Drug metabolism or biotransformation
© Oldřich Farsa 2012
Drug metabolism or biotransformation
● reactions that are responsible for the conversion of drugs or
other xenobiotics into another products (metabolites) within the
body before and after they have reached their sites of action
● it usually occurs by more than one route
● their end products are normally pharmacologically inert
compounds that are more easily excreted than the original drug
● classified for convenience as Phase I reactions which either
introduce or unmask functional groups that are believed to act
as a centre for Phase II reactions; product of Phase I are often
more water soluble and so more readily excreted than the
parent drug
● Phase II reactions produce compounds that are often very
water soluble and usually form the bulk of the inactive excreted
products of drug metabolism
Schematic of biotransformation phases
PHASE I redox metabolism enzymatic
apparatus
Mixed-Function Oxidases, formed by
microsomes made out of smooth endoplasmic
reticulum (SER) folded over on itself.
• Cytochrome-P450 Enzyme Complex: Has
four required components in order to work.
• Cytochrome-P450 Enzyme
• Cytochrome-P450 Reductase
• O2
• NADPH: NADPH is the only energy source.
Types of Phase I reactions
OXIDATIVE REACTIONS: on drugs, such as aromatic
hydroxylation, aliphatic hydroxylation, N-dealkylation, Odealkylation,
S-dealkylation, N-oxidation, S-oxidation,
desulfuration etc. in most on CYP.
REDUCTIVE REACTIONS: azo, nitrile, carbamyl
HYDROLYTIC REACTIONS: ester hydrolysis, amide
hydrolysis.
OTHER REACTIONS: decarboxylation
ibuprofen
C O 2 H C O 2 H
H O
Aliphatic ω−hydroxylation: ibuprofen (NSAID)
Aliphatic (ω−1)−hydroxylation: pentobarbital (hypnotic, sedative ...)
H N
N
O
O
H
O
H N
N
O
O
H
O O H
pentobarbital
Examples: acetanilide, phenytoin, propranolol
Endogenous substrates: steroid hormones (not aromatic amino acids)
Aromatic hydroxylation
R
u n s t a b l e
a r e n e e p o x id e
in t e r m e d ia t e
n o n e
n z y m a t ic
H Y L 1
e p o x id e
h y d r o la s e
R O H
O H
R O H
O D N A , P r o t e in
t o x ic
r e a c t io n s
O
R
R
O H
R
O H
o r
●arene epoxide can be quite stable in some cases: carbamazepine and carbamazepine epoxide
N
NH2 O
5H-dibenzo[b,f]azepine-5-carboxamide
N
NH2 O
O
N
NH2 O
OH OH
H2O
carbamazepine
Carbamazepinum PhEur
Biston®
, Neurotop®
, Tegretol®
CR ...
● antiepileptic
●
blocks voltage gated Na+
channels
and thus inhibits fast and noncontrolled
impulse spreading
carbamazepine 10,11-
epoxide
●active
●stable; found in waste
water
trans-10,11-
dihydrocarbamazepine-10,11-
diol
●main metabolite excreted by
urine
antiepileptic phenytoin: aromatic hydroxylation and water addition
Arene epoxide intermediate produces multiple products
N
N
O
C Y P 2 C 8 , 9 N
N
O
O
H
H
H Y L 1
p h e n y t o in
3 ,4 - d ih y d r o d
i h y d r o x y p h e n t o in
N
N
O
H O
N
N
O
O H
N
N
O
H O O H
p a r a - h y d r o x y p h e n y t o in m e t a - h y d r o x y p h e n y t o in
O
N
O H
H
O
N
O H
H
O H
O
N
O H
H
O H
β-adrenolytic – anti-hypertensive propranolol: hydroxylation in 2 positions of
naphthalene ring
CYP3A4
terfenadine
(Seldane – not authorized in CZ)
fexofenadine
(Ewofex ® )
•cardiac adverse effects not present
NH O
O H
NH O
C O 2 H
O H
Metabolism of terfenadine: oxidation of one of methyls of tert-butyl into carboxyl
●H1
-antihistamine if the 2nd
generation developed in 1980th
●serious cardiac adverse effects including TdP arrythmias
N (or O, S)-oxidative dealkylation
R N
C H 2
C H 2
R
C H 2
O H
R N
C H 2
C H 2
R
C H 2
R N
C H 2
C H 2
R
C H 3
R N
C H 2
C H 2
R
C H 3
+
O 2
- H +- 1 e H
C H O+
R N
C H 2
C H 2
R
H
N-demethylation generates formaldehyde
Oxidative N-demethylation: ethylmorphine (antitussive)
OO O H
N
C H 3
OO O H
N
H
H C H O+
e t h y lm o r p h in e d e s m e t h y l- e t h y lm o r p h i n e
N-demethylation favored over O-deakylation
Oxidative desisopropylation: propranolol
O
