Metabolism/

Biotransformation
 DMPK – What is it and Why study it?
Drug Metabolism
The chemical alteration of a drug by a biological system with the principal
purpose of eliminating it from the system.

Pharmacokinetics
The study of the movement of drugs within the body (What the body does to the drug).

Pharmacodynamics
The study of the pharmacological response to a drug (What the drug does to the body).

Why?
Compare drug candidates –need to understand how they behave in the body in order to have confidence that they will be safe and
efficaceous.

Understand how to improve the in vivo properties of candidates during the Lead
Optimisation process.
 Typical Plasma Concentration/Time Profiles
Plasma Plasma
conc Toxic conc Toxic
MTC MTC
Therapeutic
Therapeutic Cssmax

Cssmin
Duration
MEC MEC
Ineffective Ineffective

Time Time

Understanding the DMPK of compounds allows effective prediction of appropriate

doses to give safe, therapeutic concentrations

MTC - Maximum tolerated concentration

MEC - Minimum effective concentration
Css - Steady state concentration
 DMPK Processes & Terminology
Absorption Distribution Metabolism Excretion (ADME)
For a drug which is administered orally, a number of factors affect delivery to
the site of action:

Absorption: the process by which a drug moves from its site of administration
to the systemic circulation

Distribution: the reversible transfer of a drug to and from the systemic circulation

Metabolism: any chemical alteration of a drug by the living system to enhance

water solubility and hence excretion

Excretion (Elimination): the irreversible transfer of a drug from the systemic

circulation
Absorption Distribution
BLOOD TISSUES

Elimination
 Absorption
Factors affecting absorption:
Solubility
Acid stability
Permeability
MOUTH Metabolism – gut wall / first pass metabolism

Portal vein

pH ~1
STOMACH
Relative SA ~1
Liver BLOOD
pH ~ 7
Relative SA ~ 600 INTESTINE

Metabolism
Gut wall
 Intestinal Wall Structure
Epithelium

Central capillary network

Microvilli

Apical surface
Brush Border
Membrane

Epithelial
Cell (enterocyte) Basolateral surface

Intestinal wall epithelial cells have many finger-like projections

on their luminal surface called microvilli which form the brush
border membrane
 Absorption Mechanisms
 Transcellular absorption
– Main route for most oral drugs
– Drug must be in solution at cell surface
– pKa important - drug must be unionised
– Lipophilicity important - ideal log D 1-4
– H-bonds - solvation shell needs dispersing
– Lipinski’s ‘Rule of 5’

 Paracellular absorption
– Drug passes through gaps between cells H3N
+
O
-

– Inefficient – pores have << surface area than O

O O
cellular surface O
O P O
O
-

– Restricted to low MW hydrophilic molecules O

O

 Active Transport
– Drugs carried through membrane by a transporter –
requires energy
– Many transporters exist for nutrient molecules, eg
glucose, amino acids
– SAR specific – few drugs absorbed by this route
Phosphatidylserine
 Efflux Transporters - P-glycoprotein
A number of efflux transporters act as a barrier to prevent entry of toxic compounds
into the body
P-gp (P-glycoprotein) is the most well characterised transporter
ATP dependent efflux pump with broad substrate specificity.
170 kDa protein, dimeric structure connected by a linker peptide. Each half contains
6 transmembrane domains and an ATP binding site.
P-gp found in high levels at apical surface of enterocytes. CYP3A4 (metabolising
enzyme) also expressed - can reduce absorption through efflux/metabolism.
Co-administration of compounds which inhibit P-gp can lead to increased
bioavailability of drugs O

OH N
HO
OH O N
O O
O O N N N Cl
O
O OH O
N O O
O N O
ATP O O Cl
O Ketoconazole
OH
Antifungal
Verapamil Erythromycin P-gp Inhibitor
Ca channel blocker Macrolide antibiotic
P-gp substrate P-gp substrate/inhibitor
 Distribution
Distribution: the reversible transfer of a drug to and from the systemic circulation

Absorption Distribution
BLOOD TISSUES

Compounds can distribute out of plasma into tissues:

Main factors influencing distribution are pKa, lipophilicity, plasma protein binding
(only unbound tissue is free to distribute).

