This document discusses Phase II biotransformation reactions. It provides an introduction to conjugation reactions such as glucuronidation, sulfation, amino acid conjugation, glutathione conjugation, acetylation, and methylation. It also lists factors that can affect biotransformation reactions such as chemical structure, species differences, physiological state, genetic variations, drug dosing, nutritional status, age, gender, route of administration, and enzyme induction or inhibition.

1. PHASE II Biotransformation
reaction…
Prep By:
Rajat Mahamana
M.pharm (Pharmaceutics)
Krupanidhi College Of Pharmacy
Bangalore-35
Guided By:
Dr. R.S. Thakur
M.Pharm, Ph.D.
Professor & HOD
(Pharmaceutics)

2. CONTENT:
 Introduction
 Conjugation reactions:

glucuronidation

sulphation

amino acid conjugation

glutathione

acetylation

methylation
 Factors affecting biotransformation reactions

3. Introduction
Drug metabolism (biotransformation or detoxication) is the
biochemical changes of the drugs and other foreign substances
in the body.
This is leading to the formation of different metabolites with
different effects.
Some of the compounds are excreted partially unchanged and
some are known to be converted to products, which may be
more active or more toxic than the parent compounds.
The liver is the major site of drug metabolism, but specific drugs
may undergo biotransformation in other tissues.

4. Importance Of Biotransformation:
 To convert non-polar lipophilic compounds (lipid soluble) which the
body cannot excrete into more polar hydrophilic compounds (water
soluble) which the body can excrete in short period of time.
 Because if the lipid soluble non-polar compounds are not metabolized
to the polar water soluble compounds, they will remain in the blood
and tissues and maintain their pharmacological effects for an
indefinite time.

5. Classification of metabolites:

Inactive metabolites

Metabolites retain similar activity

Metabolites with different activity

Bioactivated metabolites (prodrug technique)

6. 1-Inactive metabolites:
Some metabolites are inactive,
i.e. their pharmacological active parent compound become inactive.
Examples:
i)
Hydrolysis of procaine to p-aminobenzoic acid and
diethylethanolamine results in loss of anesthetic activity of
procaine.
ii) Oxidation of 6-mercaptopurine to 6-mercapturic acid results in loss of
anticancer activity of this compound.
6-Mercaptopurine
6-Mercapturic acid (inactive)

7. 2-Metabolites retain similar activity:
Some metabolite retain the pharmacological activity of their parent
compounds to a greater or lesser degree.
Examples:
i) Codeine is demethylated to the more active analgesic morphine.
ii) Phenacetin is metabolized to more active paracetamol.
iii) Imipramine is demethylated to the equiactive antidepressant
desipramine.

8. 3-Metabolites with different activity:
Some metabolites develop activity different from that of their parent
drugs.
Examples:
i) Iproniazid (antidepressant) is dealkylated to isoniazide
(antitubercular).
ii) Retinoic acid (vitamin A) is isomerized to isoretinoic acid (antiacne agent).

9. 4-Bioactivated metabolites
(activation of inactive drugs):
Some inactive compounds are converted to active drugs within the body.
These compounds are called prodrugs.
Prodrugs may have more stability, having better bioavailability or less side effects
and toxicity.
Examples:
i) Levodopa (antiparkinson disease) is decarboxylated in the neuron to active
dopamine.
ii) The prodrug sulindac a new non steroidal antiinflammatory drug (sulfoxide) is
reduced to the active sulfide.
iii) Benorylate to aspirin and paracetamol.
iv) The prodrug enalapril is hydrolysed to enalaprilat (potent antihypertensive).

10. Biotransformation Pathways
Drug metabolism reactions have been divided into two classes:
i)
Phase I reaction (functionalization ) and
ii)
Phase II reaction (conjugation)


Phase I reaction: Polar functional groups are either introduced into the
molecule or modified by oxidation, reduction or hydrolysis.
Or convert lipophilic molecules into more polar molecules by introducing or
exposing polar functional groups.

E.g. aromatic and aliphatic hydroxylation or reduction of ketones and
aldehydes to alcohols.

Phase I reactions may increase or decrease or leave unaltered the
pharmacological activity of the drugs.

11. 1-Oxidation:
 Addition of oxygen or removal of hydrogen.
Majority of oxidation occurs in the liver and it is possible
to occur in intestinal mucosa, lungs and kidney.
Enzyme involved in this type of oxidation is cytochrome
P450.
Increased polarity of the oxidized products
(metabolites), increases their water solubility and
reduces their tubular reabsorption, leading to their
excretion in urine.
Further undergo metabolism by phase II pathways.

