DRUG METABOLISM

DRUG METABOLISM

 Drug metabolism or biotransformation are the

chemical reactions that are responsible for the
conversion of drugs into other products (metabolites)
within the body before and after they have reached
their sites of action.
 It usually occurs by more than one route.

These routes normally consist of a series of enzyme

controlled reactions.
Consequences of metabolism

IMPLICATIONS OF DRUG METABOLISM

 Inactive products

 Active metabolites

 Metabolites which are similar in activity to the

parent drug
 It may change the usual activity of the substrate

 Toxic metabolites.

 Products which are highly polar hence easily

eliminated from the body

 Less toxic metabolites (detoxification)
Sites of metabolism

 Liver- main site of drug metabolism

 Gastro intestinal tract
 Lungs
 Plasma
 Brain
 Skin
 kidney
First pass metabolism

Bioavailability and First Pass Metabolism (1).mp4

Drug metabolism
https://youtu.be/eOlh_oJehUg

DRUG METABOLISM and Excretion Animation!.mp4

metabolic enzymes

 Metabolism is the activity of certain enzymes

which may be obtained from the liver.
 If the liver is homogenized and centrifuged,

three metabolic fractions are isolated.

i. Microsomal fraction

ii. Mitochondrial fraction

iii. The soluble fraction

Enzymes : Microsomal fraction

 Is the most significant

 The microsomal drug metabolizing system is located
in the liver in the hepatic smooth endoplasmic
reticulum.
 It consist of non-specific enzymes responsible for
most metabolic oxidative reactions.
 The most significant of these enzymes are the mixed
function oxidases, (MFO) e.g. cytochrome p 450
system.
 Lipid soluble substances such as drugs and hormones
are primarily acted upon by enzymes of this fraction.
Enzymes: Mitochondrial fraction

 Consist of enzymes that metabolize more water

soluble drugs.
They include
 Monoamine and diamine oxidases which catalyze

breakdown/degradation of biogenic or xenobiotic

amine.
 Rhodanases which act on cyanide compounds by

converting them to thiocynides

Enzymes : The soluble fraction

 Contains enzymes involved in hydrolysis and

conjugative reaction
 Also phase two reactions except glucoronide
formation occur by enzymes of this fraction.
 Enzymes in this fraction at are also present in other
body organs.
Factors affecting metabolism

The route and rate of metabolism of drugs varies

between species and between individuals and groups of
individuals (e.g. in different races).
The differences are due to a number of factors:

1. Genetic varieties
 Variations in the genetic codes of individuals can

result in the absence of enzymes, low concentrations

of enzymes or the formation of enzymes with
reduced activity.
Factors affecting metabolism

1. Genetic varieties
 These differences in enzyme concentration and

activity result in individuals exhibiting different

metabolic rates and in some cases different
pharmacological responses for the same drug.
 An individual’s inability to metabolize a drug could

result in that drug accumulating in the body.

 This could give rise to unwanted effects.
 Factors affecting metabolism

2. Physiological factors
The metabolic differences found within a species are
believed to be due to variations in age, sex, nutritional
status and diet, and diseases
Age.
 The ability to metabolize drugs is lower in the very

young (under 5) and the elderly (over 60).

 However, it is emphasized that the quoted ages are

approximate and the actual changes will vary

according to the individual and their lifestyle.
 Eg Chloramphenicol in infants-causes gray baby

syndrome
Factors affecting metabolism

2. Physiological factors
Sex.
 The metabolic pathway followed by a drug is

normally the same for both males and females.

 However, some sex related differences in the

metabolism of anxiolytics, hypnotics and a number

of other drugs have been observed.
 Pregnant women will also exhibit changes in the rate

of metabolism of some drugs.

Factors affecting metabolism

2. Physiological factors
Disease states
 Diseases that affect the liver will have a large effect

on drug metabolism.
 Diseases of organs, such as the kidneys and lungs,

that are less important centers for metabolism will

also affect the excretion of metabolic products.
Factors affecting metabolism

3. Pharmacodynamics factors such as dose,

frequency and route of administration.
 Environmental factors: Drinking, smoking and drug

abuse may all have an influence on the rate of

metabolism.
 The use of over-the-counter self-medicaments may

also affect the rate of metabolism of an endogenous

ligand or a prescribed drug.
Metabolic reactions

 Drug metabolism is divided into phase I and phase II

reactions.
Phase I reactions
 convert drugs into more soluble products through

oxidation, hydrolysis or reduction.