Tissue pH is slightly lower than plasma pH

 Basic compounds tend to distribute out of plasma into tissue more than acids.
 Plasma Protein Binding (PPB)
Rapid
Drug Drug Protein

Equilibrium
Free
Bound
Drugs can bind to macromolecules in the blood – known as plasma protein binding (PPB)
Only unbound compound is available for distribution into tissues
Acids bind to basic binding sites on albumin, bases bind to alpha-1 acid glycoprotein

0-50% bound = negligible

50-90% = moderate
90-99% = high
>99% = very high

For bases and neutrals, PPB is proportional to logD.

Acidic drugs tend to have higher PPB than neutral/basic drugs.
 Excretion (Elimination)

Absorption Distribution
BLOOD TISSUES

Elimination

Elimination: the irreversible transfer of a drug from the

systemic circulation

Major routes of elimination:

Metabolism

Renal excretion (for free drug, ie low logD)

Biliary excretion

Also lungs, sweat etc.

 Renal Excretion
Blood Nephron

Urine

1. All unbound drug in plasma is filtered

in the glomerulus. Only significant
for very polar compounds, log D < 0.

2. Some compounds are actively secreted

into urine along the proximal tubule.

3. Unionised drug can undergo passive reabsorption from

urine into blood along the length of the nephron (net excretion may be zero).

4. Drug that is bound to plasma proteins is not filtered.

 METABOLISM:
 Drug metabolism is the chemical alteration of a drug by
the body.
 Any chemical alteration of a drug by the living system to
enhance water solubility and hence excretability

 metabolism is what the body does to the drug,

Some drugs are chemically altered by the body
(metabolized). The substances that result from
metabolism (metabolites) may be inactive, or they may
be similar to or different from the original drug in
therapeutic activity or toxicity. Some drugs, called
prodrugs, are administered in an inactive form, which is
metabolized into an active form.
13
 The resulting metabolites produce the desired
therapeutic effects. Metabolites may be metabolized
further instead of being excreted from the body. The
subsequent metabolites are then excreted . The
termination of the drug effect is caused by bio
transformation and excretion .all the substance in the
circulatory system , including drugs ,metabolites ,and
nutrients will pass through the liver.

 A significant portion of the drug metabolised by hepatic

enzyme to inactive chemical.

14
 Metabolism
Definition: Any chemical alteration of a drug by the living system
Purpose: To enhance water solubility and hence excretability

Types of metabolism
– Phase I: production of a new chemical group on the molecule
– Phase II: addition of an endogenous ligand to the molecule
Sites of metabolism
– Main site of metabolism is the liver.
– Other sites include the gastrointestinal wall (CYP-450), kidneys,
blood etc.
Factors affecting metabolism
– The structure of a drug influences its physicochemical
properties.
(blocking/altering sites of metabolism can improve DMPK
properties)
– MW, LogP/LogD, pKa
– The more complex the structure, the more the potential sites for
metabolism.
 Types of metabolism
• ◦ Phase I: adding a new chemical group on the molecule
◦ Phase II: adding an endogenous ligand to the molecule
◦ Phase III: transporter assisted process
 Phase I Metabolism
O N O N
H H
OH OH

(i) Oxidation
OH
Aliphatic or aromatic hydroxylation Propranolol
(-blocker)
N-, or S-oxidation OH

N-, O-, S-dealkylation

N NH N NH

NH2 NH2

Debrisoquine
(anti-hypertensive)
(ii) Reduction
Nitro reduction to hydroxylamine/ amine H O H O
N N
Carbonyl reduction to alcohol
O2N N H2N N

(iii) Hydrolysis
Nitrazepam
Ester or amide to acid and alcohol or amine (hypnotic)
Hydrazides to acid and substituted hydrazine CO2H
CO2H

O OH

Aspirin
(Analgesic)
 Phase II Metabolism
O CHCl2
O CHCl2
(i) Glucuronidation HN
OH HN CO2H
O O
Carboxylic acid, alcohol, phenol, amine OH
HO
HO
OH
OH
O2N
O2N
(ii) Amino acids Chloramphenicol
Carboxylic acids (antibiotic)

(iii) Acetylation
Amines
O N O N
H H
OH OH
(iv) Sulfation
Alcohol, phenol, amine
OH O O
S
Prenalterol HO O
(v) Glutathione conjugation (gly-cys-glu) (-blocker)
Halo-cpds, epoxides, arene oxides, quinone-imine
 Cytochrome P450 Enzymes (CYP-
450)
2e-, 2H+
RH + O2 ROH + H2O
CYP-450

Many Phase I oxidations are mediated by cytochrome P450

enzymes.