12. 2-Reduction:
Reduction is the converse of oxidation (i.e. removal of oxygen or addition
of hydrogen).
E.g. reduction of aldehydes and ketones, reduction of nitro and azo
compounds.
It is less common than oxidation, but the aim is same to create polar
functional groups that can be eliminated in the urine.
Cytochrome P450 system is involved in some reaction. Other reactions
are catalyzed by reductases enzymes present in different sites within
the body.

13. 3-Hydrolysis:
It is the reaction between a compound and water.
The addition of water across a bond also gives more polar
metabolites.
Different enzymes catalyze the hydrolysis of drugs:
Esterase enzymes
Amidase enzymes

14. 1- Esterase enzymes:
 Present in plasma and various tissues, are nonspecific and catalyze de-
esterification. Hydrolysis of non-polar esters into two polar and more water
soluble compounds (i.e. acid and alcohol).
O
O
CH3
CH3
C OR + H2O
Ester of acetic acid
C OH + ROH
Acetic acid
Alcohol
A classical example of ester hydrolysis is the metabolic conversion of aspirin
(acetylsalicylic acid) to salicylic acid and acetic acid.
COOH
OCOCH3
H2O
COOH
OH
+
CH3COOH
Acetic acid
Aspirin
Salicylic acid

15. 2-Amidase enzymes:
It is the hydrolysis of amides into amine and acid, and this is called
Deamination. Deamination occurs primarily in the liver.
O
O
NH2
C R
Amide
+ H2O
Water
R
C OH + NH3
Acid
Ammonia
• Amide drugs are more resistant to hydrolysis (or they are not hydrolyzed until
they reach the liver) than ester drugs which they are susceptible to plasma
esterase.
• The duration of actions of ester drugs are less than the amide analogues.
 Example:
 Procaine (ester type) injection or topical is usually shorter acting than its
amide analogue procainamide administered similarily.

16. Why phase II biotransformation…??
 When phase I reactions are not producing sufficiently
hydrophilic (water soluble) or inactive metabolites, the
drugs or metabolites formed from phase I reaction
undergoes phase II reactions.
 These are capable of converting metabolites to more
polar and water soluble products.
 These reactions require both a high-energy molecule
and an enzyme.
 The enzymes that catalyzed conjugation reactions are
called transferases, found mainly in the liver and to a
lesser extent in the intestines and other tissues.

17. Conjugating molecules:
o 1- Glucuronic acid conjugation:
o It forms O-glucuronides with phenols Ar-OH, alcohols R-OH, hydroxylamines
H2N-OH,and carboxylic acid RCOOH.
o It can form N-glucuronides with sulfonamides, amines, amides, and Sglucuronides with thiols.
• Sulfation and glucuronidation are competing pathways:
– Sulfation predominates at low substrate concentrations
– Glucuronidation predominates at higher concentrations
o 2-Sulfate conjugation:
o It is less common.
o It is restricted to phenols, alcohols, arylamines, and N-hydroxyl compounds.
o But primary alcohols and aromatic hydroxylamines can form unstable sulfate
conjugates which can be toxic.

19. 1)-Glucuronic acid conjugation:
HOOC
O
HOOC
H
+
HO
HO
R X
HO
HO
OH O UDP
Glucuroinc acid UDP
O
H
OH X R
X = OH, NR2, CO2H, SH, acidic carbon atom
Glucuronyl Transferease catalyses this conjugation reaction
2)-Sulfate conjugation
O
HO
S
O
O
O
P
OH
O
Adenine
+
O
R
X
HO
S
O
H2O3PO
HO
PAPS
X = OH, arylamine, NHOH
Reaction with 3'-Phosphoadenosine-5'-phosphosulfate(PAPS)
Sulfotransferease catalyses this conjugation reaction
X
R

20. Drug Metabolism - Glucuronidation
 N-glucuronidation:
 Occurs with amines (mainly aromatic )
 Occurs with amides and sulfonamides

21.  O-glucuronidation:
 Occurs by ester linkages with carboxylic acids
 Occurs by ether linkages with phenols and alcohols

22.  3-Amino acid conjugation:
 By the formation of peptide link. With glycine or
glutamine.
 4- Glutathione conjugation:
 It reacts with
epoxides, alkylhalides, sulfonates, disulfides, radical
species.
 These conjugates are converted to mercapturic acid
and mostly are excreted in bile. It is important in
detoxifying potentially dangerous environmental
toxins.

23. 3)-Amino acid conjugation:
O
H
C
R
S
CoA
+
H2N
O
Y
CO2H
R
C
H
N
Y
CO2H
H
Acyl coenzyme A
Y = H or CH2CH2CO2H
N-acyltransferase catalyses the conjugation reaction
4)-Glutathione conjugation
O
H
C
R
S
CoA
Acyl coenzyme A
+
H2N
O
Y
CO2H
R
C
H
N
Y
CO2H
H
Y = H or CH2CH2CO2H
Glutathione S-transferase catalyses this conjugation reaction

24. Methylation and acetylation reactions:
 These decrease the polarity of the drugs except
tertiary amines which are converted to polar
quaternary salts.
 Groups susceptible for these reactions are
phenols, amines, and thiols.
 O-methylation of meta-phenolic OH in
catecholamines
 Does not generally increase water solubility but
serve mainly to terminate or reduce
pharmacological activity (they are usually
pharmacologically inactive).

