicinal Chemistry

Metabolism II

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