Download BIOTRANSFORMATION

 When
toxic chemicals enter the body they
eventually end up in the liver for processing

Most of these chemicals are difficult to
move out of the body so they need to be
transformed in such a away that enables
them to be eliminated
 This
is done through an elaborate
system called PHASE I and PHASE II
detoxification mechanism
Two major sets of pathways (enzymatic)
•nonsynthetic reactions - Phase I
•synthetic reactions - Phase II
Liver SER
smooth
endoplasmic
reticulum
3
Phase I and phase II reactions
PHASE I
PHASE II
Expose or add
functional group
XENOBIOTIC
Oxidation
Reduction
Hydrolysis
PRIMARY
PRODUCT Conjugation
SECONDARY
PRODUCT
EXCRETION
LIPOPHILIC
HYDROPHILIC

Phase I reactions include:




oxidations
reductions
hydrolysis reactions
they introduce a functional group (e.g., -OH)
that serves as the active center for sequential
conjugation in a phase II reaction.
2.

Phase II Reactions that include conjugation
reactions, which involve the enzyme-catalyzed
combination of a drug (or drug metabolite) with
an endogenous substance.
Phase II reactions require:
 a functional group—an active center—as the
site of conjugation with the endogenous
substance.
Drug metabolism reactions :
•
•
•
•
introduction of functional group
hydrophilicity increases slightly
major player is CYP or mixed function oxygenase
(MFO) system in conjunction with NAD(P)H
location of reactions is smooth endoplasmic
reticulum
Nonsynthetic Reactions
oxidation
reduction
hydrolysis
•hydroxylations
aromatic, aliphatic, nitrogen
•dealkylations(N-, S-, P)
•deaminations
•N-, S-, P- oxidations
oxidoreductases
•S-replacements
oxidases
monoamine oxidases
•epoxidations
mixed function oxidases
•others
•azo reduction
•nitro reduction
•disulfide reduction
•others
•esters
•amides
•hemiacetals,acetals,
hemiketals, ketals
oxidoreductases
reductases
esterases
amidases
peptidases
lipases
hydrolases
9

Enzymes catalyzing phase I biotransformation
reactions include:
cytochrome P-450
 aldehyde and alcohol dehydrogenase
 deaminases
 esterases
 amidases
 epoxide hydratases

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
The principal reaction of drug/toxin metabolism is OXIDATION.
The enzymes responsible are oxido-reductases; called mixed-function
oxidases.
cytochrome P 450
Most prominent and
important among these
is the
cytochrome P450
system.
consists of
Cyt P 450 and
Cyt P 450 reductase
Fe3+
protoporphryrin
ring system
11
BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
S-replacement
O
S
CH 3CH2O
Parathion
P
C H3C H2O
O
Paraoxon
OC H2C H3
P
O
OCH2CH3
epoxidation
Cl
Cl
Cl
Cl
CCl2
CCl2
CH2
CH2
O
Cl
Cl
Cl
Aldrin
Both compounds are
insecticides.
Cl
Dieldrin
12
Nonsynthetic Reactions
oxidation
reduction
hydrolysis
•hydroxylations
aromatic, aliphatic, nitrogen
•dealkylations(N-, S-, P)
•deaminations
•N-, S-, P- oxidations
oxidoreductases
•S-replacements
oxidases
monoamine oxidases
•epoxidations
mixed function oxidases
•others
•azo reduction
•nitro reduction
•disulfide reduction
•others
•esters
•amides
•hemiacetals,acetals,
hemiketals, ketals
oxidoreductases
reductases
esterases
amidases
peptidases
lipases
hydrolases
13
BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION
azo reduction
H2N
H2N
N
Prontosil®
N
SO 2NH2
NH2
+
H2N
NH2
H2N
SO 2NH2
sulfanilamide
nitro reduction
NO2
chloramphenicol
(antibacterial)
NO
NO2
NHOH
NH2
NH2
CHOH
CHOH
HOCH2 CHNHCCHCl2
HOCH2 CHNHCCHCl2
O
O
14
BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION
disulfide reduction
S
CH3CH2
N C
CH3CH2
S
SS
CH2CH3
C N
2
CH2CH3
Disulfiram (Anatabuse®)
S
CH3CH2
N C
CH3CH2
SH
other reductions
C C
C C
O
OH
C
C
As 5+
As 3+
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Nonsynthetic Reactions
oxidation
reduction
hydrolysis
•hydroxylations
aromatic, aliphatic, nitrogen
•dealkylations(N-, S-, P)
•deaminations
•N-, S-, P- oxidations
oxidoreductases
•S-replacements
oxidases
monoamine oxidases
•epoxidations
mixed function oxidases
•others
•azo reduction
•nitro reduction
•disulfide reduction
•others
•esters
•amides
•hemiacetals,acetals,
hemiketals, ketals
oxidoreductases
reductases
esterases
amidases
peptidases
lipases
hydrolases
16
BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS
esters
COOH
OCCH3
COOH
OH
+ H2O
HOCCH 3
+
O
O
acetylsalicylic acid
amides
H2N
O
S
O
RCNH
CH3
RCOH
S
CH3
N
O
penicillins
CH3
COOH
+H2O
N
CH3
O
COOH
O
also
RCNH
S
hemiacetals
hemiketals
acetals
ketals
CH3
HOOC
N
H
CH3
COOH
17
• conjugation with endogenous molecules
(GSH, glycine, cystein, glucuronic acid)
• hydrophilicity increases substantially
• neutralization of active metabolic intermediates
• facilitation of elimination
• location of reactions is cytoplasm
 Phase
II - combines functional group
compound with endogenous substance
of
E.g. Glucuronicacid, Sulfuric acid, Amino Acid,
Acetyl.
 Products
usually very hydrophilic
 The final compounds have a larger molecular
weight.