 The product obtained may have lesser activity.

 Phase I reactions are functionalization reactions

i.e. there is a minor change on the functional groups.

 New functional groups may be introduced or older

functional groups replaced or removed.

Metabolic reactions

Phase I reactions
 This functionalization provide a site for phase II

reaction.
 Certain drugs may be eliminated from the body at

this position without going to phase II

Metabolic reactions

Phase II reactions
 Also referred to as synthetic or conjugation

reaction
 In this phase the metabolites from phase I or a drug

that has by – passed phase I is coupled or conjugated

with a polar endogenous molecule to give a larger
(more water soluble) or polar and usually inactive
product.
Metabolic reactions

Assignment;
Is metabolism the same as detoxification? 5mks

 NB: The mechanism of drug metabolism/

biotransformation are similar in all animals because
they use similar enzymes, however the rate at which the
enzymes act and the route involved in metabolism
would vary from compound to compound and from
species to species.
Metabolism Phases

Pharmacokinetics animation Phases Of Drug Metabolism.mp4

PHASE I REACTIONS

They involve;
 Hydrolysis

 Oxidation

 Reduction

Minor or miscellaneous phase I reactions include

 Hydration

 Dethioacetylation

 Isomerization.
 Oxidative reactions

A.Oxidative reaction microsomal

 Microsomal oxidative of many drugs and

steroid hormones are mediated by mixed

function oxidases/ mono oxidases e.g. CYP
450.
Oxidation is
 Addition of oxygen

 Removal of hydrogen

 Loss of electrons
 Oxidative reactions

 Oxidation is the most important of metabolic

reactions.
 The MFO particularly the CYP 450 catalyze a large
proportion of oxidative metabolism.
 CYP 450 is a group of closely related heme
containing isoenzymes or isoforms that combine with
carbon dioxide to give a complex that absorbs U.V
radiation at 450nm.
 There are about 100 iso-enzymes that have been
identified.
Oxidative reactions

A. Oxidative reaction microsomal

Most drugs are oxidized by four of this iso-enzymes
which are;
 Cyp 3A4/5

 Cyp 2D6

 Cyp 2C8/9

 Cyp 1A1/2
 Oxidative reactions

Cyp 3A4/5 (the number 3 stands for the family, A for the
subfamily while 4 and 5 represent individual
isoenzymes).
 This accounts for about 40% of all oxidations.

 Drugs that inhibit or induce these isoforms are

candidates for drug-drug and drug – food interactions.

Cyp 2D6.
 Oxidizes about 20% of drugs including tricyclic

antidepressants, neuroleptics, b-blockers and selective

serotonin re-uptake inhibitors.
Oxidative reactions

Cyp 2C8/9
 Acts on warfarin and phenytoin which have narrow

safety margins.

Cyp 1A1/2
 Responsible for activation of several procarcinogens.
Oxidative reactions

 MFOs require the reduced form of nicotinamide

adenine dinucleotide phosphate (NADPH) and
molecular oxygen for activity.
 The CYP 450 system consists of a heme protein CYP
450 that acts as the substrate and oxygen binding site,
and a flavoprotein (NADPH-CYP 450, CYP
reductase) that serves as an electron carrier.
 Oxidative reactions

The rate of oxidation depends on the following factors;

I. The concentration of CYP 450 and its affinity for the
drug. The concentration of CYP 450 differs between
species and individuals and so does the affinity
II. The species, nutritional status or diet and age. With
aging, there is decline in no of hepatocyte and
enzyme activity.
III. The concentration of CYP 450 which differs from

species to species and individual to individual.

 Oxidative reactions

Iv. The ease of dissociation or break down of the CYP

oxygen drug complex. This is the rate determining step.
At this juncture many other compounds may compete
with the drug for that complex formation e.g. carbon
monoxide which readily forms a complex with the drug
reduced CYP 450 and can displace O2 and the CYP 450
drug carbon monoxide is highly stable and does not
dissociate to release CYP 450.
v. Competing endogenous and exogenous substrates.
vi. Lipophilicity of the compound/drug
 Oxidative reactions

Drug oxidation takes various forms;

I. Side chain aliphatic oxidation/ aliphatic
hydroxylation
 An alkyl chain is hydroxylated at the carbon next to
the last carbon atom (most commonly) or at the last
carbon of the chain.
 Where there is a methyl group attached to an
aromatic group, then it is the methyl group that is
oxidized.
 Oxidative reactions: Side chain aliphatic
oxidation/ aliphatic hydroxylation
 The products of this side chain oxidation are alcohols
which will later undergo further phase II reactions
ii. Aromatic ring hydroxylation
The product formed are phenols.

