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 reaching
general circulation
First-pass metabolism:
Xenobiotic metabolized before reaching general circulation
A) Metab. lungs (inhaled subst)
Intestine mucosa, GI bacteria
B)
G e n e ra l
C irc u la tio n
GI
L iv e r
A b s o rb u n d e r tu n g
A b s o rb fro m re c tu m
Pathways of metabolism
Phase 1: Biotransformation
Attachment of new functional groups, transformation of exist. funct. groups
oxidation, 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
N
CH3
NH
NH
C o n ju g a tio n
g lu cu ro n ic a cid
D e m eth y la tio n
HO
O
M o rp h in e
OH
P h a se 1
HO
O
OH
P h a se 2
HO
O
OH
HO
O
O
OH
C O 2H
Phase 1
Metabolism 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
C ystein
S
N
F e 3+
N
N
N
H
O
CHEMICAL REVIEWS
Volume 104, Issue 9 (September 8, 2004)
3947-3980 Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes
Bernard Meunier, Samuël P. de Visser, and Sason Shaik
<http://dx.doi.org/10.1021/cr020443g>http://dx.doi.org/10.1021/cr020443g
H
F la v o p ro tein
NAD
o x . fo rm
FM NH 2
red . fo rm
F la v o p ro tein
FM N
o x. form
NADH
red . fo rm
H em e p ro tein
F e 3+
S u b strate-red . form
o x . fo rm
H em e p ro tein
F e 2+
S u b stra te-o x.fo rm
red . fo rm
F lavin m on on u cleotid e
N ico tin am id ad en in e d in u cleotid e
O
NH2
O
N
O
O
P
HO
N
N
N
O P O
OH
NH2
N
NAD
H 3C
N
O P O
OH
N
O
OH
HO
NADH
H 3C
H
N
H 3C
N
O
NH
NH
H
OH
H
N
H
OH
H
OH
H
OH
OH
H
OH
H
N
FM N
N
N
N
OH
N
OPO3
P
O
O
HO
OH
HO
HO
NH2
O
O
O
OH
HO
H 3C
NH2
N
O
O
O
H H
O
2-
OPO3
O
2-
FM NH2
CYP450 families and sub-families
Family 1:
CYP1A1
Aromatic hydrocarbon hydroxylase, metabol. PAH etc.
OH
O
OH
Nu
M o re h yd ro p h ilic
n o t tox.
OH
Nu
CYP1A2
Ox of arylamines, nitrosamines, aromatic hydrocarbons
Family 2:
Family 3:
CYP2A6
CYP3A4
CYP2B6
CYP2C
CYP2D6: Often enantioselective, lipophil. amines
CYP2E1: Halogenated hydrocarbons, other org solvents
CYP450 / Mechanisms of metobolic transformations
Hydroxylation of alkane
Dehydrogenation of alkane
R -O -H
R -H
+
Fe
H 2O
3+
C Y P 450
F e 3+
F e 4+ O H
F e 3+ O H
H
C Y P 450
C Y P 450
C Y P 450
R
R -H
NA DPH
R
=
H
F e 5+ O
C Y P 450
F e2+
R -H
C Y P 450
R -H
H 2O
O2
2H
F e3+
O2
F e2+
C Y P 450
O2
R -H
C Y P 450
e
F e3+
O2
C Y P 450
R -C H 3
C Y P 450
O ther enzym es
R -C H 2 O H
R -C O 2 H
R -H
R -H
A llylic alch oh ol
Proposed mech. ox of alkenes
F e 3+
OH
R ad ical
 -com p lex
A lk en e, arom atic
 -com p lex
H
R
R
R
F e4+ O
O
Fe
Fe
5+
3+
O
O
1,2 m igration
R
alkynes
R
O
R
O
F e3+ O
F e 3+
R
R
F e 3+ O
H 2O
R
O
O
R
OH
Proposed mech. ox of aromatics
R
O
R
F e3+
F e 3+
R
R
R
R
R
F e4+ O
R
taut
R=H
F e3+ O
O
F e 5+ O
F e3+
H
H
HO
Proposed mech. react. on heteroatom cont. compounds
H
O
H
X
X
R
R
Fe
5+
O
Fe
4+
X
X
F e4+ O H
i.e. N oxid e
R
R
F e3+
OH
F e 3+
OH
O
+ H X -R
X
R
i.e. h em iacetal
N, O, S dealkylation
cleav. of small alkylgroups (Me)
Dehalogenation: HX + carbonyl comp.
Proposed mech. react. on heteroatom cont. compounds
H
O
H
N
N
R
N
N
R
R
R
sulfide ox, see FMO
N -oxid e
Fe
5+
O
Fe
4+
F e4+ O H
F e 3+
OH
O
R ''
N
R
S tab le if R , R ' R ''° H
R'
H
OH
O
H
N
R'
O
OH
H
N
H
R earrang.