Enzymes catalyzing phase II biotransformation
reactions include:
 glucuronyl transferase (glucuronide conjugation)
 sulfotransferase (sulfate conjugation)
 transacylases (amino acid conjugation)
 acetylases
 ethylases
 methylases
 glutathione transferase.
How We Get To Phase 2
• Most of the drugs do not become polar
upon phase 1 reactions.
• Goal of Phase 2 : Make substances more
soluble that couldn’t be done in the Phase
1 reactions.
KLECOP, Nipani
03-12-2010
21
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
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.
KLECOP, Nipani
03-12-2010
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Synthetic Reactions
glucuronide formation
OH
gluconic acid
H CO2H
HO
OH
H
OH
O OH
COOH
OH
HO
H CO2H
HO
OH
HO
HO
HO
OH
glucuronic acid
salicylic acid
H
OH
H
H
UDP
O
UDP
O
COH
OH
H
UTP
OPO32-
PPi
O OH
OH
OH
H
HO
OH
glucose
HO
HO
COOH
OH
+
H CO2H
HO
HO
HO
H
H
OH
H
UDP-glucuronide
UDP
O
O
H CO2H
C
HO
HO
HO
H
H
O
OH
H
a glucuronide
derivative
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OH
Major route of biotransformation for aromatic amines,
hydrazine.
 Generally decreases water solubility
 Enzyme: - N- Acetyltransferase (NAT)
R – NH2
R – NH – COCH3
BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION
acetylation
high energy bond
O
O
SO2NH2 + CH3C SCoenzyme A
NH2
sulfanilamide
H3C
Acetyl Coenzyme A
C HN
SO2NH2
+ CoenzymeA
sulfate ester formation
NH2
HO
+
N
N
O
O
Sulfates are carried as
O S O PO
phosphoadenosineO
phosphosulfate derivatives
O
(PAPS) - a high energy form.
N
N
O
O
SO
O
O
HO O
O P O
The enzymes catalyzing
this type of reaction are
called sulfotransferases.
O
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 Generates
more polar (water soluble), inactive
metabolites
 Readily excreted from body
 Metabolites may still have potent biological
activity (or may have toxic properties)
 Generally applicable to metabolism of all
xenobiotics as well as endogenous compounds
such as steroids, vitamins and fatty acids
Biotransformation is a major mechanism for drug /
TOXIN elimination
Biotransformation of drug is defined as the conversion
from one chemical form to another
Many drugs undergo
 several sequential biotransformation reactions.
 Biotransformation is catalyzed by specific
enzyme systems
Sites of biotransformation:
 The liver: the major site
 other tissues.
XENOBIOTIC
BLOOD
EXCRETION
Somatic
effect
BIOTRANSFORMATION
DNA
damage
Non-toxic
metabolite
Activation
Detoxification
Toxic
metabolite
Definition
Biotransformation is the sum of all processes,
whereby a compound is transformed chemically
within a living organism
Metabolism of acetaminophen to harmless conjugates or to toxic metabolites.
nontoxic phase II conjugates
nontoxic phase II conjugates
phase I Reaction
toxic
nontoxic phase II conjugates
BIOTRANSFORMATION
Results of biotransformation
more potent
active
active
Drug
or Poison
inactive
biotransformed
Drug or Poison
less potent
TOXIC
inactive
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BIOTRANSFORMATION
Acetylsalicylic Acid
ACTIVE analgesic
Salicylic acid
ACTIVE analgesic
CO2H
CO2H
OCCH3
OH
O
Codeine
ACTIVE narcotic analgesic
H
N
Morphine
ACTIVE (more potent) narcotic analgesic
CH3
H
H
H3CO
O
N
CH3
H
OH
HO
O
Methanol
ACTIVE CNS depressant
CH3OH
OH
Formaldehyde
TOXIC ( )
HCH
Formic Acid
TOXIC ( )
HCOH
O
O
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Drug metabolizing organs
• Liver is the heart of metabolism
• Because of its relative richness of enzymes in
large amount.
• Schematic chart
(decreasing order)
of
metabolizing
organ
• Liver > lungs > Kidney > Intestine > Placenta >
Skin > Brain > Testes > Muscle > Spleen
KLECOP, Nipani
03-12-2010
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 One
of these chemicals are converted they
are carried to the gastrointestinal tract via
the bile.
 Bile
has two major functions in the body:
(1) help the body absorb fats and fat soluble
nutrients,
(2) help the body eliminate toxins and
wastes.
Biotransformation of drugs can be affected by many
parameters, including:
A.
B.
C.
D.
E.
F.
G.
prior administration of the drug in question or of
other drugs
diet
hormonal status
genetics
disease (e.g., decreased in cardiac and pulmonary
disease)
age and developmental status
liver function
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
• They have to be converted to simpler hydrophilic
compounds so that they are eliminated and their
action is terminated
KLECOP, Nipani
03-12-2010
37
Important phase I enzymes
Enzyme
Co-factor
Substrate
Mixed-function oxidases
(cytochrome P-450)
NADPH
(NADH)
Most lipophilic substances
with M.wt < 800
Carboxyl esterases
Unknown
Lipophilic carboxyl esters
’A’ esterases
Epoxide hydrolases
Ca++
Unknown
Organophosphate esteres
Organic epoxids
Reduktases
NADH
NADPH
Organic nitrous compounds
Organic halogens
Mixed function oxidase enzymes (P450) are
located in the endoplasmic reticulum (SER)