The reaction takes place at the least deactivated or

most activated of the molecule usually at the para

position of aromatic ring
Oxidative reactions

iii. N- dealkylation
 It involves removal of an alkyl chain attached to the

N of an amide converting the amine from 30 to 20 to

10

 The alkyl group leaves as aldehydes and the

remaining product maybe 20 or 10 amine

 These products can undergo further phase I and II

reactions.
Oxidative reactions

iii. N- dealkylation
1 – RNH2
 0

20 – R-NH-CH
3
Oxidative reactions

N- dealkylation occurs for 30 and 20,

10 amines do not undergo N- dealkylation.
30 amines undergo the reaction more readily than 2 0

amines
The reaction also occurs more readily when the

alkyl chain is short.

If the alkyl group is a methyl group, the reaction

may be termed as N- demethylation

N- dealkylation is one of the most important

oxidation reactions.
Oxidative reactions

iii. N- dealkylation
iii. N- dealkylation
 Oxidative reactions: iv. Oxidative deamination

 Primary and secondary amines undergo this reaction

Primary amines more readily undergo the reaction than

secondary amines.
Products are either ketones or aldehydes depending on

the carbon to which the N is attached i.e if the alpha

carbon is unsubstituted, the product is and aldehyde,
otherwise ketone will be produced.
The ketones or aldehydes formed then undergo further

phase I reactions or phase II reactions (rarely)

iv. Oxidative deamination
 iv. Oxidative deamination

If the carbon is primary (unsubstituted) – we get aldehyde

Substituted or secondary carbon – we get a ketone
The ammonia is converted to urea and eliminated through

urine
Secondary and tertiary amines rarely undergoes this


reaction, but in most they preferably (30 and 20 ) undergo

N-dealkylation to secondary and tertiary amines. i.e. N
dealkylation has to be complete before deamination takes
place.
The two process i.e. deamination and N- dealkylation


occurs concurrently.
iv. Oxidative deamination

Amphetamine in some species e.g. dogs and rats

preferably undergo ring ring hydroxylation as the
principle metabolism where ring hydroxylation and
oxidative deamination takes place concurrently.
v. O – dealkylation

 It occurs in aliphatic ethers but more common in

aromatic ethers.
 Products formed are alcohols and an aldehyde which
may undergo further phase I and phase II reactions.
vi. S – dealkylation

 It occurs in thioethers but more in aromatic

thioethers, but also takes place in aliphatic thioethers.
 The products are aldehyde and thiols.

R is purine molecule.
vii. Sulfoxide formation/sulfoxidation

 Also called sulfide oxidation. A sulfide is oxidized to

a sulfoxide which may itself be converted to a
sulfone.
 Sulfoxidation is more catalyzed by flavin
monoxygenases which are not part of the cyp 450.
 Oxidative desulfuration

The reaction substitutes an S atom with an O atom i.e.

thiol to alcohol and thioketone to ketone
The reaction is more common in insects than in man.
N- oxidation and N- Hydroxylation

Tertiary amines are converted to N oxides while

secondary amines are converted to hydroxy amines/
hydroxylamines.
 NON MICROSOMAL OXIDATIVE REACTIONS.

This oxidation is catalyzed by enzymes of the



mitochondrial and soluble fraction of the liver.

 Is not catalyzed by CYP 450.
1. Oxidative deamination non microsomal.
Is catalyzed by monoamine oxidases and diamine


oxidases in the liver mitochondria and other tissues.

This reaction involves deamination of primary and


secondary amines and is faster in the former i.e.

primary amines.
 NON MICROSOMAL OXIDATIVE REACTIONS.

Secondary and tertiary amines preferably



undergo N- dealkylation to a primary amine by

oxidative deamination.
The enzymes metabolize primarily amines next
to a non substituted methylene group i.e -CH 2-
NON MICROSOMAL OXIDATIVE REACTIONS.

