Metabolism

•Chemical transformaion of xenobiotics•Occurs in mostly in liver (enzymatic prosesses)•Convertion into more hydrophil. subst. - excretion urine•May convert procarciogenics into cytotox., muthagenic compounds•Different persons may have differences in methabolism (genetic diff., physiol. factors)•Methabolism of one xenobiotic may influence metab. of amother

Xenobiotics•Drugs•Other foreign non-essential compounds

Metabolism in non-hepatic tissue•Intestine mucosa•Kidney•Lung•Bacteria in GI-tract

First-pass metabolism:Xenobiotic metabolized before reachinggeneral circulation

First-pass metabolism:Xenobiotic metabolized before reaching general circulation

A) Metab. lungs (inhaled subst)Intestine mucosa, GI bacteria

B)

GI Liver

General Circulation

Absorb under tung

Absorbfrom rectum

Pathways of metabolismPhase 1: Biotransformation

Attachment of new functional groups, transformation of exist. funct. groupsoxidation, reduction, hydroxylation, hydrolysis etc.

Phase 2: Conjugation. Masking of an exist. funct. group by for instance acetylation, glycosylation, attachment amino acid etc

More hydrophilic drug

Renal excretion

O OHHO

N CH3

Morphine

Demethylation

Phase 1 O OHHO

NHConjugationglucuronic acid

Phase 2 O OHO

NH

O OHCO2H

OHHO

Phase 1Metabolism by cytochrome P450 enzyme systhem (CYP450)

•Located in endoplasmatic reticulum (liver and other cells)•Electron transport systhem - oxidation, monooxygenase•Heme protein + flavoprotein•Capablee of oxidation - many differen xenobiotics

N

NN

N

Fe3+

S

Cystein

H O H

CHEMICAL REVIEWSVolume 104, Issue 9 (September 8, 2004)3947-3980 Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 EnzymesBernard Meunier, Samuël P. de Visser, and Sason Shaik<http://dx.doi.org/10.1021/cr020443g>http://dx.doi.org/10.1021/cr020443g

Heme protein Fe3+

Heme protein Fe2+

FMN

FMNH2

Flavoprotein

Flavoprotein

ox. formred. form

red. form ox. formNAD

NADHred. form

ox. form

Substrate-red. form

Substrate-ox.form

N

N

N

NHH3C

H3C

O

OH OH

OHOHH

H

OPO3 2-

Flavin mononucleotide

FMN

N

HN

NH

NHH3C

H3C

O

OH OH

OHOHH

H

OPO3 2-

FMNH2N

N N

NNH2

O

OHHO

OP

O

OOH

P

OO

OHHO

N

OHO

O

NH2

NAD

N

N N

NNH2

O

OHHO

OP

O

OOH

P

OO

OHHO

N

OHO

O

NH2

NADH

H H

Nicotinamid adenine dinucleotide

CYP450 families and sub-familiesFamily 1:

CYP1A1Aromatic hydrocarbon hydroxylase, metabol. PAH etc.

O OHOH

More hydrophilicnot tox.Nu

OHNu

CYP1A2Ox of arylamines, nitrosamines, aromatic hydrocarbons

Family 2:CYP2A6CYP2B6CYP2CCYP2D6: Often enantioselective, lipophil. aminesCYP2E1: Halogenated hydrocarbons, other org solvents

Family 3:CYP3A4

CYP450 / Mechanisms of metobolic transformations

CYP450

Fe3+

CYP450

Fe3+

CYP450

Fe2+

R-H

CYP450

Fe2+

R-H

O2

CYP450

Fe3+

R-H

O2

R-H

e

O2

NADPH

CYP450

Fe3+

R-H

O2

2 H

H2O

CYP450

Fe5+

R-H

O

CYP450

Fe4+

R

OH

R-O-H

R-HCYP450

Fe3+

R

OHH

H=

+H2O

Hydroxylation of alkaneDehydrogenation of alkane

R-CH3CYP450 R-CH2OH

Other enzymesR-CO2H

Fe5+ O

Alkene, aromaticπ-complex

Fe4+ O

Radicalσ-complex

Fe3+

H

OH

Allylic alchohol

Fe3+ O

RR

O

R

R

O

1,2 migration

Proposed mech. ox of alkenes

alkynes

OH2O R

OOH

R

R

Fe3+

Fe3+ O

R

Fe3+ O

R RO

Proposed mech. ox of aromatics

Fe5+ O

Fe4+ OFe3+ O

Fe3+

O

R R

R R

RR

R RR=H

Fe3+

Fe3+

O H H

taut

HO

Fe5+ O Fe4+ OH

XR

H

XR

XR

Fe3+

XR

OH

i.e. hemiacetal

O+ HX-R

Fe4+ OH Fe3+

XR

H Oi.e. N oxide

Proposed mech. react. on heteroatom cont. compounds

N, O, S dealkylationcleav. of small alkylgroups (Me)

Dehalogenation: HX + carbonyl comp.