N
R
N
O
R
R'
R'
h yd roxylam in e
O
H
N
n itroso com p .
h em iam in al
H N
R
R'
CYP450 Induction / inhibition by xenobiotics
Xenobiotics may enhance metabol. of them selvs as well as other comp. taken at the same time
Induce transcript CYP450 mRNA - Synth. CYP450 enzymes (enzyme induction)
•Drugs
•Ethanol
•Organic solvents
•Components in cig. smoke
•
OH
N
H
OH
O
OH
R
HO
R=H:
H y p e ric in
HO
R=OH
P s e u d o h y p e ric in
St. Johns Worth
(Johannesurt, prikkperikum)
OH
O
OH
CYP450 Inhibitors
Reversible CYP enzyme inhibitors: Several drugs
ex. antimycotic azoles
S q u a le n e
e p o k s id s y k la s e
S q u a le n e
e p o k s id a s e
HO
O
S q u a le n e
A n tim y c o tic
a lly la m in e s
H
L a n o s te ro l
C Y P 4 5 0 : L a n o s te ro l
1 4 a -d e m e ty la s e
K lo trim a z o le
C a n e s te n e tc
A n tim y c o tic a zo le s
N
N
Cl
HO
HO
E rg o s te ro l
H
C e ll w a ll c o m p o n e n t fu n g i
H
H
HO
K o le s te ro l
H
CYP450 Inhibitors
Complexation inhibitors
ex. metabolites from alkylamines
C YP 450
R -N M e 2
A lk y la m in e
N o in h ib . a c tiv ity
R -N = O
N itro s o a lk a n e
Irre v e rs ib le c o m p le x a tio n w ith
F e (II) h e m e in C Y P s u b fa m ilie s
Mechanism based inhibitors (suicide inhib)
ex. alkynes
O
H 2O
R
F e O 3+
R
R
R
O
C Y P 45 0
OH
H
H
H
HO
E th y n y l e stra d io l
OH
O
N H 2 -E n zym e
R
N
H
E n zym e
Phase 1 react. not involving CYP450
Other microsomal enzymes
Azoreductase
O
O
H 2N
H 2N S
N
N
O
H 2N S
NH2
P ron tocil
NH2
O
Su lfanilam ide
Nitroreductase
R NO2
R NH2
Flavinmonooxygenase-FMO (N and S-ox.)
Peroxidases
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.
Flavinmonooxygenase-FMO
Cont. FAD
F la v in m on on u cleotid e
F la vin ad en in e d in u cleotid e
O
H 3C
N
H 3C
NH
N
N
H
OH
H
OH
H
OH
O
NH2
N
O
O P O
OH
N
O
NH
N
R
N
O
N
HO
N
N
N
O P O
OH
P
O
N
OR
O P O
OH
NAD
OH
H
OH
H
OH
H
OH
H
OH
2-
OPO3
N
H 3C
N
N
NH
N
H
OH
H
OH
H
OH
OH
HO
A d en in e
(V it. B 4)
N
O
OR
N
O
HO
O
N
N
N
N
O
H 3C
N icotin ic acid / N iacin
(V it. B 3)
HO
C O 2H
NADH
O
2-
FM N H 2
NH2
N
R = P h o sp h ate: N A D P + , N A D P H
N
NH
H
OH
N
O
H 3C
O
H
O
NH2
HO
R=H
OH
HO
HO
NH2
O
O
O
O
H
N
N
H
OH
N
R ib oflavin
(V it B 2)
NH2
OH
H 3C
FM N
OH
H H
O
P
N
NH
OPO3
NH2
O
H 3C
N
O
HO
N
O
N ico tin am id ad en in e d in u cleo tid e
O
H 3C
OH
HO
FAD
H 3C
O
N
O
O
H
N
FA D H 2
N
O
H 3C
Flavinmonooxygenase-FMO
Ox of soft Nu
O
R
H
P eroxide
P eracid
S u b strate
+
O Nu
O
ROH
N -oxid e
Su lfoxid e
Nu
S u b strate-O
OH
O
H 3C
H
N
H 3C
N
O
NH
N
O
H 3C
H
N
H 3C
N
OH
O
H 2O
NH
N
O
O
H 3C
N
H 3C
N
R
R
R
FA D
H 3C
H 3C
O2
H
N
N
R
O
NH
N
H
FADH2
•Amine: ox. to N-oxide / hydroxylamine
•Sulfide: ox to sulfoxide , furter to sulfone
O
•Thiol:
R -SH
R -S-S -R
R -S -S-R
N A D PH
O
N A D P+
NH
N
O
Non-microsomal enzymes
•Enzymes in mitokondria
•Enzymes in soubile tissue fractions
A lch o h o l d eh y d ro g en a se
(N A D co n t.)
a ld eh y d e / k eto n e
R -O H
O
A ldeh yde d ehydrogenase
O
R
R
OH
H
p rim o r sec.