Endogenous catecholamines are metabolized



by this reaction
 Catecholamines
 adrenaline
 noradrenaline
 dopamine
NON MICROSOMAL OXIDATIVE
REACTIONS.
 NON MICROSOMAL OXIDATIVE
REACTIONS

products of oxidative deaminations are aldehydes +

NH3 which may undergo to further phase I reactions
including reduction to alcohols or reduction/ oxidation
to COOH esp. aldehydes.
NON MICROSOMAL OXIDATIVE REACTIONS.

2. Alcohol and aldehyde oxidation / alcohol

dehydrogenation.
 Oxidation of alcohols in the body is catalyzed by
liver alcohol dehydrogenase (LAD) which requires
NAD coenzyme.
 Alcohol are oxidized to aldehydes (i.e. primary
alcohols) or ketones (secondary alcohols).
 The aldehydes/ketones formed undergo further phase
I reactions.
NON MICROSOMAL OXIDATIVE REACTIONS.

2. Alcohol and aldehyde oxidation / alcohol

dehydrogenation.
 Oxidation of primary alcohols is faster i.e. primary>
secondary> tertiary.
 Tertiary alcohols lack an alpha hydrogen that is
involved in the reaction.
 NON MICROSOMAL OXIDATIVE REACTIONS.

3.Purine Oxidation
 Xanthine oxidase found in the liver cytosol
metabolizes endogenous and exogenous non
methylated or non-alkylated purine to uric acid.
 The purines are oxidized from their hypoxanthine to
their xanthine derivatives and finally to uric acid.
 The uric acid is eliminated through urine but
sometimes accumulates in joints causing gout.
 Reaction can be blocked by xanthine oxidase inhibitors
eg allopurinol, febuxostat
NON MICROSOMAL OXIDATIVE REACTIONS.
NON MICROSOMAL OXIDATIVE REACTIONS.

4. Beta Oxidation
The alkyl chain of alkyl carboxylic acids is oxidized to
an alcohol at the beta position.
NON MICROSOMAL OXIDATIVE REACTIONS.

5. Aromatization reaction
 Aliphatic cyclic compounds are converted to
aromatic derivatives. Reaction occurs only in pigs
NON MICROSOMAL OXIDATIVE REACTIONS.

6. Oxidative dehalogenation
 It affects halogen containing compounds like

insecticides and anesthetics.

 It is a slow reaction, not common in man but in

insects.
 It involves removal of halogen from a compound.

 The reaction is done by a group to compounds found

in the soluble fraction which are named according to

the halogen they eliminate e.g. those that eliminate
chlorine are called dechlorinases. The enzymes
require glutathione
 NON MICROSOMAL OXIDATIVE REACTIONS.

 The ease of halogenation depends on the halogen in

the question.

 C-F is the strongest bond hence most difficult to

dehalogenate, followed by C- Cl > C-I

 This information is used in drug design to produce

more potent drugs with long duration of action.
 NON MICROSOMAL OXIDATIVE REACTIONS.

 If R is Cl, the bond is hard to break hence the drug is

not easily metabolized hence it has a long duration of
action.
REDUCTION
REACTIONS
 REDUCTION REACTIONS

They may be microsomal or non microsomal, majority

being microsomal.
I. MICROSOMAL REDUCTION REACTIONS
i. Nitro reductions
 Occur thro’ stepwise reduction process
 Reduce the nitro group on aromatic compounds
through the nitroso then hydroxyl amine intermediates
and finally to the corresponding amine (primary
amine)
 1. MICROSOMAL REDUCTION
REACTIONS

i. Nitro reductions
 The reaction does not occur in developed (especially
in man) unlike in less developed organisms like
bacteria.
 The primary amine can undergo further phase I or II
reactions.
1. MICROSOMAL REDUCTION
REACTIONS

E.g. Chloramphenicol
 1. MICROSOMAL REDUCTION REACTIONS

Examples of compounds that undergo this reactions are:

 Nitrobenzene
 Para-nitrosulfathiazole
The coenzymes required are NADPH
1. MICROSOMAL REDUCTION REACTIONS

