Drug Metabolism - Phase II

Phase II reactions are conjugation reactions or synthetic reactions.

Phase I reactions introduce or expose a functional group to the drug molecule, i.e., either a polar functional group is placed in the molecule or polar constituent is revealed.

Once the polar part is exposed then Phase II reaction may occur

DRUG

Phase I

Phase II

Metabolite

Phase IIMetabolite

Conjugating reagents are derived from biochemical compounds involved in carbohydrate, fat and protein metabolism.

Phase II reaction produce water soluble compounds that can be excreted through urine or bile.

1. GLUCURONIDATION Quantitatively the most important phase II pathway for

drugs and endogenous compounds (bilirubin). It is the conjugation reaction between Uridine

diphosphate glucuronic acid (UDPGA) [a-glucuronide] and drugs containing –OH, -COOH, -NH2 and –SH.

Reaction is mediated by microsomal enzyme glucuronyltransferase (b-glucuronide is formed).

O-glucuronide conjugation excreted through bile to gutb-glucuronidase break the conjugate

possible reabsorption of drug Entro-hepatic circulation of drug long elimination half life.

Ester-glucuronide get hydrolyzed in urine or plasma to form parent drug. E.g. clofibrate

N-glucuronidation of sulfanilamide

◦ Occurs with amines (mainly aromatic ), amides and sulfonamides

O-glucuronidation of Morphine

◦ O-glucuronidation occurs by ester linkages with carboxylic acids, phenols and alcohols

S-glucuronidation◦ Example glucuronidation of Disulfiram

Conjugation with other sugarConjugation with glucose, xylose, ribose and

arabinose are also possible.Glucuronidation is the major sugar

conjugation.

Example:2-hydroxy nicotinic acid conjugates with ribose

All sugar conjugates are water soluble and excreted through urine.

2. SULFATION Common for phenols, alcohols, amines and lesser

extent to thiols. Conjugating agent (i.e., sulfate donor) is 3’-

phosphosadenosine-5’phosphosulfate (PAPS) Enzymes involved are

◦ Non-specific – phenol, alcohol and arylamine transferases◦ Specific – steroid transferases

Sulfate conjugates are water soluble and have high renal clearance.

MINOXIDIL

(inactive)

MINOXIDIL N-O-SULFATE

(active metabolite) N

N

NH2O

H2N N

N

N

NHO

H2N N

S

O

HO

O

Minoxidil Minoxidil-sulfate

3. Methylation Non-specific methyltransferases methylate

drugs with the help of S-adenosylmethionine (SAM) to form methyl-conjugates.

SAM is produced from L-methionine and ATP under the influence of enzyme L-methionine adenosyltransferase.

methyl-conjugates are less polar and thus the metabolite is not easily excreted from body.

N

N

O

HS

THIOURACIL

H

N

N

O

S

H

H3C

S-METHY-THIOURACIL

4. Acetylation Common for aromatic amines and

sulfonamides, requires Acetyl-CoA and enzyme N-acetyl transferases.

It can take place mainly in Kupffer cells of liver. This reaction can also take place reticuloendothelial cells of spleen, lungs and gut.

Acetylated product are less polar than parent compound. This will increase the elimination half life of the metabolite, also renal toxicity (e.g. : acetylatedsufonamide).

In the case of active metabolite, this long half life is important. (procainamide N-acetylprocainamide).

N-acetyltransferase exhibit genetic polymorphism.◦ E.g. Isoniazid inactivation

N

CO-NH-NH2

N

CO-NH-NH-CO-CH3

ISONIAZID ACETYL ISONIAZID

5. AMINO ACID CONJUGATION It is a special form of acetylation. Aromatic acids are activated by combining

with ATP to form CoA derivative then it undergoes the amino acid conjugation.

Amino acid involved are glycine, glutamine, ornithine, arginine and taurine.

Bile acids also undergo amino acid conjugation.

RCOOH + CoA-SH RCO-S-CoA

RCO-S-CoA + NH2CH2COOHRCONHCH2COOHN-acyltransferase

ATP

GlycineGlycine conjugate

(mitochondria)

COOH

+ CoA-SH

CO-S-CoA

CO-S-CoA

+ NH2CH2COOH

CO-NH-CH2-COOH

BENZOIC ACID

HIPPURIC ACID

GLYCINE

6. GLUTATHIONE CONJUGATION Glutathione, glutamyl-cysteine-glycine is

recognized as a protective device against toxic electrophilic compounds.

It reacts non-enzymatically and enzymatically via glutathionine-S-transferase, through nucleophilic sulfhydryl group with electrophilic oxygen intermediates.

Compounds conjugate with glutathione includes epoxides, haloalkanes, nitroalkanes, alkenes and aromatic halo- and nito- compounds.

Glutathione-S-transferase are found in liver, kidney, gut and other tissues.

Reaction occurs in 4 steps1. Drug+Glutathione forms drug conjugate2. Removal of glutamyl by glutamyl transpeptidase3. Removal of glycine by peptidase4. N-acetylation of cysteine conjugate forms N-

acetylated cysteine(mercapturic acid)

Glutathione conjugate undergoes biliary secretion, but in gut it breaks down by C-S lyase produced by intestestinal microflora. This results –SH transfer and its methylation. Methylated drug is reabsorbed and reaches liver. In liver oxidation occurs ant methylthio-derivative is excreted.◦ Example : caffeine

Saturation of this pathway will lead to accumulation of electrophilic intermediates. These are capable of binding with hepatic cellular macromolecules and will cause cell damage.

N-acetylcysteine, which containing sulfhydral group is used as antidote for toxicity.◦ Acetaminophen

C

C

H COOR

COORH

C

C

H COOR

COORGlutathione

Esters of maleic acid Glutathione conjugate

Phase I Phase II

7. FATTY ACID CONJUGATION This involved in the conjugation of stearic

and palmitic acids. 11-hydroxy-D9-tetrahydrocannabinol

undergoes this type conjugation.

8. CONDENSATION REACTIONS These reactions are purely chemical and

common among aldehydes and amines. Dopamine + 3,4-

dihydroxyphenyacetaldehyde tetrahydropapaveroline (potent dopamine antagonist)

Phase II reactions produces more polar product, which can be excreted easily.

Major reactions are Glucuronide conjugation, Acetylation and Sulfation.

Glucuronide conjugation may increase half-life.

Products are generally more water soluble.

These reactions products are ready for (renal) excretion.

Phase II reactions are possible only for compounds having polar group.

This is important for lipophilic drug, determines the rate of excretion.

Reaction EnzymeFunctional

groupExample

GlucuronidationUDP-

glucuronyltransferase-OH, -COOH, -

NH2, -SH Morphine

Sulfation Sulfotransferase-OH, SO2NH2, -

NH2

Minoxidil

Methylation Methytransferase -OH, -NH2 Thiouracil

Acetylation Acetyltransferase-OH, SO2NH2, -

NH2, -COOHIsoniazid

Amino acid conjugation

---- -COOH Benzoic acid

Glutathione conjugation

Glutathione-S-transferase

Epoxides, organic halides

Maleic acid

Fatty acid conjugation

---- -OH11-hydroxy-D9-

tetrahydro-cannabinol

Condensation ----Aldehydes and

aminesDopamine