BIOTRANSFORMATION

Prof. Dr. Basavaraj K. Nanjwade M. Pharm., Ph. D

Department of Pharmaceutics
KLE Universitys 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
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 Introduction:
Important terms:

Detoxication (detoxification)

Prodrugs

Active Metabolite
Reactive Metabolite

Metabolism
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Xenobiotics
 Definition:
Biotransformation of drug is defined as the
conversion from one chemical form to another.
the term is used synonymously with metabolism.

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 Biotransformation leads to:
Pharmacologic Inactivation of Drug
Ex: Active Drug Inactive Drug
Salicylic Acid Salicyluric Acid

Active Metabolite From An Inactive Drug

Ex. Inactive(Prodrug) Active
Aspirin Salicylic Acid

No Change in Pharmacologic Activity

Ex. Active Active Drug
Codeine Morphine
03-12-2010 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

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 Drug Metabolizing
Enzymes
Non -
Microsomal Microsomal

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 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
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Microsomal Enzymes Non-microsomal
Non-synthetic/ Phase I enzymes
reactions Non-synthetic/ Phase I
reactions
Most oxidation and
reduction Most hydrolysis
Some hydrolysis Some oxidation and
reduction
Synthetic/ Phase II Synthetic/ Phase II reactions
reactions ALL except Glucuronide
ONLY Glucuronide conjugation
conjugation

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Microsomal Enzymes
Inducible
Drugs, diet, etc.

Non-microsomal
enzymes
Not inducible
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 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.

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 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.
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 Metabolic reaction:

Phase I reaction Phase II reaction

Oxidation
Conjugation
Reduction

Hydrolysis
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 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
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system known as
KLECOP, Nipani
cytochrome P450. 15
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 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

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 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)

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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.

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 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.

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 Oxidation of aromatic carbon atoms
(aromatic hydroxylation):
R

OH

Arenol (major)

R R R OH
H2O
O
epoxide hydrase

OH
Arene oxide
Arene (highly reactive electrophile) Dihyrdrodiol
GSH
5-epoxide transferase

R
R OH OH

SG OH
Tissue toxicity in instances
when glutathione is depleted. Glutathione conjugate Catechol(min. Pro.)
(min.pro.)
E.g. Epoxides of Bromobenzene and Benzopyrene.
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 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 1,2-
dihydrodiols.

O HO OH

H2O

N
N epoxide hydrase N
CONH2 CONH2 CONH2

Carbamazepine Carbamazepine-10,11 Trans-10,11

epoxide dihydroxy
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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.

CH2OH CHO COOH

CH3
Alcohol
dehydrogenase

SO2NHCONHC4H9 SO2NHCONHC4H9
Corresponding Corresponding
aldehyde carboxylic acid.
Tolbutamide Prmary carbinol
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 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 H3C
O O O O

HN N HN N

CH3 CH3

O O

Hexobarbital 3'-Hydroxy Hexobarbital

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 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

Diazepam 3-Hydroxy diazepam

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 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 CH3 OH CH3

H3C C C C COOH H3C C C C COOH

H2 H H2 H
CH3 CH3

Tertiary alcohol metabolite

Ibuprofen

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 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 N

O N N 4' O N N OH

H2N
H2N

Minoxidil 4'-Hydroxy Minoxidil

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 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 carbon-
heteroatom bond.

E.g. N-, O- and S- dealkylation, oxidative

deamination and desulfuration.

2. Oxidation of the heteroatom itself.

E.g. N- and S- oxidation.

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 Oxidation of Carbon-Nitrogen System:
N-Dealkylation:
Mechanism of N-dealkylation involve oxidation of -
carbon 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.
H OH
O C
NH
N C N C +

H H
Carbonyl
N-Dealkylated
Carbinolamine metabolite
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Intermediate
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 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

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 N-Hydroxylation:-
Converse to basic compounds that form N-oxide, N-
hydroxy formation is usually displayed by non-basic
nitrogen atoms such as amide Nitrogen.