Membrane bound proteins - found on the endoplasmic

reticulum.

Heme-containing proteins – porphyrin ring co-ordinating iron

at the active site.

Many iso-forms with different substrate specificities:

Major human CYP’s: 1A2, 2C9, 2C19, 2D6, 3A4

CYP inhibition/induction: issues in exposure + drug-drug

interactions. N ON
N N
Fe
N
Fe .
N
+
N N

HO2C S CO2H HO2C S CO2H

Cys Cys

Iron(III) porphyrin Active oxygen Fe (IV) species

 Sites of metabolism
• Main site of metabolism is the liver.
Other sites include the gastrointestinal wall, kidneys, blood,
etc.
Chemical factors affecting
biotransformation

• ◦ Chemical structure of the drug

*Functional groups that are prone to
metabolism
**Soft spots
*Secondary reactions
**Hydroxyamine
**Aldehyde (alcohol oxidation)
**Chloride (dehalogenation)
◦ The more complex the structure, the more the
potential sites for metabolism.
 Phase 1 Transformation
• ◦ Polar functional groups are introduced into drug molecules
◦ Lipophilic molecules are transformed into more polar molecules by
introducing or exposing polar functional groups.
--For example, 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
--Toxic metabolites
--Active metabolites of prodrugs
◦ Drugs can be subjected to several Phase I pathways
◦ These reactions create functional groups that place the drugs in a correct
chemical state to be acted upon by Phase II conjugative mechanisms
◦ Main function of phase I reactions is to prepare chemicals for phase II
metabolism and subsequent excretion
◦ Phase II is the true "detoxification" step in the metabolism process.
Types of Phase I reactions

• ◦ Oxidation
--Oxidation involving CYP450
--Oxidation involving other enzymes
◦ Reduction
◦ Hydrolysis
◦ Hydration
◦ Dethioacetylation
◦ Isomerisation
 Oxidation
• is the addition of O or removal of H
◦ Normally the first and most common step involved in the drug
metabolism
◦ Majority of oxidation occurs in the liver and it is possible to occur in
intestinal mucosa, lungs and kidney.
◦ Most important 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.
◦ These oxidation metabolites are more polar than their parent
compounds and might undergo further metabolism by phase II
pathways
 Oxidative enzymes
• ◦ Cytochrome P450s
• A hemeprotein containing an iron atom
• Ferriprotoporphyrin-9 (F-9)
* Core of the CYP450s
* Iron is anchored by 4 nitrogen atoms and 1
sulfur atom from cystein
* An electron acceptor
--Ferric (Fe+++) to ferrous (Fe++)
CYP450 catalyzed oxidation

• Inserting an O to the drug molecule

RH+O2-->ROH+H2O

• Epoxidation (Oxidation by CYP450)

• ◦ Epoxide is typically not stable, possibly the first

step of hydroxylation
-result is one or two -OH groups bound to ring
Aromatic hydroxylation (Oxidation by
CYP450)

• addition of -OH to aromatic ring

-Common to drugs with aromatic rings
-occurs at various positions on rings
-The more ELECTRON RICH aromatic ring is
preferentially hydroxylated (ring with EDG,
NOT EWG like Cl)
Hydroxylation (oxidation) of non-
aromatic double bonds (Oxidation by
CYP450)
• E.g. Carbamazepine has larger ring in
between two aromatic rings that
undergoes epoxidation, resulting in 2 -OH
groups
Hydroxylation of benzylic carbon
(Oxidation by CYP450)
• ◦ Benzylic carbon - carbon attached to aromatic
ring
--Hydroxylated to carbinol (CH2OH)
--Carbinol further oxidized to aldehyde and
carboxylic acid (primary alcohol), or ketone (2nd
alcohol)
Hydroxylation of allylic carbon
(Oxidation by CYP450)
• ◦ Allylic carbon - carbon attached to a double bond

