Phase-II Drug Metabolism

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

N

NN

N

NH2

O OH

OHS

-OOC

NH2

SAM

Glutathione

Sulfate

Acetyl CoA

Glucuronic acid

Glycine

Methyl source

Phase-II Drug MetabolismInvolves the following conjugation reactions

that are catalyzed by transferase enzymes:

Glucuronidation.Sulfation.Amino acid conjugation.Methylation.Acetylation.

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.

Most of the time gives metabolite that is:

More polar

Non-toxic

Pharmacologically inactive

Phase-II Drug Metabolism

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

Sometimes:Phase-II gives:

Less polar conjugate: Methylated drug Acetylated drug

Toxic metabolite: Some sulfate conjugates Some Acetylated metabolites.

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

OHO

HOHO

OH

OP

O

OOH

P

O

OOH

UridineUDP-Glucose

OHO

HOHO

OH

OP

O

OOH

Uridine triphosphate phosphorylase

Glucose-6-phosphate

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

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

Examples

Estradiol

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

Example of C-glucuronidation

N N

O

O

H S

Sulfinpyrazone

N N

O

O

S

HO

O

HOHO

OH

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

Possible groups for glucuronide conjugation

Possible groups for glucuronide conjugation

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

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

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

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

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

Examples of sulfation

HO

HO

COOH

NH2

-methyldopaHO

HO

OHHN

Salbutamol

HO

OHHN

OH

Terbutaline

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

N

OH

O

O3SO N

O

O

N-acetyl imidoquinone

Microsomal proteins

HN

O

O

Microsomal proteins

Hepato and Nephrotoxicity

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.

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.

Example of active acetylated metabolite

H2N

NH

O

N

ProcainamideHN

NH

O

N

O

N-acetylprocainamidepharmacologically active

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

Example of toxic acetylated metabolite

H2N

SNH

O OS

N

Sulfathiazole

NH

SNH

O OS

N

O

N-acetylsulfathiazole

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

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

Examples of acetylation

N

HN

H2N

HistamineNH2

O

OH

P-aminosalicylic acid

N

N

HNNH2

Hydralazine

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