A ldeh yde d ehyd rogenase
A lcohol deh ydrogen ase
C H 2O
C H 3O H
H C O 2H
rel. fast
Slow
EtO H com etitive substr.
T ox. effects:
A cidosis
B lind ness
D ietary folic acid
O
O
HN
O
N
H
H
N
N
H
H C O 2H
C O 2H
N
H
N
H
O
O
C O 2H
HN
O
T etrahyd rofolate
N
H
N
H
H
N
N
H
N
H
O
10-F orm yl-T H F
deh ydrogen ase
C O 2H
C O 2H
CO2
Non-microsomal enzymes (Phase 1)
Molybdenum Hydroxylases
•Aldehyde oxidase
•Xantine oxidase
•Xantine dehydrogenase
Xanthine oxidase
Electron transfer:
Cont. Mo in cat. site
Cont FAD and 2 Fe/s clusters
Use H2O not O2
FAD - Fe2S2ˇI - Fe2S2ˇII - Moco - Substrate
Active form
•Aldehyde oxidase
O
R
O
H
R
N
N
OH
H 2N
O
A ld ehyd e oxid ase
E sterase / hyd rolysis
N
N
H 2N
N
N
H
N
HO
AcO
OH
N
HO
A cO
N
N
HN
O
F am ciclovir
A n tiviral (H erpes etc)
P ro-drug
A za h eterocycle
•Xantine oxidase
xanthine oxidoreductase
•Xantine dehydrogenase
O
O
(requires NAD+)
N
N
HN
O
HN
N
N
H
O
N
H
HN
N
H
O
X anth in e
H ypoxan thin e
O
HN
N
N
N
H
A llop urin ol
HN
O
N
N
H
H
N
O
N
H
N
H
U ric acid
T reatm ent of gout (p odagra)
O
P enciclovir
N
H
A lloxanth in e
Inh ib. enzym e
Non-microsomal enzymes (Phase 1)
Oxidative deamination of amines
•Monoamine oxidase (MAO)
•Diamine oxidase (DAO)
R-CH 2 -NH 2
+
O2
O
NH 2
HO
MAO
R-CHO
[R-CH=NH]
HO
N
H
N
H
Serotonin
5-Hydroks otryptamin (5-HT)
+
Serotonine:
•Neurotransmittor; temp. control, mood
•Depresion: Low serotonine activity
•MAO Inhibitors - Older antidepresants
(low selectivity)
Moklobemid
Aurorix® Moklobemid®
O
O
N
N
H
Cl
O th e r M A O s u b s tra te s :
OH
HO
HO
H
N
N o t M A O s u b s tra te s (s u b s t a t  -C ):
HO
H
NH2
NH2
HO
N o ra d re n a lin e
D o p a m in e
(S )-a m fe ta m in
Low dopamine conc. ≈ Parkinston
NH 4 +
Non-selective monoamine re-uptake inhib.
Tricyclic antidepressants
SSRI (selective serotonine re-uptake inhib.)
“Lykkepiller” Prozac etc (Fontex)
 -Arylam
X
H2 N
Active transport re-uptake transmittor
(not Acetylcholine)
- -Arylamin
Y
R

Z
H

O
F3 C
N
Stor gruppe / sterisk hinding
Hindrer reopptak
 -Arylaminer
HO
HO

HO
OH
NH2

DA
HO
HO
NH 2
NH2
NE
N
H
5-HT
N
Non-microsomal enzymes (Phase 1)
Oxidative deamination of amines
•Monoamine oxidase (MAO)
•Diamine oxidase (DAO)
HN
Oxidize diamines, histamine
N
NH2
MAO like enzymes in plants
NH2
HN
N
N
N
N
H
N
N
N
BAP
C Y to k in in (P la n t g ro w th h o rm o n e )
N
H
N
N
+
N
O
N
H
N
N
N
N
H
N
N
N
C K a n a lo g s , A n d e rs B rå th e
N
H
Non-microsomal enzymes (Phase 1)
Miscellaneous react.
