BIOTRANSFORMATION
Prof. Dr. Basavaraj K. Nanjwade M. Pharm., Ph. D
Department of Pharmaceutics
KLE University’s College of Pharmacy
BELGAUM – 590010, Karnataka, India
Cell No: 0091 9742431000
E-mail: bknanjwade@yahoo.co.in
03-12-2010
KLECOP, Nipani
1
Contents:Introduction
Phase I reaction
Phase II reaction
Factors affecting Biotransformation
Reference
03-12-2010
KLECOP, Nipani
2
 Introduction:
Important terms:
Detoxication (detoxification)
 Prodrugs
Active Metabolite
Reactive Metabolite
 Metabolism
03-12-2010
Xenobiotics
KLECOP, Nipani
3
Definition:
 “Biotransformation of drug is defined as the
conversion from one chemical form to another”.
the term is used synonymously with metabolism.
03-12-2010
KLECOP, Nipani
4
 Biotransformation
leads to:
• Pharmacologic Inactivation of Drug
• Ex: Active Drug
Salicylic Acid
Inactive Drug
Salicyluric Acid
• Active Metabolite From An Inactive Drug
• Ex. Inactive(Prodrug)
•
Aspirin
Active
Salicylic Acid
•No Change in Pharmacologic Activity
• Ex.
03-12-2010
Active
Codeine
Active Drug
Morphine
KLECOP, Nipani
5
Drug metabolizing organs
• Liver is the heart of metabolism
• Because of its relative richness of enzymes in large
amount.
• Schematic chart of metabolizing organ (decreasing
order)
• Liver > lungs > Kidney > Intestine > Placenta >
Skin > Brain > Testes > Muscle > Spleen
03-12-2010
KLECOP, Nipani
6
Drug Metabolizing
Enzymes
Non Microsomal
Microsomal
03-12-2010
KLECOP, Nipani
7
Microsomal Enzymes
• Found predominately in the
smooth Endoplasmic
Reticulum of liver
• Other areas:
– Kidney
– Lungs
– Intestinal mucosa
Non-microsomal
enzymes
• Found in the cytoplasm and
mitochondria of hepatic cells
• Other tissues including plasma
03-12-2010
KLECOP, Nipani
8
Non-microsomal
enzymes
Microsomal Enzymes
• Non-synthetic/ Phase I
reactions
– Most oxidation and
reduction
– Some hydrolysis
• Synthetic/ Phase II
reactions
–
ONLY Glucuronide
conjugation
03-12-2010
• Non-synthetic/ Phase I
reactions
– Most hydrolysis
– Some oxidation and
reduction
• Synthetic/ Phase II reactions
– ALL except Glucuronide
conjugation
KLECOP, Nipani
9
Microsomal Enzymes
• Inducible
– Drugs, diet, etc.
Non-microsomal
enzymes
• Not inducible
03-12-2010
KLECOP, Nipani
10
Why Biotransformation?
• Most drugs are excreted by the kidneys.
• For renal excretion drugs should:
– have small molecular mass
– be polar in nature
– not be fully ionised at body pH
• Most drugs are complex and do not have these
properties and thus have to be broken down to
simpler products.
• Drugs are lipophilic in nature.
03-12-2010
KLECOP, Nipani
11
• Strongly bound to plasma proteins.
• This property also stops them from getting
eliminated.
• They have to be converted to simpler
hydrophilic compounds so that they are eliminated
and their action is terminated.
When and How?
• Biotransformation occur between absorption
and elimination from kidneys.
• Drugs administered orally can biotransform in
the intestinal wall.
03-12-2010
KLECOP, Nipani
12
03-12-2010
KLECOP, Nipani
13
Metabolic reaction:
Phase I reaction Phase II reaction
Oxidation
Conjugation
Reduction
Hydrolysis
03-12-2010
KLECOP, Nipani
14
 Phase I:
 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 called as
functionalization reactions.
Phase I reactions are Non-synthetic in nature.
The majority of Phase I metabolites are
generated by a common hydroxylating
enzyme
system
known
as
cytochrome
P450.