Proposed mech. react. on heteroatom cont. compounds

Fe5+ O Fe4+ OH

NR

H

NR

NR

Fe4+ OH Fe3+

NR

H O

NR''

R

R'

O

Stable if R, R' R''≠H

NH

H

R'

ON

OH

H

hydroxylamine

N-oxide

NH

R

R'

ORearrang.

N

OHRR'

hemiaminal

ON RR'

H

N

OH

nitroso comp.

sulfide ox, see FMO

CYP450 Induction / inhibition by xenobiotics

Xenobiotics may enhance metabol. of them selvs as well as other comp. taken at the same timeInduce transcript CYP450 mRNA - Synth. CYP450 enzymes (enzyme induction)•Drugs•Ethanol•Organic solvents •Components in cig. smoke•

NH

OH

St. Johns Worth (Johannesurt, prikkperikum)

OH

HO

O OH

R

O OHOH

HO

R=H: Hypericin

R=OH Pseudohypericin

CYP450 InhibitorsReversible CYP enzyme inhibitors: Several drugsex. antimycotic azoles

Squalene

Squalene epoksidase

O

Squalene epoksid-syklase

HOLanosterol

Lanosterol14a-demetylase

Antimycotic azoles

HOErgosterol

HO

H

H

HO

KolesterolH

H

H

Antimycoticallylamines

Cell wall component fungi

N N

Cl

KlotrimazoleCanesten etcCYP450:

CYP450 InhibitorsComplexation inhibitorsex. metabolites from alkylamines

R-NMe2

AlkylamineNo inhib. activity

CYP450R-N=O

NitrosoalkaneIrreversible complexation with Fe(II) heme in CYP sub families

Mechanism based inhibitors (suicide inhib)ex. alkynes

CYP450

FeO3+ O

NH2-EnzymeR N

H

OEnzyme

H2O ROOH

R

R

R

HO

H

HH

OH

Ethynyl estradiol

Phase 1 react. not involving CYP450

microsome: Artefactual spherical particle, not present in the living cell, derived from pieces of the endoplasmic reticulum present in homogenates of tissues or cells: microsomes sediment from such homogenates when centrifuged at 100 000 g and higher: the microsomal fraction obtained in this way is often used as a source of mono-oxygenase enzymes.

Other microsomal enzymesAzoreductase

Nitroreductase

Flavinmonooxygenase-FMO (N and S-ox.)

Peroxidases

NN NH2

SO

OH2N

H2NNH2S

O

OH2N

Prontocil Sulfanilamide

R NO2 R NH2

Flavinmonooxygenase-FMOCont. FAD

N

N

N

NHH3C

H3C

O

OH OH

OHOHH

H

OPO3 2-

Flavin mononucleotide

FMN

N

HN

NH

NHH3C

H3C

O

OH OH

OHOHH

H

OPO3 2-

FMNH2

N

N

N

NHH3C

H3C

O

OH OH

OHOHH

H

O P OO

OH

O

OHO O

HO OH

NN

N

N

NH2

FAD

N

HN

N

NHH3C

H3C

O

ORFADH2

Flavin adenine dinucleotide

N

N

N

NHH3C

H3C

O

OH OH

OHOHH

H

OH

Riboflavin(Vit B2)

N

N N

NNH2

O

ORHO

OP

O

OOH

P

OO

OHHO

N

OHO

O

NH2

N

N N

NNH2

O

ORHO

OP

O

OOH

P

OO

OHHO

N

OHO

O

NH2

NADH

H H

Nicotinamid adenine dinucleotide

NADR=H

R=Phosphate: NADP+, NADPH

N

N N

NNH2

O

OHHO

HO

Adenine(Vit. B4)

N

CO2H

Nicotinic acid / Niacin(Vit. B3)

Flavinmonooxygenase-FMO

N

N

N

NHH3C

H3C

O

OR

H2O

N

HN

N

NHH3C

H3C

O

OR

OH

N

HN

N

NHH3C

H3C

O

OR

OOH

FAD

Substrate Substrate-O

N

HN

NH

NHH3C

H3C

O

OR

FADH2

O2

NADPH

NADP+

R OO

H

NuPeroxidePeracid

O Nu

N-oxideSulfoxide

+ ROH

•Amine: ox. to N-oxide / hydroxylamine•Sulfide: ox to sulfoxide , furter to sulfone

•Thiol:

Ox of soft Nu

R-SH R-S-S-R R-S-S-R

O

Non-microsomal enzymes•Enzymes in mitokondria•Enzymes in soubile tissue fractions

R-OH

prim or sec.