E.g. Lidocaine
CH3
CH3
O C2H5
O C2H5

N C C N
N C C N H2
H H2
OH C2H5
C2H5
CH3
CH3

N- Hydroxy Lidocaine
Lidocaine
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 Oxidation of Carbon-Sulfur Systems:
S-Dealkylation:
The mechanism of S-Dealkylation of
thioethers is analogous to N-dealkylation .IT
proceed via -carbon hydroxylation.
The C-S bond cleavage results in formation of
a thiol and a carbonyl product.
E.g. 6-Methyl mercaptopurine.
SCH3 SCH2OH SH

N N N
N N N
+ HCHO
N N N
N N N
H H H

6-Methyl Hydroxylated
intermediate 6-Mercaptopuri9ne
Mercaptopurine
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 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
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 S-Oxidation:

Apart from S-dealkylation, thioethers can also undergo S-

oxidation reaction to yield sulfoxides which may be further
oxidized to sulfones several phenothiazines.

E.g. Chlorpromazine undergo S-oxidation.

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 Oxidation of Carbon-Oxygen Systems:
O-Dealkylation:

This reaction is also similar to N-Dealkylation and

proceeds by -carbon hydroxylation to form an
unstable hemiacetal or hemiketal intermediate.

Which spontaneously undergoes C-0 bond cleavage

to form alcohol and a carbonyl moiety.
OH H

R-O-CH2R' R-O-CH-R' R-OH + O=C-R'

Alcohol Aldehyde/ketone
Hemiacetal
Ether
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 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 CH3COOH
CH3CH2OH
dehydrogenase
dehydrogenase
Ethanol Acetaldehyde Aceticacid
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 Miscellaneous oxidative reactions:
Oxidative aromatization /dehydrogenation:

E.g. Metabolic aromatization of drugs is nifedipine.

COOCH3 COOH
NO2 NO2 CH2OH
CH3

NH N

CH3 CH3
COOCH3 COOCH3

Nitedipine Pyridine metabolite

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 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 O
oxi. Covalent binding to tissues.
Cl Cl Cl Cl
-HCL
H
Chloroform Phosgene
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 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 Trichloroethanol

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 Aliphatic ketones: E.g. Methadone.

O OH
C 2 H5 C 2 H5
H2 C H CH3 H2 C H CH3
C N C N
H3 C CH3 H3 C CH3

Methadone Methadol

Aromatic Ketone: E.g. Acetophenone.

O OH
CH3 C CH3
H

Acetophenone Methyl phenyl carbinol

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 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 OH

Bencyclane Bencyclanol Benzylidine cycloheptane

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 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.

H O
H O N
N

H2 N N
O2N N

Nitrazepam 7-Amino metabolite.

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 Reduction of azo compounds yield primary amines
via formation of hydrazo intermediate which
undergo cleavage at N-N bond.
R-N=N-R' R-NH-NH-R' RNH2 + NH2R'

Azo Hydrazo Amines

E.g. Prontosil.
NH2 NH2

H2N N N SO2 NH2 H2N NH2 + H2N SO2 NH2

Prontosil 1,2,4-Triamino benzene sulfanilamide

It is reduced to active Sulfanilamide.

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 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
Cl
Halothane 1,1,1- Trifluroethane Trifluroacetic acid

Reduction of sulfur containing functional groups:

C 2H5 S S C 2H5 C 2H5 S E.g. Disulfuram
N S S N N SH
C 2H5 C 2H5 C 2H5

Dsulfiram Diethyldithiocarbamic acid

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 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 O
R-C-OR' R-C-OH + R'-OH
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 Organic acid esters:
Esters with a large acidic group : E.g. Clofibrate
COOC2H5 COOH
Cl O CH3 Cl O CH3 C 2H5OH
CH3 CH3
+

Clofibrate Free acid metan

Esters with a large acidic and alcoholic group:

E.g. Succinylcholine
H2C COOCH2CH2-N(CH3)2 Pseudo CH2COOH
+ 2
HOCH2CH2N(CH3)2
C COOCH2CH2-N(CH3)2 Choline- CH2COOH
H2 sterase
Suceinylcholine succinic acid choline
03-12-2010 KLECOP, Nipani 46
 Inorganic acid esters:
Phosphates: E.g. Stilbestrol diphosphate

OH C2 H5 OH C2 H5
O P O O P O HO OH + 2 H3PO4
OH C2 H5 OH C 2 H5

Stilbestrol diphosphate stilbestrol

Sulfates: E.g. Isopropyl methanesulfonate

CH3 O CH3 O
H3C O S CH3 H3C OH + HO S CH3
H O H O

Isopropyl methnesulfonate isopropanol methanesulfonic acid

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 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 C2H5
O
H2N N C C N H2N + H2N C C N
H2 H2 C2H5
H H2 H2 C 2H5
OH

