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Phase-II Drug Metabolism
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Phase-II Drug MetabolismReactions which conjugate the drug or its phase-I
metabolite with a hydrophilic, endogenous species (conjugation reactions).
These endogenous compounds are:
Glucuronic acid Sulfate group Amino acid (Glycine)Methyl group (as SAM)Acetyl group (as acetyl CoA)Glutathione (tripeptide)
N
NN
N
NH2
O OH
OHS
-OOC
NH2
SAM
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N
NN
N
NH2
O OH
OHS
-OOC
NH2
SAM
Glutathione
Sulfate
Acetyl CoA
Glucuronic acid
Glycine
Methyl source
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Phase-II Drug MetabolismInvolves the following conjugation reactions
that are catalyzed by transferase enzymes:
Glucuronidation.Sulfation.Amino acid conjugation.Methylation.Acetylation.
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Phase-II Drug Metabolism
The drug must have a group capable of forming a bond with the endogenous compound.
If the drug does not have such group, it will undergo phase-I metabolism, then phase-I metabolite will be conjugated during phase-II reactions.
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Most of the time gives metabolite that is:
More polar
Non-toxic
Pharmacologically inactive
Phase-II Drug Metabolism
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Drug
Active drug
XHEndogenous compound
GDrug X
G
Phase-II drug conjugate
Excreted in urine
X = O, N, S
Phenol, alcohol and carboxylic acidAmine (aliphatic and aromatic)Thiol
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Sometimes:Phase-II gives:
Less polar conjugate: Methylated drug Acetylated drug
Toxic metabolite: Some sulfate conjugates Some Acetylated metabolites.
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GlucuronidationInvolves conjugation of drug with glucuronic
acid.
OHO
O
HOHO
OHOH
Glucuronic acid
DRUG
Hydroxyl group is a bad leaving group
IT must be activated to be a good leaving group
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OHO
HOHO
OH
OP
O
OOH
P
O
OOH
UridineUDP-Glucose
OHO
HOHO
OH
OP
O
OOH
Uridine triphosphate phosphorylase
Glucose-6-phosphate
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OHO
O
HOHO
OH
OP
O
OOH
P
O
OOH
Uridine
OHO
HOHO
OH
OP
O
OOH
P
O
OOH
Uridine
UDP-Glucuronic acid
UDP-Glucose
UDPG-dehydrogenase
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Mechanism of conjugation
OHO
HOHO
OH
OP
O
OOH
P
O
OOH
Uridine
DRUGHX
OHO
HOHO
OHX
DRUG
Drug-Glucuronide conjugate
O
O
UDP-glucuronosyl transferase OP
O
OOH
P
O
OOH
Uridine
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Examples
Estradiol
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Examples
O
N
HO
OHMorphine
UDP-glucuronosyl transferase
O
N
O
OH
OO
HO
HO HO
OH
Major metabolite
Morphine is extensively metabolized into glucuronide conjugate
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Example of C-glucuronidation
N N
O
O
H S
Sulfinpyrazone
N N
O
O
S
HO
O
HOHO
OH
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Example of C-glucuronidationR R
OO Alkaline condition
HBase
R R
OO
Nucleophilic carbanion
OHO
HOHO
OH
OP
O
OOH
P
O
OOH
Uridine
O
R R
OO
OHO
HO HOOH
O
C-Glucuronide
Because this H-atom is acidic
Nucleophilic attack
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Possible groups for glucuronide conjugation
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Possible groups for glucuronide conjugation
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Sulfation (sulfate conjugation)
Occurs primarily for phenols and occasionally for alcohols, arylamines, and N-hydroxy compounds
catalyzed by sulfotransferase enzyme.
Sulfotransferase is available mainly in liver, but can be found in kidney, intestine and other tissues
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SulfationAlso occur for endogenous compounds, such as
steroids, thyroxin, catecholamine and heparin.