• Diazepam metabolism (Oxidation by CYP450)

• -6-hydroxyl
-demethylated
Aliphatic hydroxylation (Oxidation
by CYP450)
• ◦ Common to drugs with aliphatic side chains (C
chains)-->Generate higher alcohols
◦ Cyclohexyl ring hydroxylation-->Typically at 4
position
◦ Formation of a carbonyl group at the α- carbon
to a heteroatom
Dealkylation-demthylation (Oxidation
by CYP450)
• ◦ Drugs containing a 2 prime or 3 prime amine,
an alkoxy or an alkyl substituted thiol.
◦ Prepare the parent drug for Phase II
conjugation by exposing the heteroatoms
◦ A two-step reaction
--Attach hydroxyl group to the α-carbon
--Removal of aldehyde
N-demthylation (Oxidation by
CYP450)
• ◦ A two-step reaction
--Attach hydroxyl group to the α-carbon
--Removal of aldehyde
--Result: -NH
• O-and S-demthylation (Oxidation by CYP450)
• ◦ A two-step reaction
--Attach hydroxyl group to the α-carbon
--Removal of aldehyde
--Result: -OH or -SH
Oxidative deamination (removal of NH2
group) (Oxidation by CYP450)
• ◦ Converts primary amines to ketone
◦ Two-step reaction
--Attach hydroxyl group to the α-carbon
--Removal of ammonia (NH3)
• Demthylation and deamination in a sequence
(Oxidation by CYP450)
◦ Demethylation typically occurs before
deamination
N-oxidation (Oxidation by CYP450)

• ◦ Amines, amides, imines, hydrazines and

heterocyclic compounds
◦ Form N-oxide with tertiary (3 prime) amine
◦ Form hydroxyamine (RNHOH) with 1o and 2o
amines
◦ Hydroxyamine is not stable-->result is Nitro
(RO-N+=O)
◦ There are some stable hydroxyamines e.g. N-
Hydroxy Lidocaine
S-oxidation (Oxidation by CYP450)

• ◦ Form Sulfoxides (S=O)

◦ Sulfoxides can be further oxidized to sulfone
(O=S=O)

• Dehalogenation (Oxidation by CYP450)

• ◦ Remove of halogen atoms to yield
corresponding
alcohol or acid
Other microsomal enzymes
involved in Phase 1 metabolism
• ◦ Flavin monooxygenase (FMO)
◦ Monoamine oxidase (MAO)
◦ Alcohol dehydrogenase (ADH)
◦ Aldehyde dehydrogenase
◦ Xanthine oxidase
 Monoamine oxidase (MAO)
• ◦ Located on the outer membrane of mitochondria in all tissues and exists as MAO-A and
MAO-B.
--Equal amounts are found in the liver
--the brain contains primarily MAO-B
--MAO-A is found in the adrenergic nerve endings
◦ Catalyze oxidative deamination of endogenous biogenic amines (catecholamines) as well
as xenobiotic amines (1o and 2o amines)
--MAO-A shows preference for serotonin, catecholamines, monoamines with phenolic
aromatic rings
--MAO-B prefers non-phenolic amines
◦ Food interactions
--Substrate found in foods (tyramine)
--Causes drug/food interaction
◦ MAO inhibitors
--Selegiline, moclobemide (reversible)
--Drug-drug interactions
*SSRI, tricyclic antidepresssant
*Meperidine, tramadol
*dextromethorphan
Oxidation catalyzed by MAOs
• ◦ Remove amine group and form an aldehyde (1o amine)
or a ketone (2o amine)
• Reduction
– ◦ Removal of oxygen or addition of hydrogen
--Reduction of aldehydes and ketones, reduction of
nitro and azo compounds.
◦ Less common than oxidation, but the aim is the 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.
Reduction of azo and nitro groups
(Phase I)
• azo (RN=NR)-->NH2
nitro (NO2)-->NH2
• Reduction of heterocyclic rings and halogenated
hydrocarbons (Phase I)
• Ring connected by O-->no ring with OH
Remove H from CF3CClBrH-->F2C=CBrCl
 Hydrolysis
• ◦ It is the reaction between a compound and water. The
addition of water across a bond also gives more polar
metabolites.
--Hydrolysis of Esters and Ethers
--Hydrolysis of Amides.
--Hydrolytic cleavage of non aromatic heterocycles
--Hydrolytic Dehalogination
--Miscellaneous hydrolytic reactions.
◦ Different enzymes catalyze the hydrolysis of drugs
--Esterase enzymes
--Amidase enzymes
Ester hydrolysis (Phase 1)