2. Azo reduction
 Azo reductases convert aromatic azo aromatic
compounds into two amines per compound.
 The coenzyme is NADPH. Examples protonsil – is a
sulfanilamide, It undergoes reduction to produce a
sulfanilamide.
 The reaction is slow in mammals but fast in bacteria.
1. MICROSOMAL REDUCTION REACTIONS

This reaction was the basis for discovery of

sulfanilamide.
1. MICROSOMAL REDUCTION
REACTIONS

3. Disulfide reduction
1. MICROSOMAL REDUCTION
REACTIONS
 Sulphur – sulphur linkage/bonds undergo reactions
leading to the rapture of the S-S bonds and formation
of two sulfides.
 It is a very slow reaction that less reaction in less
developed organisms e.g. bacteria.
2. NON MICROSOMAL REDUCTION
REACTIONS.

Aldehydes and ketones can be reduced to their

corresponding alcohols using liver alcohol
dehydrogenase.
HYDROLYSIS REACTIONS

 Restricted to group of drugs containing the following

linkages.
 Esters

 Amides

 Hydrazines

 Carbamates

 Involves esterases and amidases which are widely

spread in the body e.g. blood plasma, liver, skin, GIT.
 Esterases are responsible for esters and carbamates
linkages while amides are responsible for amides and
hydrazine linkages.
HYDROLYSIS REACTIONS

For and ester → Carboxylic acid + alcohol

HYDROLYSIS REACTIONS

Amide → Carboxylic acid + amine

HYDROLYSIS REACTIONS
PHASE II
REACTIONS
 PHASE II REACTIONS

 They are conjugative or additional reactions.

 Involve addition of a polar endogenous substances to
a drug or phase I metabolites hence increasing its
polarity and eventually its water solubility.
 This makes the drug to be easily eliminated by renal
excretion.
 The endogenous molecules include:
 glucoronic acid
 Sulphate
 amino acids e.g. glycerin, acetyl COA, glutathione.
 PHASE II REACTIONS

 For conjugation to occur, the endogenous molecule

is synthesized in the body before being transferred to
the drug through enzyme catalyzed process.
 Conjugation reaction occur in the liver enzyme
fractions whereby glucoronide conjugative reactions
occurs mainly in hepatic microsomes while other
conjugative reactions occur in other liver fractions.
 The products of conjugation are usually biologically
inactive, non-toxic and readily eliminated from the
body since they are highly water soluble
PHASE II REACTIONS

1. GLUCORONIDE CONJUGATION
 Takes place in microsomal fraction.
 Enzymes involved are known as glucoronyl
transferase.
 It occurs in the liver but also to a small extent in the
kidney, intestinal tract, skin and lung.
PHASE II REACTIONS

1. GLUCORONIDE CONJUGATION
 Takes place in two stages:
a) Involves formation of the activated form of
glucoronic acid called uridine disphosphate
glucoronic acid (UDPGA) from D glucose – 1 –
phosphate.
b) The UDPGA combines with an active hydrogen from
an acid, OH, phenol, SH, NH2 in the presence of
UDP glycoronly transferase.
PHASE II REACTIONS : GLUCORONIDE
CONJUGATION

Compounds/ functional groups metabolized through

glucoronide conjugation include:
 Phenols
 OHs
 NH2
 Carboxylic acids
 Amines especially aromatic amines.
PHASE II REACTIONS
PHASE II REACTIONS
PHASE II REACTIONS

 O- glucuronides are acid stable while N and S are

labile
E.g.

PHASE II REACTIONS

Though glucuronide conjugation leads to inactive

products there is an exception with drugs are more
active biologically.
 PHASE II REACTIONS

2. SULPHATE CONJUGATION
Enzymes involved are sulphate transferases/sulfokinases
found in the cytosol i.e. soluble fraction.
The drugs that can be conjugated through this process
include OHs, phenols and NH2. The activated
transferring molecule phosphoadenosine – 5 –
phosposulphate (PAPS) is synthesized from adenosine –
5 - phosphosulphate (APS).
PHASE II REACTIONS

2. SULPHATE CONJUGATION
PHASE II REACTIONS

3. ACETYLATION
The acetyl group from acetylation is derived from the
acetyl COA, the conjugation is catalyzed by acetyl
transferases found in the cytosol/soluble fraction.
Hydrozines and primary aromatic amines, OH and SH
may undergo acetylation. i.e
PHASE II REACTIONS: 3. ACETYLATION
 PHASE II REACTIONS: 3. ACETYLATION

The amount of acetyl transferases available for



acetylation varies btn species and btn individuals.