03-12-2010
KLECOP, Nipani
15
03-12-2010
KLECOP, Nipani
16
 Oxidative reaction:
1) Oxidation of aromatic carbon atoms
2) Oxidation of olefins (C=C bonds)
3) Oxidation of Benzylic, Allylic carbon atoms
& carbon atoms alpha to carbonyl & imines
4) Oxidation of aliphatic carbon atoms
5) Oxidation of alicyclic carbon atoms
03-12-2010
KLECOP, Nipani
17
6) Oxidation of carbon-heteroatom systems:
A. Carbon-Nitrogen system
 N- Dealkylation.
 Oxidative deamination
 N-Oxide formation
 N-Hydroxylation
B. Carbon-Sulfur system
 S- Dealkylation
 Desulfuration
 S-oxidation
C. Carbon-Oxygen systems(O- Dealkylation)
03-12-2010
KLECOP, Nipani
18
7) Oxidation of Alcohol, Carbonyle and Acid functios.
8) Miscellaneous oxidative reactions.
 Reductive reactions:
1) Reduction of Carbonyl functions.(aldehydes/ketones)
2) Reduction of alcohols and C=C bonds
3) Reduction of N-compounds (nitro,azo & N-oxide)
4) Miscellaneous Reductive reactions.
03-12-2010
KLECOP, Nipani
19
 Hydrolytic reactions
1) Hydrolysis of Esters and Ethers
2) Hydrolysis of Amides.
3) Hydrolytic cleavage of non aromatic heterocycles
4) Hydrolytic Dehalogination
5) Miscellaneous hydrolytic reactions.
03-12-2010
KLECOP, Nipani
20
 Oxidation of aromatic carbon atoms
(aromatic hydroxylation):
R
OH
Arenol (major)
R
R
R
OH
H2O
O
Arene
epoxide hydrase
OH
Arene oxide
(highly reactive electrophile)
Dihyrdrodiol
GSH
5-epoxide transferase
R
OH
SG
Tissue toxicity in instances
when glutathione is depleted.
Glutathione conjugate
(min.pro.)
R
OH
OH
Catechol(min. Pro.)
 E.g. Epoxides of Bromobenzene and Benzopyrene.
03-12-2010
KLECOP, Nipani
21
 Oxidation of olefins (C=C bonds):
 Oxidation of non-aromatic C=C bonds is
analogous to aromatic hydroxylation. i.e. it
proceeds via formation of epoxides to yield 1,2dihydrodiols.
OH
HO
O
H2O
N
CONH2
Carbamazepine
03-12-2010
N
CONH2
Carbamazepine-10,11
epoxide
KLECOP, Nipani
epoxide hydrase
N
CONH2
Trans-10,11
dihydroxy
carbamazepine
22
 Oxidation of Benzylic Carbon Atoms:
 Carbon atoms attached directly to the aromatic ring
are hydroxylated to corresponding Carbinols.
 If the product is a primary carbinol, it is further
oxidized to aldehydes and then to carboxylic acids,
 E.g.Tolbutamide
 A secondary Carbinol is converted to Ketone.
CHO
CH3
CH2OH
SO2NHCONHC4H9
SO2NHCONHC4H9
Tolbutamide
03-12-2010
COOH
Alcohol
dehydrogenase
Prmary carbinol
Corresponding
aldehyde
KLECOP, Nipani
Corresponding
carboxylic acid.
23
 Oxidation of Allylic carbon Atoms:
 Carbon atoms adjacent to Olefinic double bonds (are
allylic carbon atoms) also undergo hydroxylation in a
manner similar to Benzylic Carbons.
 E.g. Hydroxylation of Hexobarbital to 3`-hydroxy
Hexobarbital.
Allylic carbon atom
OH
3'
2'
H3C
H3 C
O
O
O
HN
O
HN
N
N
CH3
CH3
O
O
3'-Hydroxy Hexobarbital
Hexobarbital
03-12-2010
KLECOP, Nipani
24
 Oxidation of Carbon Atoms Alpha to
Carbonyls and Imines:
 Several Benzodiazepines contain a carbon atom (C-3)
alpha to both Carbonyl (C=0) and imino (C=N)
function which readily undergoes Hydroxylation.
 E.g. Diazepam
O
N
N
3
OH
N
N
IC
IC
3-Hydroxy diazepam
Diazepam
03-12-2010
KLECOP, Nipani
25
 Oxidation of Aliphatic Carbon Atoms
(Aliphatic Hydroxylation):

Terminal hydroxylation of methyl group
yields primary alcohols which undergoes
further oxidation to aldehydes and then to
carboxylic acid.