Procanamide PABA
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 Secondary amide with aromatic Substituent on N-atom:
E.g. Lidocaine
CH3 CH3
O C 2H5 C 2H5
N C N NH2 + HOOC C N
H H2 C 2H5 H2 C 2H5
CH3 CH3

Lidocaine 2,6 Xylidine N, N- Diethylglycine

Tertiary amide: E.g. Carbamazepine

N N
CONH2 H

Carbamazepine Iminostilbene
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 Hydrolytic cleavage of non
aromatic heterocyclics:
Four membered lactams: E.g. Penicillins
O O
H H
C N S CH3 C N
H2 S CH3
H2
N CH3 HO N CH3
O
COOH O
COOH

Penicillin G Penicinoic acid metabolite

Five member lactams: E.g. Succinimides

O
O N O COOH
CH3 NH2

Phensuximide Phenyl succinamic acid

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 Hydrolytic dehalogenation:
Chlorine atoms attached to aliphatic carbons are
dehalogenated easily.
E.g. Dichloro diphenyl trichloro ethane
H -HCL H
Cl Cl 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
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 Phase 2 Reactions

Synthetic Conjugation
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 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 couldnt be done in the Phase 1
reactions.

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 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
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 Phase II
Glucuronide Conjugation
Methylation
Acetylation
Sulfate Conjugation
Conjugation With Alpha Amino Acids
Glutathione Conjugation
Glycine Conjugation
Cyanide Conjugation

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 Very important Synthetic reactions carried out by
Uredine Di Phosphate Glucuronosyl Transferase.
Hydroxyl and Carboxylic acid groups are easily
combined with Glucuronic acid.

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 Glucuronide formation occurs in 2 steps:-

1. Synthesis of an activated coenzyme uridine-5- diphospho -alpha-

D- Glucuronic acid (UDPGA) from UDP- glucose (UDPG).
Pyrophosphorylase
-D-Glucose-1-phosphate + UDPG +
UTP Ppi

UDPG - Dehydrogenase
UDPG +2NAD + H2O UDPGA
+2NADH + 2H+

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2. Transfer of the glucuronyl moiety from UDPGA to
the substrate RXH in presence of enzyme UDP-
UDP-Glucuronyl transferase
glucuronyl transferase to form the conjugate.

UDPGA + RXH RX
Glucuronic Acid +UDP
Where,
X = O, COO, NH or S

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 COOH COOH COOH Benzoic acid

OH OH
OH UDP OH
OH
OH
UDPGA Benzoic Acid Glucuronide Benzoic acid

Ex.
Chloramphenicol, Morphine, Salicylic Acid, Paracetamol
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 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 L-
methionine and ATP.
Methionine Adenosyl Transferase
L Methionine + ATP 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
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Major route of biotransformation for aromatic amines,
hydrazine.
Generally decreases water solubility
Enzyme: - N- Acetyltransferase (NAT)

R NH2 R NH COCH3

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 COOH
COOH
OH
OH

CH3COS-
COA NHCOCH3
NH 2
NH 2
N- Acetylated PAS
Paraaminosalicyclic Acid Acetyl Co enzyme

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 R O SO3

03-12-2010 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 + Ppi

APS Phosphokinase/Mg++
APS + ATP PAPS + ADP
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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

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 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.
N-Acetyl transferase
RCSCoA RCONH-RCOOH
+ NH2-R-COOH + CoA- SH
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 Glutathione-S-transferase catalyzes conjugation with
glutathione
Glutathione is tripeptide of Glycine, Cysteine, Glutamic
acid

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 Glutathione-S-transferase

-Glutamyl
transferase
03-12-2010 KLECOP, Nipani 72
 Glycine Cysteinyl glycinase

N- acetylase

Mercapturic Acid Derivative

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 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- - rhodanese - 2-
S2O3 + CN SCN + SO3
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 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.
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 D. Age.
E. Diet.
F. Altered pharmacologic factors:
i Pregnancy.
ii Hormonal imbalance.
iii Disease state.
G. Temporal factors:
I Circadian rhythm.

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 References
Biopharmaceutics & Pharmacokinetics by D.M.
Brahmankar, S. B. Jaswal, Vallabh Prakashan, Pg-
111-158.
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

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 Cell No: 0091 9742431000
E-mail: bknanjwade@yahoo.co.in
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