1

drug molecule

Highly lipophyllic lipophilic polar hydrophylic

accumulation(fatty tissues)

phase I

polar

phase II bioinactivationconjugation

hydrophylic

extracellular mobilisation

circulation

excetion with bile excretion with urine

Steps of biotransformationSteps of biotransformation

Phase II reactionsPhase 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/aliphatic-NH2,

hidrazinok, -SO2,

NH2

Acetil coenzyme A N-, O-acetil

transferase

Cytosol

( membranes)

Methylation aromatic-OH, NH2, NH

-SH

Adenosyl-S-

metionin

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 acidSynthesis of UDP glucuronic acid

O

OOHOH

OH

COOH

UDP

pirofoszforilázglükóz-1-P + UTP UDP-glükóz glikogén + PPi

UDP-glükóz NAD+

dehidrogenáz

NADH + H+

UDP-glükuronsav

Glucuronidation of phenolGlucuronidation of phenol

OHO

COOH

OH

OH

OH

O N

OCH2O

OHOH

P

O

OHOH

O

O

P

N

O

OH

+

+O

COOH

OH

OH

OH

OUDP

Characteristics of glucuronide-conjugation 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.

cofactors xenobiotic

Mixed Function Oxidase system Mixed Function Oxidase system in the smooth endoplasmic reticulumin the smooth endoplasmic reticulum

fp1, fp2: flavoproteinsb5: 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 phenolSulphatation of phenol

sulphurilaseSO4

2- + ATP APS + PPi (pyrophosphate)

APS-PhosphokinaseAPS + ATP PAPS + ADP

OH

N

OCH2O

OHO

P+

O-

O-

O-O3S N

N

NH2

O-

O-

O P

+

OSO3H

+ 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-S- methionin Methyl-transferase Cytosol ( also in membranes)

Glutathione conjugation

epoxides, organic haloids Reduced glutathione Glutathione transferase Cytosol (also in membranes)

Glutathione conjugation of some compounds:Glutathione conjugation of some compounds:

Cl

Cl

NO2

SG

Cl

NO2

Br

O

OH

SG

GSH

GSH

3,4-dikloro-nitrobenzol

bróm-ciklohexán ciklohexén

GS = glutation csoport

Formation of mercapturic acid Formation of mercapturic acid after glutathione conjugationafter glutathione conjugation

OH

HH

OH

SGH

S

H

OH

H

CH2

NH

COOH

O

CH3

SCH2

NH

COOH

O

CH3

NADPH

[O]

GSH

H+

- H2O

Non conjugation reactions of phase IIReactions 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 ionsFormation of carbonium és nitrenium ionsfrom sulphate conjugates from sulphate conjugates

of benzyl alcohols and hydroxamic acidsof benzyl alcohols and hydroxamic acidsOH OSO3-

R3

CH2+

R3

+ SO42-

benzil-alkoholkarbónium ion

R

O

NH OH

+ :Nu-NH

O-

R

Nu OH

R

O

Nu

NH2 OH+

NOSO3-

R2

R3

N+

R2

R3

+ SO42-

nitrénium ion

hidroxámsav

Reversion of inactivation in the urinary bladderReversion of inactivation in the urinary bladder

NH2 NOH

H

NOH

glükuronid

UDPGA

MÁJ

Hugyhólyag

NOH

glükuronid

H+

glükuronsav

NOH

H

elektrofil, reaktiv intermedier

Characteristics of the biotransformation reactionsCharacteristics of the biotransformation reactions

1.1.No strict substrate specificity,No strict substrate specificity,2.2.Induction and inhibitionInduction and inhibition3.3.Not only xenobiotics but endogenous substratesNot 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)

CGA 17020

GlutathionePathway

GlutathionePathway

MET 4G

MET 2G = MET 3G

COOH

MET 4U = MET 1G

COOH

MET 13U

MET 11U

COOH

MET 3U / MET 5U* = MET 5G* =MET 6G* = MET 7G* = MET 8G*

GlutathionePathway

COOH

MET 6U = MET 8U

MET 2U / MET 10dG*

MET 18U / MET 10cG*

MET 10U / MET 12U*

MET 7U

MET 1U

MET 10aG

MET 9U* = MET 9G*

MET 16U**

GlutathionePathway

COOH

MET 14U = MET 15U MET 17U

Cys: cysteineGlu: glutamic acid

* excreted as glucuronic acid derivative** excreted as hydroxymethyl-glucuronic acid conjugate

