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Steps of biotransformation
drug molecule
Highly lipophyllic
lipophilic
polar
hydrophylic
accumulation
(fatty tissues)
phase I
polar
phase II bioinactivation
conjugation
hydrophylic
extracellular mobilisation
circulation
excetion with bile
excretion with urine
1
Phase II reactions
Conjugation
reaction
Functional group
on the xenobiotic
or its metabolite
Cofactor
(endogenous
partner)
Enzyme
Place of the
reaction
Glucuronic acid
conjugation
-OH, -COOH, NH2,
-NH, -SH, -CH
UDPGA
Glucuronyl
transferase
(GT)
Smooth
endoplasmic
reticulum
Sulphate
conjugation
Aromatic OH/NH2
-COOH
PAPS
Sulfotransferase
(ST)
Cytosol
Glycine
conjugation
aromatc-NH2, COOH
CoenzymeA
Glycine
Amino acid
aciltranszferase
Mitochondria
Acetylation
aromatic/aliphaticNH2,
hidrazinok, -SO2,
NH2
Acetil coenzyme A
N-, O-acetil
transferase
Cytosol
( membranes)
Methylation
aromatic-OH, NH2,
NH
-SH
Adenosyl-Smetionin
Metiltransferase
Cytosol
( membranes
Glutathione
conjugation
epoxide, organic
haloids
Reduced
glutathione
Glutathione
transferase
Cytosol
( membranes
II. Non conjugation reaction enzymes: epoxide hydrolases, glyoxalases, carboxylesterases
-OH, -COOH, -NH2,
-NH, -SH, -CH
UDPGA
Glucuronic acid
conjugation
Smooth endoplasmic reticulum
Synthesis of UDP glucuronic acid
glükóz-1-P + UTP
pirofoszforiláz
UDP-glükóz
glikogén + PPi
NAD+
UDP-glükóz
dehidrogenáz
NADH + H+
UDP-glükuronsav
COOH
O
OH
HO
O
OH
UDP
Glucuronidation of phenol
OH
COOH
OH
N
O
+
O
O
OH
O
P
O
P
OH
OH
OH
O
CH2
COOH
+
OH
O
OH
OH
O
OH
OH
O
N
UDP
OH
O
Characteristics of glucuronideconjugation






It is fast (coupled to phase I reactions in the
endoplasmic reticulum),
It is common,
It can not be saturated,
The same xenobiotic molecule can conjugate with
several glucuronides,
The enzyme is polymorphic,
The effect is mostly inactivation, but activation can
also happen.
Mixed Function Oxidase system
in the smooth endoplasmic reticulum
cofactors
xenobiotic
fp1, fp2: flavoproteins
b5:
ciytochrome b5
Glucuronidation can produce a more
active or a less active molecule

Glucuronids of morphine

6-glucuronide- morphine is much more
effective than the parent molecule
3-glucuronide morphine is totally uneffective

Sulphate
conjugation
Aromatic OH/NH2
-COOH
PAPS
Sulphotransferase
Cytosol
Sulphatation of phenol
sulphurilase
2-
SO4 + ATP
APS + PPi (pyrophosphate)
APS-Phosphokinase
PAPS + ADP
APS + ATP
NH2
N
O
-O 3S
OH
O
-
+
P
O
O
CH2
N
O
N
OSO 3H
-
+
+
O
O
HO
P
O
O
-
-
PAP
The characteristics of sulphate conjugation






There is some substrate specificity
It can be saturated
It is the second most freguent phase II
reaction
It takes place in the cytosol
The enzyme sulphotransferase is
polymorphic
The effect is mostly inactivation, but
activation can also happen.
Glycine conjugation (amino
acid conjugaton)





aromatic-NH2, -COOH
CoenzymeA
Glycine (or other amino acid)
Aminoacid-acyltransferase
Mitochondria
Acetylation





aromatic/aliphatic-NH2,
hydrazins, -SO2, NH2
Acetyl coenzyme A
N-, O-acetyl transferase
Cytosol ( also in membranes)
Methylation






aromatic-OH, NH2, NH
-SH
Adenosyl-Smethionin
Methyl-transferase
Cytosol ( also in membranes)
Glutathione conjugation




epoxides, organic haloids
Reduced glutathione
Glutathione transferase
Cytosol (also in membranes)
Glutathione conjugation of some compounds:
Cl
SG
Cl
Cl
GSH
NO 2
GS = glutation csoport
NO 2
3,4-dikloro-nitrobenzol
OH
Br
O
SG
GSH
bróm-ciklohexán
ciklohexén
Formation of mercapturic acid
after glutathione conjugation
H
H
O
SG
H
NADPH
H
GSH
OH
[O]
O
O
NH
CH2
NH
CH3
CH2
S
COOH
H
H
OH
CH3
S
+
H
- H2O
COOH
Non conjugation reactions of phase II
Reactions catalysed by:
 Epoxide hydrolases
 Glyoxalases
 Carboxylesterases
Detoxication of epoxides by hydratation


The reaction is catalysed by an epoxide
hydrolase enzyme.
From bromobenzene 3,4-oxide
bromobenzene 3,4-dihydrodiol is formed. It is
assumed that the first step is that the enzyme
deprotonates water rather than activating the
epoxide ring.