• Ester (ROC=O)
◦ Procaine hydrolysis by plasma esterase (non-
specific)
*Ester hydrolysis is more rapid than amide
hydrolysis
• Amide hydrolysis (Phase I)
• Amide (HNC=O)
◦ Monoethylglycylxylidide (an active
metabolite of
lidocaine) hydrolyzed by liver amidase
(specific)
 Phase 2
• ◦ When phase I reactions are not producing sufficiently
hydrophilic (water soluble) or inactive metabolites to be
eliminated from the body, the drugs or metabolites
formed from phase I reaction undergoes phase II
reactions.
◦ Generally phase I reactions provide a functional group
or handle in the molecule that can undergo phase II
reactions. Thus, phase II reactions are those in which the
functional groups of the original drug (or metabolite
formed in a phase I reaction) are masked by a
conjugation reaction.
Phase 2 conjugation reactions
• ◦ Produce more polar and water soluble metabolites
for elimination
--More soluble to stay in urine and minimize re-
absorption
◦ Produce larger and heavier metabolites to be
eliminated in bile and feces
◦ Terminate pharmacological action
--Conjugated metabolites cannot bind to target
--Many conjugated metabolites cannot cross cell
membranes due to high polarity
---Methylated and acetylated metabolites are exception.
They are usually pharmacologically inactive.
Conjugation reactions
• ◦ Attaching small, polar, and ionizable endogenous
molecules such as glucuronic acid , sulfate, glycine,
glutamine and glutathione to the phase I metabolites or
parent drugs
--Glucuronidation: adding glucuronic acids
--Sulfation: adding sulfate groups
--Amino acid conjugation
--Glutathione conjugation
--Methylation
--Acetylation
--Cholesterol conjugation
--Fatty acid conjugation
Glucuronidation (Phase 2)
• ◦ Add glucuronic acid to the parent drug or Phase I metabolites
and form glucuronide
--Dugar molecule attaches through the -OH on the anomeric
carbon
◦ Catalyzed by UDP (uridine diphosphate)
glucuronosyltransferases (UTGs)
--Subfamilies UTG1A and UTG2B
--Found in many tissues, but the greatest concentration in liver
◦ Formation of O-glucuronides
--Phenol, alcohol, hydroxyamine, aryl acid, aryl alkyl acid
◦ Formation of N-glucuronides
--Aryl amine, alkyl amine, amide
◦ Formation of S-glucuronides
◦ Formation of C-glucuronides (rare)
Sulfation (Phase 2)
• ◦ Addition of sulfate groups (SO4-)
--Less frequently than glucuronidation
◦ Catalyzed by sulfotransferases (SULTs)
--Subfamilies SULT1 and SULT2
◦ Attach to phenol group. Phenolic sulfating
dominates
*Exception: Paracetamol: SO3H adds to N on
phenol; Minoxidil: SO4 adds to NO
Methylation (Phase 2)
• ◦ Addition of a methyl group
◦ Increases drug lipophilicity, but is responsible
for
DNA regulation
◦ S-adenosyl methionine (SAM) is the carrier of
the methyl group
◦ Catalyzed by methyltransferases
◦ N-methylation: amines
◦ O-methylation
◦ S-methylation
Acetylation (Phase 2)
• ◦ Addition of an acetyl group (CH3C=O)
◦ Increases lipophilicity and decreases water
solubility
◦ Regulates cell homeostasis (e.g., NDA
transcription and hormones)
◦ Catalyzed by acetyl transferase
◦ Uses acetyl Co enzyme A as a co-factor
◦ Amines, aromatic amines, alcohols and thiols
Glutathione conjugation (Phase
2)
• ◦ Glutathione (GSH)
--A three AA tripeptide with a thiol group
--Found in virtually all mamalien cells
--Thiols are very reactive
--De-activated by forming a dimer (GSSG)
through a disulfide linkage
Glutathione S-transferases (GSTs)
• ◦ Catalyze glutathione conjugation
◦ Exist as homodimers (most commonly)
--H site (C-terminus) is the binding site
--G site (N-terminus) binds GSH
◦ SGT classes
--Alpha class
--Mu class
--Pi class
--Theta class
--Omega class
Glutathione conjugation
reactions
• ◦ Most complicated chemically
--Rearrangement
--Further metabolism of the conjugate
◦ Common to epoxides, haloalkanes,
nitroalkanes,
alkenes, and aromatic halo- and nitro-
compounds
Conjugation with alkenes (Phase
2)
• Michael addition-->addition of Glutathione to a
C=C