H
H3C
C
CH3
OH
CH3
C
H2
C
H
COOH
C
CH3
C
H2
C
H
COOH
Tertiary alcohol metabolite
Ibuprofen
03-12-2010
H3C
CH3
KLECOP, Nipani
26
 Oxidation of Alicyclic Carbon Atoms
(Alicyclic Hydroxylation):
 Cyclohexane (alicyclic) and piperidine (non-aromatic
heterocyclic) rings are commonly found in a number
of molecules.
 E.g. Acetohexamide and minoxidil respectively.
 Such rings are generally hydroxylated at C-3 or C-4
positions.
H2N
H2N
N
O
N
N
N
O
4'
N
N
OH
H2N
H2N
4'-Hydroxy Minoxidil
Minoxidil
03-12-2010
KLECOP, Nipani
27
 Oxidation of Carbon-Heteroatom Systems:
 Biotransformation of C-N, C-0 and C-S system
proceed in one of the two way:
1. Hydroxylation of carbon atom attached to the
heteroatom and subsequent cleavage at carbonheteroatom bond.
E.g. N-, O- and S- dealkylation, oxidative
deamination and desulfuration.
2. Oxidation of the heteroatom itself.
 E.g. N- and S- oxidation.
03-12-2010
KLECOP, Nipani
28
 Oxidation of Carbon-Nitrogen System:
 N-Dealkylation:
 Mechanism of N-dealkylation involve oxidation of αcarbon to generate an intermediate carbinolamine
which rearranges by cleavage of C-N bond to yield
the N dealkylated product and the corresponding
carbonyl of the alkyl group.
OH
H
O
N
C
H
03-12-2010
N
C
NH
C
+
H
Carbinolamine
Intermediate
KLECOP, Nipani
N-Dealkylated
metabolite
Carbonyl
29
 A tertiary nitrogen attached to different alkyl groups
undergoes dealkylation by removal of smaller alkyl
group first.
Example:
 Secondary aliphatic amine E.g. Methamphetamine.
 Tertiary aliphatic amine
E.g. imipramine
 Tertiary alicyclic amine
E.g. hexobarbital
 Amides
E.g. Diazepam
03-12-2010
KLECOP, Nipani
30

N-Hydroxylation:-
Converse to basic compounds that form N-oxide, Nhydroxy formation is usually displayed by non-basic
nitrogen atoms such as amide Nitrogen.
E.g. Lidocaine
CH3
CH3
O
N
H
C
O
C2H5
C
H2
N
N
C
H2
N
OH
C2H5
C2H5
CH3
CH3
N- Hydroxy Lidocaine
Lidocaine
03-12-2010
C
C2H5
KLECOP, Nipani
31
 Oxidation of Carbon-Sulfur Systems:
 S-Dealkylation:
The mechanism of S-Dealkylation of
thioethers is analogous to N-dealkylation .IT
proceed via α-carbon hydroxylation.
 The C-S bond cleavage results in formation of
a thiol and a carbonyl product.
 E.g. 6-Methyl mercaptopurine.
SCH2OH
SCH3
N
N
N
N
N
N
N
N
H
N
H
6-Methyl
Mercaptopurine
03-12-2010
SH
Hydroxylated
intermediate
KLECOP, Nipani
N
N
+ HCHO
N
H
6-Mercaptopuri9ne
32
 Desulfuration:
 This reaction also involves cleavage of carbon-sulfur bond
(C=S).
 The product is the one with C=0 bond. Such a desulfuration
reaction is commonly observed in thioamides such as
thiopental.
Thiopental
KLECOP, Nipani
Pentobarbital
33
 S-Oxidation:
 Apart from S-dealkylation, thioethers can also undergo Soxidation reaction to yield sulfoxides which may be further
oxidized to sulfones several phenothiazines.
 E.g. Chlorpromazine undergo S-oxidation.
03-12-2010
KLECOP, Nipani
34
 Oxidation of Carbon-Oxygen Systems:
 O-Dealkylation:
 This reaction is also similar to N-Dealkylation and
proceeds by α-carbon hydroxylation to form an
unstable hemiacetal or hemiketal intermediate.
 Which spontaneously undergoes C-0 bond cleavage
to form alcohol and a carbonyl moiety.