postulated intermediate

oxalic acid derivatives

CH3

CH3

N

CH2-CH2-O-CH3

CO-CH2Cl

CH3

CH3

N

CH2-CH2-O-CH3

CO-CH2-S-Cys-Glu

CH3

CH3

N

CH2-CH2-O-CH3

CO-CH2-S-CH2-CH|

NH2

|

CH3

CH3

N

CH2-CH2-O-CH3

CO-CH2-S-CH2-CH|

NH-CO-CH3

|

CH3

CH3

N

CH2-CH2-O-CH3

CO-CH2-SO-CH3

CH3

CH3

NH CO-CH2-S-CH2-CH|

NH-CO-CH3

|

CH3

CH3

N

CH2-CH2OH

CO-CH2Cl

CH3

CH3

N

CH2-CH2OH

CO-CH2-S-CH2-CH|

NH-CO-CH3

|

CH3

CH3

N

CH2-CH2OH

CO-CH2-S-CH3

CH3

CH3

N

CH2-CH2OH

CO-CH2-SO-CH3

CH3

CH3

N

CH2-CH2OH

CO-CH2-SO2-CH3

CH3

CH3

N

CH2-COOH

CO-CH2OH

CH3

CH3

N

CH2-CONH2

CO-CH2OH

CH3

CH3

N

CH2-COOH

CO-CH3

CH2OH

CH3

N

CH2-CH2-O-CH3

CO-CH2Cl

CH2OH

CH3

N

CH2-CH2OH

CO-CH2Cl

CH2OH

CH3

N

CH2-CH2OH

CO-CH2-S-CH2-CH|

NH-CO-CH3

|

CH2OH

CH3

N

CH2-CH2OH

CO-CH3

CH3

CH3

N

CH2-CH2-O-CH3

CO-CH2OH

Factors influencing biotransformation of a

xenobiotic Species Intra-species genetic variations Age Physiological status Other xenobiotics

Ratio of glucuronidation and sulphatation Ratio of glucuronidation and sulphatation in some speciesin some species

glucuronidation (%)

Sulfatation

(%)

cat 0 87

human 23 71

rat 25 68

rabbit 46 45

pig 100 0

Biotransformation of Amphetamine Biotransformation of Amphetamine in rabbits, rats, guinea pigs and dogsin rabbits, rats, guinea pigs and dogs

NH2

CH3

NH2

CH3

OH

O

CH3

OHO

OH

CH3

nyúl

tengerimalac

patkány

kutya

konjugált fenolok

tengerimalac

nyúl

konjugátumok

konjugátumok

Genetic polymorphism of biotransformation Genetic polymorphism of biotransformation enzymesenzymes

Differences Differences •in the base sequence of the DNAin the base sequence of the DNA•In the amino acide squence of an enzymeIn the amino acide squence of an enzyme•In the reaction kineticsIn the reaction kinetics

•% of fast acetilation in some human populations% of fast acetilation in some human populations

•EuropeansEuropeans 40%40%•AsiaticsAsiatics 80%80%•InuitsInuits 96%96%

Consequences of acetylation kinetics:Consequences of acetylation kinetics:different side effectsdifferent side effects

•Isoniazid (drug against tuberculosis)Isoniazid (drug against tuberculosis)

slow acetilation: neurotoxic effectsslow acetilation: neurotoxic effectsfast acetilation: liver problemsfast acetilation: liver problems

• Hidralazin (drug against high blood pressure)Hidralazin (drug against high blood pressure)

slow acetilation: Lupus eritomatosusslow acetilation: Lupus eritomatosusfast acetilation: no specific side effectfast acetilation: no specific side effect

Differences between sexesDifferences between sexes

Hormones influence the lipid environment of Hormones influence the lipid environment of enzymes.enzymes.Sex-differences in the activity of CYP P450 enzymes.Sex-differences in the activity of CYP P450 enzymes.

(The hypothalamus is releasing a feminizing factor, (The hypothalamus is releasing a feminizing factor, which results in „feminine liver” having somewhat which results in „feminine liver” having somewhat lower metabolising capacity in general than the liver lower metabolising capacity in general than the liver of males. of males.

::

The role of age in biotransformationThe role of age in biotransformation

The influence of the physiological status The influence of the physiological status of the individual:of the individual:

Diseases,Diseases,Fasting.Fasting.

The influence of other xenobioticsThe influence of other xenobiotics

Enzyme induction by PAHs,Enzyme induction by PAHs,Enzyme inhibition by heavy metals,Enzyme inhibition by heavy metals,Dietary factors,Dietary factors,Smoking, consumption of alcohols, illegal drugs…Smoking, consumption of alcohols, illegal drugs…

Effects of exposure before birth 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: superoxide glutathione- conjugation

human: Superoxideglutathion conjugation

Differences of age: foetuses have a limited (or missing) metabolising capacity