• Conjugates further metabolized

• ◦ Produce a cysteine conjugate-->cysteine AA
with R group CH2S is acetylated at the NH
3 Phase 2 reactions that INCREASE
polarity:
• 1. Glucuronidation
2. Sulfation
3. Glutathione conjugation
• 2 Phase 2 reactions that DECREASE
polarity:
• 1. Methylation
2. Acetylation
Stereo-selective metabolism
• ◦ Inversion of enantiomers (R-->S and vice
versa)
◦Different metabolism of enatiomers (R
and S undergo different metabolism
processes)
◦ Warfarin enantiomers=has R and S, but S
i metabolized faster than R
Stereoselectivity in Phase II
metabolism
• ◦ Propranolol is supplied as a racemic mixture
◦ Metabolized via glucuronidation
◦ UGT1A9 favors (S)-isomer and UGT1A10 favors
(R)- isomer
◦ Albuterol is supplied as a racemic mixture
◦ Metabolized through sulfation particulary after
oral dosing
--Greater amount of unmetabolized S-isomer
excreted in urine than the R-isomer
Phase I and II biotransformation is
not mutually exclusive
• ◦ Parent drugs can be metabolized by either
Phase I or Phase II reactions
◦ Phase I metabolites can be transformed by
Phase II conjugation
• Phase I, II and II biotransformation works in
• a concerted fashion.
 Case study: beta agonists
 ß-agonists (e.g. salbutamol) are used to control asthma
by causing activation of the ß2 receptors in the lung. This
causes the airways to dilate.
 These compounds are taken by inhalation, so most of the
drug stays in the lung.
 If the patient takes too much medicine,
the levels in the systemic circulation rise
and can now affect the ß2 receptors in
the heart causing palpitations.
OH
H
N

HO
salbutamol
HO
Formation of reactive metabolites
 We don’t want chemically reactive medicines!
What functional groups might we want to avoid?
e.g. R
O H O
N O
R Cl R Cl
R

 These are all electrophiles, which means that they

can covalently bind to nucleophiles in the body, e.g.
in proteins and DNA which lead to toxic effects.
 Most common effects are hepatotoxicity (liver) &
genotoxicity (DNA).
 But don’t forget that in the body, chemicals are
metabolised so we need to consider the fate of our
new medicine – will any of the metabolites be
chemically reactive?
 Some unwanted groups!
OMe
H
N
Cl N O
O O
N HO
CN
O
Cl
N OH

S CH2Cl
S

NH2
N
O H
NH2 O

N NH
O2N N
O
Cl
S N N
O
O O O
 Some unwanted groups!
OMe
Aziridines H Michael acceptors
N
Cl N Certain O
O Electrophilic esters
Electophilic phenols O
N HO
aromatics CN Mono-substituted
O
Cl furans and
N Disulphides OH thiophenes
Chloroamines
S CH2Cl
S Alkylhalides
Terminal NH2 Hydrazines
N
acetylenes O H
NH2 O
Epoxides Anilines Masked anilines
N NH
O2N N
Nitro O Azo Cl
S N N Acylating agents
O
Alkylsuphohonate O O O
esters Isocyanates
 Case study: paracetamol H
normal phase II H N O
H metabolism N O
N O
O O and Glucuronide
S O
O O
HO
paracetamol

phase 1 oxidation
H
N O
Urinary
reaction with
N O glutathione excretion
HO
S
O Glutathione