25. Methylation
acetylation

26. CH3
S
HO2C
+
Adenine
O
NH2
+
H2O3PO
HO
R
R X
X CH3
X = OH, NH2, SH
SAM
Methyltransferase catalyses this conjugation reaction
O
O
C
H3C
S
CoA
Aceyl CoA
+
R X
R
C
X
R
Y =NH2, NHNH2, SO2NH2, CONH2
N-acyltransferase catalyses the conjugation reaction

27. Summary:
REACTION
ENZYME SYSTEM
FUNCTION GROUPS
Glucuronidation
Glucoronyl Transferase
-OH,-COOH,
-NH2, -SO2NH2
Sulfation
Sulfotransferase
-OH, -NH2,
-SO2NH2, -NHOH
Glutathione
Glutathione-S-transferase
Alkyl Halides,
Alkyl Nitrates,
Epoxides,
Lactones
(electrophilic centers)
Acetylation
Acetyl transferase
-OH, -SO2NH2,
-NHNH2, -NH2,
-NHOH
Amino Acid Conjugation
Acyl transferase
-COOH,
-NH2
Methylation
Methyl transferase
-OH,
-NH2,
-SH

28. Factors influencing Drug Metabolism
1-Chemical Structure :
The chemical structure (the absence or presence of certain
functional groups) of the drug determines its
metabolic pathways.
2-Species differences (Qualitative & Quantitative):
Qualitative differences may result from a genetic
deficiency of a certain enzyme while quantitative
difference may result from a difference in the enzyme
level.

29. 3-Physiological or disease state:
1-For example, in congestive heart failure, there is decreased
hepatic blood flow due to reduced cardiac output and
thus alters the extent of drug metabolism.
2-An alteration in albumin production can alter the fraction of
bound to unbound drug, i.e., a decrease in plasma
albumin can increase the fraction of unbound free drug
and vice versa.
3-pathological factors altering liver function can affect hepatic
clearance of the drug.

30. 4-Genetic variations:
Isoniazid is known to be acetylated by Nacetyltransferase into inactive metabolite.
The rate of acetylation in asian people is higher or faster
than that in eurpoean or north american people.
Fast acetylators are more prone to hepatoxicity than
slow acetylator.
5-Drug dosing:
1- An increase in drug dosage would increase drug
concentration and may saturate certain metabolic
enzymes.
2- when metabolic pathway becomes saturated, an
alternative pathway may be pursued.

31. 6-Nutritional status:




1-Low protein diet decreases oxidative reactions or
conjugation reactions due to deficiency of certain
amino acids such as glycine.
2-Vitamin deficiency of A,C,E, and B can result in a
decrease of oxidative pathway in case of vitamin C
deficiency , while vitamin E deficiency decreases
dealkylation and hydroxylation.
3-Ca, Mg, Zn deficiencies decreases drug metabolism
capacity whereas Fe deficiency increases it.
4-Essential fatty acid (esp. Linoleic acid) deficiency
reduce the metabolism of ethyl morphine and
hexobarbital by decreasing certain drug-metabolizing
enzymes.

32. 7-Age:
1- Metabolizing enzymes are not fully developed at
birth, so infants and young children need to take
smaller doses than adults to avoid toxic effects.
2-In elderly, metabolizing enzyme systems decline.
8-Gender (sex):
Metabolic differences between females and males have
been observed for certain compounds
Metabolism of Diazepam, caffeine, and paracetamol is
faster in females than in males while oxidative
metabolism of lidocaine, chordiazepoxide are faster in
men than in females

33. 9-Drug administration route:

1-Orally administered drugs are absorbed from the GIT
and transported to the liver before entering the
systemic circulation. Thus the drug is subjected to
hepatic metabolism (first pass effect) before reaching
the site of action.

2-Sublingually and rectally administered drugs take
longer time to be metabolized than orally taken drugs.
Nitroglycerine is ineffective when taken orally due to
hepatic metabolism.

3-IVadministration avoid first pass effect because the
drug is delivered directly to the blood stream.

34. 10-Enzyme induction or inhibition
Several antibiotics are known to inhibit the activity of
cytochrome P450.
Phenobarbitone is known to be cytochrome P450 enzyme
inducer while cimetidine is cyt. P450 inhibitor.
If warfarin is taken with phenobarbitone, it will be less
effective.
While if it is taken with cimetidine, it will be less metabolized
and thus serious side effects may appear.