N
O H
H
O
N
O H
H
O H
O
N
O H
H
O H
O
N
O H
H
H
- CH3
COCH3
●also 2´and 7´hydroxylated metabolites have been reported
Oxidative S-demethylation: 6-methylthiopurine = 6-methylsulfanylpurine
N
N
N
N
S
C H 3
N
N
N
N
S H
H C H O+
6-methythiopurine
●prodrug
●not used
6-mercaptopurine
●active form normaly
originated from
antineoplastic and
antirheumatic
azathioprin
Examples: chlorpheniramine, trimethylamine
Examples: chlorpromazine, cimetidine
N - O x id a t io n
R N H 2
R N H O H
R N
R
R
R N
+
R
R
O
_
S - O x id a t io n
S
R
R 2
S
R
R 2
O
chlorpromazine - antipsychotic
chlorpheniramine - H1
-antihistamine
S
N C l
N
S
N C l
N
O
H
N
N
CH3
CH3
Cl
H
N
N
+
CH3
CH3
Cl
O
-
[3-(4-chlorophenyl)-3-(pyridin-2-yl)propyl]
dimethylamine oxide
R C H C H 3
N H 2
R C
N H 2
C H 3
O H
R C C H 3
O
+ N H 3
Oxidative deamination of primary amines
Examples: amphetamine, diazepam (after benzodiazepine ring opening)
amphetamine - central stimulant, indirect adrenergic
N H 2 O
N H 3+
2-phenylpropane-2-on
2-amino-3-phenylpropane
amphetamine
PHASE I hydrolytic metabolism enzymatic
apparatus
● hydrolases
● esterases – have also some amidase activity
– cholinesterases: acetylcholiesterase, butyrylcholinesterase
– pseudocholinesterase
– lipases
● peptidases – naturally cleave the peptidic bond, but are
capable to cleave also other amide bonds
– exopeptidases – cleave peptide bonds of terminal amino acid
rests
● carboxypeptidases – from C-terminal
● aminopeptidases – from N-terminal
– endopeptidases – cleave peptide bonds inside peptide chain
● in general are all the types of peptidases capable to
cleave anilides, naphtylamides etc.
Hydrolysis Reactions
Esters
Example: acetylosalicylic acid (others include procaine, clofibrate)
+ R 2 O HR 1 O H
O
R 1 O
R 2
O
C O 2 H
O C O C H 3
C O 2 H
O H
Hydrolysis Reactions
Amides
Example: lidocaine; others include peptide drugs
R 1 N H
R 2
O
R 1 O H
O
N H 2R 2+
N
H
O
N
N H 2N
O H
O
+
Hydrolysis reactions in local anaesthetics: a difference between esters and amides
NH2
O
N
CH3
O
CH3
NH2
NH
N
CH3
O
CH3
H2O
hydrolase
fast NH2
OH
O
N
CH3
CH3OH
+
NH2
OH
O
N
CH3
CH3NH2
+
H2O
hydrolase
slow
procaine
procainamide
●procaine does not act as an antidysrrhythmic after i.v. administration because of its fast
hydrolysis by esterases in blood it does not reach the myocardium tissue in enough
concentration while isosteric procainamide does because the amide bond is hydrolyzed
much more slowly due to its higher stability and low activity of esterases in hydrolysis of
this bond
Hydrolysis reactions in local anaesthetics: stereoselectivity
Local anaesthetics of anilide series: prilocaine
●anaesthetic activity of R and S enantiomers does not markedly
differ
NH
CH3
NH
CH3
O
CH3
S-(+)
NH2
CH3
NH
CH3
O
CH3
OH
(2R)-2-(propylamino)propanoic acid
R-(-)
hydrolase
aromatic ring
hydroxylation
NH2
CH3OH
NH2
CH3
OH
+
+
H2O
●toxic metabolites
●methemoglobinemia
●administration of the pure S-(-)
enantiomer can eliminate the toxicity
Decarboxylation reaction
α-methyldopa – antihypertensive, α-
adrenolytic
Dopegyt ® contains (-)-(S) sesquihydrate
●stereoselectivity of enzyme reaction: (-)-(S)isomer
only undergoes the decarboxylation
and thus is active
OH
O
OH OH
CH3
NH2
OH
O
OH OH
CH3
NH2
2-amino-3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid
(-)-(S) (+)-(R) - inactive
decarboxylase
OH OH
CH3
NH2
H
β -hydroxylase
OH OH
CH3
NH2
H
OH
- CO2
α-methylnoradrenaline – metabolite active as α1
antagonist
α-methyldopamine
PHASE II metabolic routes:
conjugation reactions
●involve the attachement of a group or a molecule to the drug or
metabolite
●may occur at any point in the metabolism of a drug or xenobiotic but
they are often the final step in the metabolic pathway before excretion
●conjugates are usally inactive with some exceptions
●in most cases markedly more hydrophilic than the parent compound
but with frequent exceptions
●excreted from body in most in form of salts (Na+
...)