Alchohol dehydrogenase(NAD cont.)

aldehyde / ketoneR

O

H

Aldehyde dehydrogenaseR

O

OH

Alcohol dehydrogenase Aldehyde dehydrogenaseCH3OH HCO2HCH2O

SlowEtOH cometitive substr.

rel. fastTox. effects:AcidosisBlindness

HN

NH

NH

HN

O

O

NH

Tetrahydrofolate

NH

O CO2H

CO2HHN

NH

NH

HN

O

O

N

NH

O CO2H

CO2H

OH

HCO2H

Dietary folic acid

10-Formyl-THF dehydrogenase

CO2

Non-microsomal enzymes (Phase 1)

Molybdenum Hydroxylases•Aldehyde oxidase•Xantine oxidase•Xantine dehydrogenase

Xanthine oxidaseElectron transfer: FAD - Fe2S2ˇI - Fe2S2ˇII - Moco - Substrate

Active form

Cont. Mo in cat. siteCont FAD and 2 Fe/s clustersUse H2O not O2

•Aldehyde oxidase

R H

O

R OH

O

N

Aza heterocycle

N OH NH

O

N

N N

N

H2NAcO

AcO

FamciclovirAntiviral (Herpes etc)Pro-drug

Aldehyde oxidaseEsterase / hydrolysis HN

N N

N

H2NHO

HO

O

Penciclovir

•Xantine oxidase•Xantine dehydrogenase (requires NAD+)

xanthine oxidoreductase

HN

N

O

NH

N

Hypoxanthine

HN

NH

O

NH

N

Xanthine

OHN

NH

O

NH

HN

Uric acid

OO

HN

N

O

NH

N

Allopurinol

HN

NH

O

NH

N

AlloxanthineInhib. enzyme

O

Treatment of gout (podagra)

Non-microsomal enzymes (Phase 1)

Oxidative deamination of amines•Monoamine oxidase (MAO)•Diamine oxidase (DAO)

R-CH2-NH2 + O2 [R-CH=NH] R-CHO + NH4+

NH

NH2

HO

Serotonin5-Hydroksotryptamin (5-HT)

MAONH

O

HOSerotonine:

•Neurotransmittor; temp. control, mood•Depresion: Low serotonine activity•MAO Inhibitors - Older antidepresants(low selectivity)

N NH

O O

Cl

MoklobemidAurorix® Moklobemid®

Other MAO substrates:

HN

OH

HO

HO H

Noradrenaline

NH2HO

HODopamine

Not MAO substrates (subst at α-C):

NH2

(S)-amfetaminLow dopamine conc. ≈ Parkinston

Active transport re-uptake transmittor(not Acetylcholine)

Non-selective monoamine re-uptake inhib.Tricyclic antidepressants

SSRI (selective serotonine re-uptake inhib.)“Lykkepiller” Prozac etc (Fontex)

NH2HO

HO

NH2HO

HO

OH

DA NE

HO

NH

NH2

5-HTα

β

β-Arylaminer

Y

X

R Z

N

αβ

β-Arylam

≈ β-Arylamin

Stor gruppe / sterisk hindingHindrer reopptak

O

F3C

NHH2N

Non-microsomal enzymes (Phase 1)

Oxidative deamination of amines•Monoamine oxidase (MAO)•Diamine oxidase (DAO)

Oxidize diamines, histamine NH2

HN

N

MAO like enzymes in plants

N

N NH

NHN

BAPCYtokinin (Plant growth hormone)

N

N NH

NNH2

O

+ N

N NH

N N

N NH

N N

N NH

N

CK analogs, Anders Bråthe

Non-microsomal enzymes (Phase 1)

Miscellaneous react.