Enzymes catalyzing phase II biotransformation
reactions include:
 glucuronyl transferase (glucuronide conjugation)
 sulfotransferase (sulfate conjugation)
 transacylases (amino acid conjugation)
 acetylases
 ethylases
 methylases
 glutathione transferase.

Location of these enzymes:



Subcellular locations include:




numerous tissues
some are present in plasma.
cytosol
mitochondria
endoplasmic reticulum
Only those enzymes located in the endoplasmic
reticulum are inducible by drugs
a.
General features

A large number of families (at least 18 in
mammals) of cytochrome P-450 (abbreviated
“CYP”) enzymes exists
each member of which catalyzes the
biotransformation of a unique spectrum of
drugs
some overlap in the substrate specificities.



This enzyme system is the one most frequently
involved in phase I reactions.
Cytochrome P-450 catalyzes numerous
reactions, including:

aromatic and aliphatic hydroxylations
dealkylations at nitrogen, sulfur, and oxygen
atoms
 heteroatom oxidations at nitrogen and sulfur
atoms
 reductions at nitrogen atoms
 ester and amide hydrolysis


b.
Localization

The primary location of cytochrome P-450 is the liver,

Other tissues, including:




the adrenals
ovaries and testis
tissues involved in steroidogenesis and steroid metabolism.
The enzyme's subcellular location is the endoplasmic
reticulum.
Mechanism of reaction
In the overall reaction:
c.
1.



the drug is oxidized
oxygen is reduced to water.
Reducing equivalents are provided by nicotinamide
adenine dinucleotide phosphate (NADPH), and
generation of this cofactor is coupled to cytochrome
P-450 reductase.
Metabolism of phenytoin :