Phase II reactions mean bioinactiovation in
most of the cases.
There are some exceptions, where they
result in bioactivation.
Formation of carbonium és nitrenium ions
from sulphate conjugates
of benzyl alcohols and hydroxamic acids
+
CH2
OSO 3-
OH
+
R3
benzil-alkohol
R3
O
O
+
R
NH
:Nu-
OH
O
NH
Nu
hidroxámsav
karbónium ion
-
R
+
R
OH
Nu
R2
R2
N
N
+
OSO 3-
+
R3
SO42-
R3
nitrénium ion
SO42-
H2N
OH
Reversion of inactivation in the urinary bladder
MÁJ
NH2
H
glükuronid
N
N
OH
OH
UDPGA
Hugyhólyag
glükuronid
H
N
N
OH
OH
H+
glükuronsav
elektrofil, reaktiv intermedier
Characteristics of the biotransformation reactions
1. No strict substrate specificity,
2. Induction and inhibition
3. Not only xenobiotics but endogenous substrates
Metabolism studies


In vitro studies
in vivo studies with labelled xenobiotics
(different doses, single and repeated
treatment)
Metabolic pathways of the pesticide dimethachlor
(18 metabolites identified)
CH3
CH2-CH2-O-CH3
N
CO-CH2Cl
CH3
CGA 17020
Glutathione
Pathway
CH3
Glutathione
Pathway
CH3
CH2-CH2-O-CH3
CH2-CH2OH
|
|
CO-CH2-S-Cys-Glu
CH3
MET 4G
NH-CO-CH3
CH2-CH2-O-CH3
COOH
CO-CH2-S-CH2-CH
CH3
CH2-COOH
|
|
CH3
NH-CO-CH3
MET 6U = MET 8U
CH3
MET 7U
MET 10aG
CH2-CONH2
N
N
CO-CH2OH
CO-CH2-S-CH3
CO-CH2-S-CH2-CH
|
NH-CO-CH3
CO-CH2Cl
CH3
CH3
|
CH3
CO-CH3
CH3
CH2-CH2OH
CH2-CH2-O-CH3
COOH
N
CH2-CH2OH
N
MET 16U**
CH3
CH3
CH3
CH2OH
CH2-COOH
N
CO-CH2OH
CO-CH2-S-CH2-CH
NH2
MET 2G = MET 3G
CH3
N
COOH
N
|
CO-CH2OH
CH3
MET 9U* = MET 9G*
postulated intermediate
CH3
CH2-CH2OH
|
N
CO-CH2Cl
MET 3U / MET 5U* = MET 5G* =
MET 6G* = MET 7G* = MET 8G*
Glutathione
Pathway
CH3
CH2-CH2-O-CH3
N
CH3
CH3
CH3
CH2-CH2-O-CH3
CO-CH2Cl
MET 11U
CH3
CH2OH
N
NH CO-CH2-S-CH2-CH
N
N
CH3
COOH
CH3
MET 2U / MET 10dG*
Glutathione
Pathway
oxalic acid derivatives
CH2OH
CH3
CH2OH
CH2-CH2OH
CH2-CH2OH
COOH
N
MET 1U
N
|
CO-CH2-S-CH2-CH
MET 4U = MET 1G
CO-CH3
|
CH3
CH3
MET 14U = MET 15U
CH2-CH2OH
CH3
N
CH2-CH2-O-CH3
N
CO-CH2-SO-CH3
NH-CO-CH3
CH3
MET 17U
MET 18U / MET 10cG*
CO-CH2-SO-CH3
CH3
CH3
MET 13U
Cys: cysteine
Glu: glutamic acid
CH3
CH2-CH2OH
N
MET 10U / MET 12U*
CO-CH2-SO2-CH3
CH3
* excreted as glucuronic acid derivative
** excreted as hydroxymethyl-glucuronic acid conjugate
Factors influencing biotransformation
of a xenobiotic


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
Species
Intra-species genetic variations
Age
Physiological status
Other xenobiotics
Ratio of glucuronidation and sulphatation
in some species
glucuronidation
(%)
Sulfatation
(%)
cat
0
87
human
23
71
rat
25
68
rabbit
46
45
pig
100
0
Biotransformation of Amphetamine
in rabbits, rats, guinea pigs and dogs
konjugátumok
tengerimalac
nyúl
O
tengerimalac
CH3
NH2
CH3
OH
nyúl
O
konjugátumok
patkány
CH3
kutya
OH
OH
CH3
NH2
konjugált fenolok
Genetic polymorphism of biotransformation
enzymes
Differences
•in the base sequence of the DNA
•In the amino acide squence of an enzyme
•In the reaction kinetics
•% of fast acetilation in some human populations
•Europeans 40%
•Asiatics
80%
•Inuits
96%
Consequences of acetylation kinetics:
different side effects
•Isoniazid (drug against tuberculosis)
slow acetilation: neurotoxic effects
fast acetilation: liver problems
• Hidralazin (drug against high blood pressure)
slow acetilation: Lupus eritomatosus
fast acetilation: no specific side effect
Differences between sexes
Hormones influence the lipid environment of
enzymes.
Sex-differences in the activity of CYP P450 enzymes.
(The hypothalamus is releasing a feminizing factor,
:
which results in „feminine liver” having somewhat
lower metabolising capacity in general than the liver
of males.
The role of age in biotransformation
The influence of the physiological status
of the individual:
Diseases,
Fasting.
The influence of other xenobiotics
Enzyme induction by PAHs,
Enzyme inhibition by heavy metals,
Dietary factors,
Smoking, consumption of alcohols, illegal drugs…
Effects of exposure before birth
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

Thalidomide
1953 synthesis, Chemie Grünenthal
1957. putting the drug on the market
Thalidomide babies
Thalidomide S: sedative effect
R: teratogenic
Cause of the tragedy: thalidomide was tested
on adult mice only….

Differences in species:
mouse:
glutathione- conjugation
superoxide
human: Superoxide
Differences of age:
foetuses have a limited (or missing)
metabolising capacity
glutathion conjugation