H
OH
R-O-CH2R'
Ether
03-12-2010
R-O-CH-R'
Hemiacetal
R-OH
Alcohol
KLECOP, Nipani
+
O=C-R'
Aldehyde/ketone
35
 Oxidation of Alcohol, Carbonyl and
Carboxylic Acid
 In case of ethanol, Oxidation to acetaldehyde is
reversible and further oxidation of the latter to
acetic acid is very rapid since Acetaldehyde is
highly toxic and should not accumulate in body.
Aldehyde
alcohol
CH3CHO
CH3CH2OH
dehydrogenase
Ethanol
03-12-2010
Acetaldehyde
KLECOP, Nipani
CH3COOH
dehydrogenase
Aceticacid
36
 Miscellaneous oxidative reactions:
 Oxidative aromatization /dehydrogenation:
 E.g. Metabolic aromatization of drugs is nifedipine.
COOH
COOCH3
NO2
NO2
CH3
N
NH
CH3
COOCH3
CH3
COOCH3
Nitedipine
03-12-2010
CH2OH
Pyridine metabolite
KLECOP, Nipani
37
 Oxidative Dehalogenation:
 This reaction is common with halogen containing drugs
such as chloroform.
 Dehalogenation of this drug yields phosgene which
may results in electrophiles capable of covalent
binding to tissue.
Cl
Cl
oxi.
Cl
H
Chloroform
03-12-2010
O
Cl
Cl
Covalent binding to tissues.
-HCL
Phosgene
KLECOP, Nipani
38
 Reductive reaction:
 Bioreductions are also capable of generating polar
functional group such as hydroxy and amino which can
undergo further biotransformation or conjugation.
 Reduction of carbonyls:
 Aliphatic aldehydes :
E.g. Chloral hydrate
Cl3C-CHOH2O
Cl3C-CH2OH
Chloral hydrate
03-12-2010
Trichloroethanol
KLECOP, Nipani
39
Aliphatic ketones:
E.g. Methadone.
O
OH
C 2 H5
C 2 H5
H2 C
H
C
N
CH3
H2 C
CH3
H3 C
H3 C
Methadone
Aromatic Ketone:
N
CH3
CH3
Methadol
E.g. Acetophenone.
OH
O
C
H
CH3
CH3
Methyl phenyl carbinol
Acetophenone
03-12-2010
H
C
KLECOP, Nipani
40
 Reduction of alcohols and C=C:
 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.

Example – bencyclane . (antispasmodic)
O
(CH2)3 N(CH3)2
Bencyclane
03-12-2010
OH
Bencyclanol
KLECOP, Nipani
Benzylidine cycloheptane
41
 Reduction
of N-compounds:
 Reduction of nitro groups proceeds via formation of
nitro so and hydroxyl amine intermediates to yield
amines.
RNO2
R-N=O
R-NHOH
RNH2
Nitro
Nitroso
Hydroxylamine
Amine
 For
E.g. Reduction of Nitrazepam.
N
O2N
H2 N
Nitrazepam
03-12-2010
O
H
N
O
H
N
N
7-Amino metabolite.
KLECOP, Nipani
42
 Reduction of azo compounds yield primary amines
via formation of hydrazo intermediate which
undergo cleavage at N-N bond.
R-N=N-R'
RNH2
R-NH-NH-R'
Azo
 E.g. Prontosil.
Hydrazo
N N
Prontosil
NH2R'
Amines
NH2
NH2
H2N
+
SO2NH2
H2N
NH2
+
H2N
SO2NH2
1,2,4-Triamino benzene sulfanilamide
 It is reduced to active Sulfanilamide.
03-12-2010
KLECOP, Nipani
43
 Miscellaneous reductive reactions:
 Reductive Dehalogenation:
This reaction involves replacement of halogen attached
to the carbon with the H-atom.
E.g. Halothane.
Br
CF3
H
CF3-CH3
CF3-COOH
1,1,1- Trifluroethane
Trifluroacetic acid
Cl
Halothane
 Reduction of sulfur containing functional groups:
C2H5
S
N
C2H5
Dsulfiram
03-12-2010
S
S S
N
C2H5
C2H5
C2H5
E.g. Disulfuram
S
N
SH
C2H5
Diethyldithiocarbamic acid
KLECOP, Nipani
44
 Hydrolytic
reactions:
1. The reaction does not involve change in the state of
oxidation of substrate.
2. The reaction results in a large chemical chain in the
substrate brought about by loss of relatively large
fragments of the molecule.