OH
O
HO
OOH
Prednisolone
HO
HO
OHHN
Adrenaline
O
I
I
COOHH2N
ThyroxineI
IHO
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The first step is the bioactivation of inorganic sulfate by enzyme called ATP sulforylase to give the coenzyme 3'-phosphoadosine-5’-phosphosulfate (PAPS)
Sulfation
OS
O
O O
ATP sulforylase
ATP PPiO
H2O3PO OH
OP
O
OS
O
O
O Adenine
OH
PAPS
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The second step here is the transfer of sulfate group from the coenzyme PABS to the acceptor drug by nucleophilic attack:
Sulfation
O
H2O3PO OH
OP
O
OS
O
O
O Adenine
OH
PAPSDRUG____OH
DRUG___ OS
O
O
O
O
H2O3PO OH
OP
O
O Adenine
OH
PAP
Drug sulfate conjugate
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As in all conjugation reactions:The endogenous polar group must be
activated and converted into electrophilic derivative.
Then the drug nucleophilic group will attack the reactive form get the polar, ionizable endogenous molecule.
Endogenous molecule
Endogenous molecule
Good Leaving Group
DRUG
XH
DRUG
XEndogenous molecule
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Examples of sulfation
HO
HO
COOH
NH2
-methyldopaHO
HO
OHHN
Salbutamol
HO
OHHN
OH
Terbutaline
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Most of the time, sulfate conjugation will give non toxic, polar and easily excreted metabolite.
Sometimes, the sulfate conjugate will be converted into toxic metabolite when the sulfate leaves the compound leaving a highly electrophilic intermediate.
HN
OH
O
HN
OSO3
O
N
OH
O
HO
N
OH
O
O3SO
O-sulfate conjugate
Non toxic easily excreted O-sulfate conjugate
Toxic metabolie
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N
OH
O
O3SO N
O
O
N-acetyl imidoquinone
Microsomal proteins
HN
O
O
Microsomal proteins
Hepato and Nephrotoxicity
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AcetylationIs a reaction of amino groups involving the
transfer of acetyl group to:An aromatic or aliphatic primary amineAmino acidsHydrazineHydrazideSulfonamides
Secondary and tertiary amines are not acetylated.
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AcetylationThe acetylated drug is generally inactive and
noon toxic.
In contrast to other metabolic transformations, acetylation ends with less polar metabolite compared to the parent drug
In some cases, the acetylated metabolite will be as active as the original drug.
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Example of active acetylated metabolite
H2N
NH
O
N
ProcainamideHN
NH
O
N
O
N-acetylprocainamidepharmacologically active
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Example of toxic acetylated metabolite
N
OHN
NH2
N
OHN
NH
O
N-acetylizoniazid
Hydrolysed H2NNH
O
N
O OH
N-oxidation
OCovalent binding
with liver proteins
O
Proteins
Highly reactive specie
Liver damage
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Example of toxic acetylated metabolite
H2N
SNH
O OS
N
Sulfathiazole
NH
SNH
O OS
N
O
N-acetylsulfathiazole
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Mechanism of acetylationAcetyl CoA is the activated carrier for acetyl
group
The reaction catalyzed by the soluble acetyltransferase enzyme:
Mainly found in liverMight found in lung, spleen, GIT and red blood
cells
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Mechanism of acetylation
NH
S
O
NH
O
OH
OP
O
HO O P
O
OHO
O
OH
O3PO
N
NN
NH2N
O
Acyl CoA
NH2
HN
OCoA-SH
N-acetyltransferase
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Examples of acetylation
N
HN
H2N
HistamineNH2
O
OH
P-aminosalicylic acid
N
N
HNNH2
Hydralazine
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AcetylationThe rate of acetylation is mainly affected by the
existence of genetic polymorphism.Two acetylator phenotypes:
Slow acetylators: tend to accumulate higher blood concentrations of un-acetylated drugs...toxicity (isoniazid… peripheral nerve damage and liver damage)
Fast acetylators: eliminate drugs more rapidly, at the same time can form toxic metabolite very fast.
Egyptians and western Europeans are slow acetylator
Eskimos and Asians are rapid acetylator