ReductionsS S RR R-SH

R-SO-R R-S-R (i.e.DMSO)

R-CO-R R-CHOH-R

R-NO-R R-NH-R

R-CH=CH-R R-CH2-CH2-R

R-OH R-H

Ar-OH Ar-H

β-OxidationR-CH2-CH2-CO2H R-CH2-CH2-CO-S-CoA R-CO2H

+

CHa-CO-S-CoA

Acetyl-CoA

R-CO-S-CoA

N

N N

NNH2

O

OHPO3

OPOPOHN

O OO OOH

O

HNSO

O

Acetyl-CoA

Nu

Hydrolysis - EsterasesO

OR R'

O

NR R'

R''

O

OHR

Esters as pro-drugs

Pathways of metabolismPhase 1: Biotransformation

Attachment of new functional groups, transformation of exist. funct. groupsoxidation, reduction, hydroxylation, hydrolysis etc.

Phase 2: Conjugation. Masking of an exist. funct. group by for instance acetylation, glycosylation, attachment amino acid etc

More hydrophilic drug

Renal excretion

O OHHO

N CH3

Morphine

Demethylation

Phase 1 O OHHO

NHConjugationglucuronic acid

Phase 2 O OHO

NH

O OHCO2H

OHHO

Phase 2: Conjugation Most comp. excreted as cojugates, ionic, hydrophilic groups added,

most common glucuronation

•Glucuronic acid conjugation•Sulfate conjugation•Conjugation with amino acids•Acetylation•Glutathione conjugation•Methylation

Phase 2: Conjugation •Glucuronic acid conjugation

Substrates:

•Alchohols

•Phenols

•Amines

•Sulfides

•Carboxylic acids

•1,3-Dicarbonyls

OHO

OHOHOH

OH

Glucose

OCO2H

OHOOH

OH

UDP-Glucuronate

P P O

HO OH

N

NO

O

RXH

OCO2H

OHOHOH

X R

RXH: Xenobiotic / Phase 1 metabolite

OCO2H

OHOHOH

X R

Kidney Urive excretion

Bile (galle)

RXH

OCO2H

OHOHOH

X RIntestine

RXH

Poor reabsorb.

OCO2H

OHOHOH

X R

Reabsorb

Entro-hepatic recyclingImportant for many hormones etc

OCO2H

OHOOH

OH

UDP-Glucuronate

P P Uridine

OH

OCO2H

OHOHOH

OR

O

R

O

Relatively labile

HydrolysisO

H R

O

HNu Macromolecule

Nu MacromoleculeR

O

Altered protein (hapten)Unwanted immune responce / allergic react.

Acyl migr.

ex. NSAIDs

Ar-NH2Phase 1

Ar-NH-OH Ar-NH-OH-Glu

UrinepH<7

Ar-NH-OH

H2O

Ar-N

Nitrene(c.f. carbene)

Bladder cancer

Phase 2: Conjugation •Sulfate conjugation: Phenols, (alcohols, N-compounds)

ATPO NPO

O

OHSO

OO

HO3P OH

PAPS

N

N

N

NH2 HO

OSO

O

O

•Conjugation with amino acids (Most ofthen Gly): Carboxylic acids

R-CO2H

O

R S CoAH2N CO2H O

R NH

CO2H

No tox. conjugates known

Phase 2: Conjugation •Acetylation: N-compounds

O

H3C S CoANH NO

•Glutathione conjugation: Electrophilic species

HS NH

HN

O CO2H

ONH2

CO2HR-X + S NH

HN

O CO2H

ONH2

CO2HR

Glutathione conjugate

S OH

HN

O

O

R

Mercapturic acid der.

•Alkylhalides•Epoxides•Michael acceptors etc

may otherwisealkylate biomolecules

Br

BrRSH

Br

SS R

Nu(i.e. DNA)

HN

O

OHParacetamol

Phase 1CYP450

H2O

N

O

O

HS-R

O

HN

O

SRH

HN

O

OHSR

H2N Liver Protein

Liver dammage

Phase 2: Conjugation •Methylation (O and N- compd) Prod. may be more lipophilicReact. mainly aimed at converting endogenic compouds

O.Metylation by COMT (catecol O-methyl transferase)

ATPO N

HO OH

N

N

N

NH2 HO

OH3C

SAM (S-adenocyl methionine)

SH3C

CO2HH2N

HO

HO

SAM may also methylate N-comp.

HO

HOOH

NH2

Noradrenaline(Norepinephrine)

MAO

HO

HOOH

CHOAldehyde Dehydrogenase

HO

HOOH

CO2H

COMT

HO

H3COOH

NH2

COMT

HO

H3COOH

CO2HMAO Aldehyde Dehydrogenase

OHNH2

EphedrineAdrenerg agonist