 Hydrolysis of esters and ethers:
Esters on hydrolyisis yield alcohol and carboxylic acid.
The reaction is catalyzed by esterases.
O
R-C-OR'
03-12-2010
O
R-C-OH
KLECOP, Nipani
+
R'-OH
45
 Organic acid esters:
Esters with a large acidic group :
E.g. Clofibrate
COOC 2H5
Cl
O
CH3
COOH
Cl
O
CH3
CH3
+
CH3
Clofibrate
C 2H5OH
Free acid metan
 Esters with a large acidic and alcoholic group:
E.g. Succinylcholine
H2C COOCH2CH2-N(CH3)2
Pseudo
C COOCH2CH2-N(CH3)2 CholineH2
sterase
Suceinylcholine
03-12-2010
CH2COOH
CH2COOH
succinic acid
KLECOP, Nipani
+
HOCH2CH2N(CH3)2
2
choline
46
 Inorganic acid esters:
 Phosphates:
E.g. Stilbestrol diphosphate
C2 H5
OH
C2 H5
OH
O P O
O P O
C2 H5
OH
CH3
H3C
E.g. Isopropyl methanesulfonate
CH3
O
H3C
O
Isopropyl methnesulfonate
03-12-2010
2 H3PO4
stilbestrol
O S CH3
H
+
C2 H5
OH
Stilbestrol diphosphate
 Sulfates:
OH
HO
OH
H
isopropanol
KLECOP, Nipani
O
+
HO S CH3
O
methanesulfonic acid
47
 Hydrolysis
of amides:
The reactions catalyzed by amides, involves C-N
cleavage to yield carboxylic acid and amine.
O
O
R-C-NHR'
R-C-OH
+
R'NH2
 primary amide with aliphatic substituent on N-atom:
E.g. Procainamide
C2H5
O
H2N
N C C N
H H2 H2
C2H5
Procanamide
03-12-2010
C2H5
O
+
H2N
H2N
C C N
C2H5
H2 H2
OH
PABA
KLECOP, Nipani
48
 Secondary amide with aromatic Substituent on N-atom:
E.g. Lidocaine
CH3
CH3
O
N
H
CH3
C 2H5
NH2
C N
H2
C 2H5
+
C 2H5
HOOC
C N
C 2H5
H2
CH3
Lidocaine
 Tertiary amide:
2,6 Xylidine
N, N- Diethylglycine
E.g. Carbamazepine
N
N
CONH2
H
Carbamazepine
Iminostilbene
03-12-2010
KLECOP, Nipani
49
 Hydrolytic cleavage of non
aromatic heterocyclics:
 Four – membered lactams:
O
C
H2
E.g. Penicillins
O
H
N
S
N
C
H2
CH3
CH3
O
CH3
N
CH3
O
Penicillin G
COOH
Penicinoic acid metabolite
 Five – member lactams:
N
E.g. Succinimides
O
Phensuximide
O
COOH
NH2
CH3
03-12-2010
S
HO
COOH
O
H
N
Phenyl succinamic acid
KLECOP, Nipani
50
 Hydrolytic
dehalogenation:
Chlorine atoms attached to aliphatic carbons are
dehalogenated easily.
 E.g. Dichloro diphenyl trichloro ethane
H
Cl
Cl
H
-HCL
Cl
Cl
CCl3
CCl2
DDT
DDE
 Miscellaneous hydrolytic reactions:
Include hydration of epoxides and arene oxides,
hydrolysis of Sulfonylureas, Carbamates, Hydroxamates
and alpha Glucuronide and sulfate conjugates
03-12-2010
KLECOP, Nipani
51
Phase 2 Reactions
Synthetic Conjugation
03-12-2010
KLECOP, Nipani
52
Phase II
• Phase II - combines functional group of compound
with endogenous substance
E.g. Glucuronicacid, Sulfuric acid, Amino Acid, Acetyl.
 Products usually very hydrophilic
 The final compounds have a larger molecular weight.
03-12-2010
KLECOP, Nipani
53
Enzymes
•
•
•
•
Glucuronosyl Transferases
Sulfotransferases (ST)
Acetyltransferase
Methylases
03-12-2010
KLECOP, Nipani
54
How We Get To Phase 2
• Most of the drugs do not become polar upon
phase 1 reactions.
• The Body is left with a plan to further
metabolize the Drugs
• Goal of Phase 2 : Make substances more
soluble that couldn’t be done in the Phase 1
reactions.