Reductions
R
S
S
Hydrolysis - Esterases
R
R -S H
O
R -S O -R
R -S -R (i.e .D M S O )
R
O
R'
O
R -C O -R
R -C H O H -R
R -N O -R
R -N H -R
R
O
R
N
OH
R'
R ''
R -C H = C H -R
R -C H 2 -C H 2 -R
R -O H
R -H
A r-O H
A r-H
Esters as pro-drugs
-Oxidation
R -C H 2 -C H 2 -C O 2 H
R -C H 2 -C H 2 -C O -S -C o A
R -C O 2 H
+
NH2
C H a -C O -S -C o A
N
N
A c etyl-C o A
O
S
H
N
OH
H
N
O
O O O O
P
P
O
O
O
N
N
O
O
Nu
PO3
A c e ty l-C o A
OH
R -C O -S -C oA
Pathways of metabolism
Phase 1: Biotransformation
Attachment of new functional groups, transformation of exist. funct. groups
oxidation, 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
N
CH3
NH
NH
C o n ju g a tio n
g lu cu ro n ic a cid
D e m eth y la tio n
HO
O
M o rp h in e
OH
P h a se 1
HO
O
OH
P h a se 2
HO
O
OH
HO
O
O
OH
C O 2H
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:
RXH: Xenobiotic / Phase 1 metabolite
•Alchohols
•Phenols
X
C O 2H
HO
OH
•Sulfides
O
N
O
O
OH
OH
•Amines
R
H
OH
OH
G lu c o s e
OH
O
P
O
OH
P
N
O
C O 2H X
O
OH
R
OH
OH
U D P -G lu c u ro n a te
HO
OH
•Carboxylic acids
•1,3-Dicarbonyls
RXH
C O 2H X
O
OH
R
K id n e y
U riv e e x c re tio n
OH
OH
OH
OH
P o o r re a b s o rb .
B ile (g a lle )
R e a b s o rb
Entro-hepatic recycling
Important for many hormones etc
RXH
C O 2H X
O
OH
C O 2H X
O
OH
OH
OH
R
In te s tin e
R
O
O
H y d ro ly sis
H
R
O
O
O
H
C O 2H
R
C O 2H O
O
OH
O
OH
OH
O
OH
P
P
U rid in e
R
HNu
OH
M a c ro m o le c u le
OH
U D P -G lu c u ro n a te
R e la tiv e ly la b ile
A c y l m ig r.
Nu
M a c ro m o le c u le
R
O
A lte re d p ro te in (h a p te n )
U n w a n te d im m u n e re s p o n c e / a lle rg ic re a c t.
e x . N S A ID s
U rin e
pH <7
Phase 1
A r-N H 2
A r-N H -O H
A r-N H -O H -G lu
A r-N
A r-N H -O H
H 2O
N itre n e
(c .f. c a rb e n e )
B la d d e r c a n c e r
Phase 2: Conjugation
•Sulfate conjugation: Phenols, (alcohols, N-compounds)
NH2
N
O
ATP
O
O
O
N
N
S O P
HO
O
O
O
OH
H O 3P
O
S
N
O
OH
PAPS
•Conjugation with amino acids (Most ofthen Gly): Carboxylic acids
O
R -C O 2 H
R
H 2N
S
C oA
O
C O 2H
R
N
H
C O 2H
No tox. conjugates known
Phase 2: Conjugation
•Acetylation: N-compounds
O
H 3C
NH
S
O
C oA
N
•Glutathione conjugation: Electrophilic species
•Alkylhalides
•Epoxides
•Michael acceptors etc
O
R -X
+
HS
O
C O 2H
N
H
S
N
H
HN
HN
H S -R
SR
OH
H 2O
O
M e rca p tu ric a cid d e r.
O
O
N
OH
HN
NH2
O
G lu ta th io n e c o n ju g a te
O
P hase 1
C Y P450
S
C O 2H
NH2
O
HN
O
R
HN
HN
O
C O 2H
R
C O 2H
may otherwise
alkylate biomolecules
O
O
H
SR
OH
P a ra c e ta m o l
Br
H 2N
L ive r P ro te in
S
RSH
S
Br
R
Br
L iv e r d a m m a g e
Nu
(i.e . D N A )
Phase 2: Conjugation
•Methylation (O and N- compd)
Prod. may be more lipophilic
React. mainly aimed at converting endogenic compouds
O.Metylation by COMT (catecol O-methyl transferase)
HO
C O 2H
H 2N
NH2
N
N
N
ATP
S
O
H 3C
HO
HO
HO
N
OH
S A M (S -a d e n o c y l m e th io n in e )
SAM may also methylate N-comp.
O
H 3C
OH
HO
OH
NH2
MAO
HO
HO
OH
CHO
A ld e h yd e
D e h yd ro g e n a s e
HO
HO
C O 2H
HO
N o ra d re n a lin e
(N o re p in e p h rin e )
COMT
COMT
OH
OH
H 3C O
MAO
NH2
A ld e h y d e
D e h y d ro g e n a se
H 3C O
HO
HO
OH
NH2
E p h e d rin e
A d re n e rg a g o n is t
C O 2H