The human population can be divided into fast and slow


acetylators.
The slow acetylators are prone to toxicity where drugs
that are metabolized primarily through acetylation
slow acetylators are more likely to have PN with
isoniazid use.
Acetylated form has low soluble and may accumulate in


urine causing crystauria.

PHASE II REACTIONS

4. AMINO ACIDS CONJUGATION

 Also known as uric acid formation.
 Glycine conjugation occurs with aromatic acids
while glutamine conjugates to aryl acetic acid.
 ASA – is metabolized to uric acid.
PHASE II REACTIONS
 PHASE II REACTIONS

5. GLUTATHIONE CONJUGATION /
MERCAPTOPURIC ACID FORMATION
Aromatic hydrocarbons and halogenated aromatic
hydrocarbons undergo glutathione conjugation.
Glutathione is a tri-peptide consisting of cysteine,
glutamic acid and glycine and conjugates with the
substrates via the mercapturic (-SH) function of cysteine.
The enzyme involved is glutathione S-transferase and the
products are mercaturic acid derivatives.
PHASE II REACTIONS

5. GLUTATHIONE CONJUGATION /
MERCAPTOPURIC ACID FORMATION
PHASE II REACTIONS

6. METHYLATION i.e. O, N, S methylation.

S-adenosylmethionine (SAM) acts as a donor
molecule for transfer of a methyl group to the N of
amines, O of phenols or S of thiols. An important
methylation reaction is O-methylation of biogenic
catecholamines by catechol-O-methyl transferase
(COMT).
PHASE II REACTIONS

NB: Some drugs act as enzyme inhibitors e.g.

cimetidine, macrolides (erythromycin), ketoconazole
and phenytoin. Propanol, warfarin, phenytoin and
theophylline should not be given/administered
together with ketoconazole.
Theophylline and ciprofloxacin should not be


administered together. Disulfurum inhibit the enzyme

alcohol dehydrogenase in treatment of alcoholism.
PRO-DRUGS

 These are drugs whose metabolisable

functional groups have been blocked by larger
chemical groups to prevent the drug from
being metabolized prematurely hence
prolonging the duration of action.
 They are drugs which exist in inactive form by
only becoming active after undergoing
metabolism i.e becomes active in vivo.
PRO-DRUGS: Importance of pro- drugs

i. To overcome formation of pharmacokinetic problems

associated with the parent drug e.g. instability.
ii. Prolonging the duration of action
iii. To enhance activity some drugs are less active and
may be converted into more active forms or active
form.
iv. To prevent the drug from undergoing the 1st pass
effect hence increasing its duration of action.
v. To increase its bioavailability.
 PRO-DRUGS

E.g. bacamphillin, pivampicillin are pro-drugs of

amphicillin and are better absorbed than ampicillin. The
esters are hydrolysed to ampicillin in vivo.
General structure of penicillin
PRO-DRUGS

For ampicillin R1 – H

For Bacampicillin R1 –

For Pivampicilin R1
PRO-DRUGS

Anticancer drug cyclophosphamide is a pro drug which

is converted to the active form phosporamidate mustard
by CYP 450. The pro-drug is less reactive and more
chemically stable in the creative form. Giving the pro-
drug reduces the toxicity associated with the active
form also allows the drug to reach its site of action
before destruction.
PRO-DRUGS

1. Erythromycin is unstable in gastric acid. It may

therefore be prepared as the more acid stable estolase
of succinate ester drug. The esters are hydrolysed
after absorption.
2. Chloramphenicol and clindamycin may be
formulated as palmitates to improve the tastes of
liquid preparations. The sweet tasting palmic acid
moiety overcomes the impalatibility of the free drug.
The esters are then hydrolyzed to the parent
compound in vivo.
Characteristics of a pro-drug

a) It should be inactive.
b) Non toxic
c) Easily converted to the parent compound in vivo
d) Should be easily synthesized.
e) Once the pro-drug moiety is removed, the moiety
should be inactive i.e. it should be eliminated without
causing any effect. It thus should be inactive, non-
toxic and readily eliminated.
Aspirin Journey through the body - 3D Animation.mp4