03-12-2010
KLECOP, Nipani
55
Synthetic Reactions / Phase II
• These reactions usually involves covalent attachments
of small polar endogenous molecules such as
Glucoronic acid, Sulfate, Glycine to either unchanged
drugs or Phase I product having suitable functional
groups as COOH,-OH,-NH2,- SH.
• Thus is called as Conjugation reactions.
• Since the product formed is having high molecular
weight so called as synthetic reactions.
• The product formed is hydrophilic in nature with total
loss of pharmacologic activity so called as a true
detoxification reaction
03-12-2010
KLECOP, Nipani
56
Phase II
•
•
•
•
•
•
•
•
Glucuronide Conjugation
Methylation
Acetylation
Sulfate Conjugation
Conjugation With Alpha Amino Acids
Glutathione Conjugation
Glycine Conjugation
Cyanide Conjugation
03-12-2010
KLECOP, Nipani
57
• Very important Synthetic reactions carried out by
Uredine Di Phosphate Glucuronosyl Transferase.
• Hydroxyl and Carboxylic acid groups are easily
combined with Glucuronic acid.
03-12-2010
KLECOP, Nipani
58
 Glucuronide formation occurs in 2 steps:-
1. Synthesis of an activated coenzyme uridine-5’- diphospho -alphaD- Glucuronic acid (UDPGA) from UDP- glucose (UDPG).
-D-Glucose-1-phosphate +
UTP
Pyrophosphorylase
UDPG +
Ppi
UDPG - Dehydrogenase
UDPG +2NAD + H2O
03-12-2010
UDPGA
+2NADH + 2H+
KLECOP, Nipani
59
2. Transfer of the glucuronyl moiety from UDPGA to
the substrate RXH
in presence of enzyme UDPUDP-Glucuronyl transferase
glucuronyl transferase to form the conjugate.
UDPGA + RXH
Where,
X = O, COO, NH or S
03-12-2010
RX
Glucuronic Acid +UDP
KLECOP, Nipani
60
COOH
COOH
COOH Benzoic acid
OH
OH
UDP
OH
OH
OH
UDPGA
OH
Benzoic Acid
Glucuronide Benzoic acid
Ex.
Chloramphenicol, Morphine, Salicylic Acid, Paracetamol
03-12-2010
KLECOP, Nipani
61
• Common, minor pathway.
• Methyltransferases
– -CH3 transfer to O, N, S, C
03-12-2010
KLECOP, Nipani
62
1. Synthesis of an activated coenzyme S- adenosyl
methionine(SAM), the donor of methyl group, from Lmethionine and ATP.
L – Methionine + ATP
Methionine Adenosyl Transferase
SAM +
PPi + Pi
2. Transfer of the methyl group from SAM to the substrate in
presence of nonmicrosomal enzyme methyl transferase.
RXH + SAM
Methyl Transferase
RX-CH3 +
S – Adenosyl Homocysteine
Ex.
Morphine, Nicotine, Histamine
03-12-2010
KLECOP, Nipani
63
Major route of biotransformation for aromatic amines,
hydrazine.
 Generally decreases water solubility
 Enzyme: - N- Acetyltransferase (NAT)
R – NH2
03-12-2010
R – NH – COCH3
KLECOP, Nipani
64
COOH
COOH
OH
OH
CH3COSCOA
NH 2
Paraaminosalicyclic Acid
Acetyl Co enzyme
NHCOCH3
NH 2
N- Acetylated PAS
Ex.
Histamine, PAS, PABA
03-12-2010
KLECOP, Nipani
65
• Sulfotransferases are widely-distributed enzymes
• Cofactor is 3’-phosphoadenosine-5’-phosphosulfate
(PAPS)
• Produce highly water-soluble sulfate esters,
eliminated in urine, bile
• R – OH
03-12-2010
R – O – SO3
KLECOP, Nipani
66
1. Synthesis of an activated coenzyme 3’-phosphoadenosine-5’phosphosulfate (PAPS) which acts as a donor of sulfate to the
substrate.
 This also occurs in two steps- an initial interaction between
the sulfate and the adenosine triphosphate (ATP) to yield
adenosine-5’-phosphosulfate (APS) followed by activation of
latter to PAPS.