The most common „conjugation partners“
OHH
O
H
H
H
H
OH
OH
OH
OH
O
NH2
H
O
NH
H
O
NH
O
OH
SH
O
OH
D-glucuronic acid
glutathione
S
O
OOH
OH
O
OH
CH3
O
OHNH2
S
OH
O
O
NH2
O O
OHOH
NH2
sulfuric acid
acetic acid
glycine
taurine
(2-aminoethanesulfonic acid)
(S)-gluatmic acid
N
H
H
H
O
P
OH
OH O
H
O
OPO
S
OH
OO
OH
O OH
N
N
NH2
N
PAPS: 3'-Phosphoadenosine-5'-phosphosulfate
●„activated form“ of sulfuric acid used as cosubstrate for sulfate conjugations
„Activated“ glucuronic acid = UDP-glucuronic acid as cosubstrate in conjugation of paracetamol
Examples of substrates of glucuronic acid
conjugation include alcohols,
phenols, 3°-amines, aromatic amines etc.
M o r p h in e
O
H O
H O
N C H 3
6
3
A m it r ip t y lin e
N
N
N
C H 3
O
C o t in in e
Glucuronate conjugations of propranolol and some its hydroxylation products
O
NH
OH
CH3 CH3
O
NH
O
CH3 CH3
O
OH
H
H
H
OH
H OH
H
O
OH
O
NH
OH
CH3 CH3
OH
O
O
OH
H
H
H
OH
HOH
H
O OH
O
NH
OH
CH3 CH3
(-)-enantiomer only
O
NH
OH
CH3 CH3
OH
O
NH
OH
CH3 CH3
O
O
OH
H
H
H
OH
HOH
H
O
OH
OHO
O CH3
O
OHO
OH
O
OH
OH
O
OH
O
OH
O OH
O
O
OHOH
OH
OH
O OH
O
O
OH
NH
OH
O
OHO
OH
OH
ASA
salicylic acid.
10 %
+ CH3COOH
O1-salicyoylglucuronic acid
5 %
O1-(2-carboxyphenyl)glucuronic acid
10 %
glucuronation
salicyluric acid
(N-salicyoylglycine)
75 %
conjugation with Gly
gentisic acid
< 1 %
hydroxylation
Metabolism of acetylosalicylic
acid
• proceeds in most in liver
•conjugations are the most
important part of its
biotransformation
•all metabolits are excreted by
urine
t1/2
= 15 min
Conjugation Reactions
Acetylation
A r N H 2
R S H
R O H
R N H 2
+
A r N
C H 3
O
H
A c e t y l t r a n s f e r a s e
C o A S
O
R N
O
C H 3H
R O
O
C H 3
R S
O
C H 3
Examples: Procainamide, isoniazid, sulfonamides, histamine
N-acetyl transferase (NAT) enzyme is found in many tissues, including liver
Acetylation leads in most cases to conjugates which are more lipophilic and
thus less soluble in water than the parent compound
Whole human population is genetically divided into fast and slow acetylators
Procainamide: participation of acetylation in its metabolism
Unchanged
in Urine, 59%
3%
24% Fast
17% Slow
Unchanged
in Urine, 85%
N-acetylprocainamide (NAPA)
0.3%
1%
H 2 N
O
N
H
N
N
O
N
H
N
O
H
H 2 N
O
N
H
N
H
N
O
N
H
N
O
H H
Antituberculotic isoniazid (INH): acetylation is an important metabolic step
NAT2
N
O HO
N
N N HO
H
H
N
N N
O
H
H
C H 3
O
minor
Isoniazid N-Acetylisoniazid
t1/2
= 70 min in fast
acetylators
t1/2
= 180 min in slow
acetylators
● N-acetyltransferase (NAT2 isoform) is in liver, gut
●the first drug which slow and fast acetylators were seen in
●periferal neuropathy seen in slow acetylators
Antituberculotic isoniazid (INH): acetylation followed with hydrolysis
                                                                                                                                                                     
                   
Glutathione conjugations on the example of a part of paracetamol metabolism
O
N
CH3
O
OH
N
CH3
O
OH
N
CH3
O
NH2
O
S
OH
N
CH3
O
O
S
O
O
O
O
O
OHOH
OH
OH
O
N
O
CH3
OH
NH
CH3
O
N
O
O
N
O
S H
N
O
OH
H
H
H
OH
OH
NH
CH3
O
NH2
O
S
N
O
OH
H
OH
N
CH3
O
NH
O
S
OH
CH3
O
N-acetyl-p-benzoquinoneimine
N-(4-oxocyclohexa-2,5-diene-1-yli
dene)acetamide
NAPQI
GSH
(glutathione)
conjugationconjugation
4 %
42 % 52 %
2% urine
paracetamol glucuronide paracetamol sulfate
urine
urine
urine
cytochrome
P-450
δ +
subst. mercapturic acid
GSH transferase
peptidase
peptidase
N-acetyltransferase