ATP Sulfurylase/Mg++
ATP + SO42-
APS + ATP
03-12-2010
APS + Ppi
APS Phosphokinase/Mg++
PAPS + ADP
KLECOP, Nipani
67
2. Transfer of sulfate group from PAPS to the substrate RXH in
presence of enzyme Sulfotransferase and subsequent liberation
of 3’- phosphoadenosine-5’-phosphate(PAP).
PAPS + RxH
Sulfotransferase
Rx-SO3 + PAP
X= O,NH
Examples of compounds undergoing sulfation are:
 Phenol
Paracetamol , Salbutamol
 Alcohols
Aliphatics C-1 to C-5
 Arylamines
Aniline
03-12-2010
KLECOP, Nipani
68
• Alternative to glucuronidation
• Two principle pathways
– -COOH group of substrate conjugated with -NH2
of Glycine, Serine, Glutamine, requiring CoA
activation
• E.g. conjugation of benzoic acid with Glycine
to form Hippuric acid
– Aromatic -NH2 or NHOH conjugated with -COOH
of Serine, Proline, requiring ATP activation
03-12-2010
KLECOP, Nipani
69
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 sulfation where the
donor coenzyme is activated and not the substrate.
Acetyl Synthetase
RCOOH + ATP
RCOAMP + H2O + Ppi
Acyl CoA Transferase
RCOAMP + CoA-SH
RCSCoA + AMP
2. Acylation of the alpha- amino acid by the acyl CoA in presence
of enzyme N-acyl transferase.
RCSCoA
+ NH2-R’-COOH
03-12-2010
N-Acetyl transferase
KLECOP, Nipani
RCONH-R’COOH
+ CoA- SH
70
• Glutathione-S-transferase catalyzes conjugation with
glutathione
• Glutathione is tripeptide of Glycine, Cysteine, Glutamic
acid
03-12-2010
KLECOP, Nipani
71
Glutathione-S-transferase
γ-Glutamyl
transferase
03-12-2010
KLECOP, Nipani
72
Glycine
Cysteinyl glycinase
N- acetylase
Mercapturic Acid Derivative
03-12-2010
KLECOP, Nipani
73
Glycine Conjugation
 Salicylates and other drugs having carboxylic acid group
are conjugated with Glycine.
 Not a major pathway of metabolism
Cyanide Conjugation
 Conjugation of cyanide ion involves transfer of sulfur
atom from thiosulfate to the cyanide ion in presence of
enzyme rhodanese to form inactive thiocyanate.
2-
S2O3 + CN
03-12-2010
-
rhodanese
KLECOP, Nipani
-
2-
SCN + SO3
74
Biotransformation-Conclusion
• Change the Xenobiotics to a form that can be
eliminated from the body
• Change the Xenobiotics to a less biologically active
form
• Bioactivation to more toxic forms can also occur
• Synthetic Phase II reactions are carried out by other
enzymes.
03-12-2010
KLECOP, Nipani
75
 Factor affection of
Biotransformation of drug:
The Therapeutic efficacy, Toxicity and Biological half life of drug
depends on metabolic rate and the factor that influence metabolic rate
are:
1) Physicochemical property of drug.
2) chemical factors:
 a. Induction of drug metabolizing enzyme.
 b. Inhibition of drug metabolizing enzyme
 c. Environmental chemicals.
3) Biological factors.
 A. Species differences.
 B. Strain differences.
 C. Sex differences.
03-12-2010
KLECOP, Nipani
76
 D. Age.
 E. Diet.
 F. Altered pharmacologic factors:
 i Pregnancy.
 ii Hormonal imbalance.
 iii Disease state.
 G. Temporal factors:
 I Circadian rhythm.
03-12-2010
KLECOP, Nipani
77
References
• Biopharmaceutics & Pharmacokinetics by D.M.
Brahmankar, S. B. Jaswal, Vallabh Prakashan, Pg111-158.
• Biopharmaceutics & Pharmacokinetics by Milo
Gibaldi, 4th edition, Pg.no. 203.
• Text book of Biopharmaceutics & pharmacokinetics,
Dr.Sobha Rani R. Hiremath, Prism Books Pvt Ltd,
Bangalore, 2000 Pg.no. 157-166.
• www.google.com
• www.pharmacology.com
03-12-2010
KLECOP, Nipani
78
Cell No: 0091 9742431000
E-mail: bknanjwade@yahoo.co.in
03-12-2010
KLECOP, Nipani
79