N-acetyl-4-benzoquinone imine

reaction with protein

Toxic effect
H
N O

HO
S
Protein
 Avoiding the problem
 Most obviously, avoid functional groups known to show
reactive metabolites (not an absolute – some are worse
than others).
SH
 Test for the presence of reactive groups O
H
N
 Look for binding to proteins or O N
H
O
glutathione - detect by mass HO HO O

spectroscopy NH 2
glutathione
 ‘Ames’ test to detect mutagenicity
 Use a genetically modified bacterium which cannot
grow in the absence of histidine.
 Expose bacteria to chemical.
 If the chemical can cause mutations, the genetic
modification can be reversed and the bacteria will grow.
 Can also be carried out in the presence of liver
enzymes to look for mutagenic metabolites.
 Factors affecting biotransformation of drugs

1. Physicochemical properties of the drug

2. Chemical factors
(a).induction of drug metabolising enzymes
(b).inhibition of drug metabolising enzymes
(c).environmental chemicals
3.Biological factors (a).species differences (b).strain
differences (c).sex differences d).age (e).diet
(f).altered physiological factors i).pregnanacy
ii).hormonal imbalance iii).disease states
(g).temporal factors i).circadian rhythm
ii).circannual rhythm
 Factors affecting biotransformation of drugs
1).Physicochemical properties of the drug
 Molecular size and shape
 pKa
 Acidity/basicity
 Lipophilicity
 Steric and electronic characteristics
2).Chemical Factors
 a).induction of drug metabolising enzymes Increased drug
metabolising ability of the enzymes by drugs and chemicals is
called as enzyme induction and the agents which brings about
such an effect known as enzyme inducers.
Two categories of inducers: 1)Phenobarbital type eg:-several
drugs & pesticides 2)Polycylic hydrocarbon type eg:- 3-methyl
cholanthrene & cigarette smoke Auto-induction or self-induction
eg:- carbamazepine, meprobamate, cyclophosphamide,
rifampicin
 Factors affecting biotransformation of drugs

b).inhibition of drug metabolising enzymes

 Decrease in the drug metabolising ability of an enzyme is called
as enzyme inhibition.
 Direct or indirect inhibition
 Direct inhibition :3 mechanisms 1).competitive inhibition 2).non-
competitive inhibition 3).product inhibition Eg:- xanthine oxidase
inhibitors(allopurinol) & MAO inhibitors(phenelzine)
 Indirect inhibition: 2 mechanisms 1).repression eg:-ethionine,
puromycin, actinomycin D, CCl4, carbon disulphide, disulfiram
2).altered physiology
c).environmental chemicals
 Halogenated pesticides & polycyclic aromatic hydrocarbons
 Organophosphate insecticides & heavy metals
 Temperature, altitude, pressure, atmosphere etc..
 Factors affecting biotransformation of drugs
3).Biological factors
a).species difference Difference in both phase I and phase II
reactions. Phase I:eg; metabolism of amphetamine and
ephedrine Phase II: eg; phenol
b).strain difference/pharmacogenetics A study of inter-subject
variability in drug response is called as pharmacogenetics. The
inter-subject variations in drug biotransformation may either be
monogenically or polygenically controlled Ethnic variations and
discontinuous variations.
c).sex difference Regulation by sex hormones and generally
observed following puberty.
d).age In neonates: microsomal enzyme system not fully
developed.eg: caffiene  In infants: same as neonates In
children: metabolise certain drugs much more rapidly than
adults In elderly persons: liver size reduced-microsomal enzyme
activity is decreased and hepatic blood flow also declines
 Factors affecting biotransformation of drugs
. e).diet: The enzyme content and activity is altered by a number
of dietary components: Low protein diet and high protein
Protein-carbohydrate ratio Fat free diet Dietary deficiency of
vitamins and minerals Grapefruit Starvation Malnutrition in
women Alcohol ingestion

f).altered physiological factors i. Pregnancy: maternal drug

metabolising ability is reduced-due to high levels of steroid
hormones ii. Hormonal imbalance: higher level of one hormone
may inhibit the activity of few enzymes while inducing that of
others iii. Disease states: enhanced half-life of almost all drugs

g).temporal factors i. Circadian rhythm: diurnal variations or

variations in the enzyme activity with light cycle are called as
circadian rhythm.
 Factors affecting biotransformation of drugs
METHODS FOR THE STUDY OFDRUG BIOTRANSFORMATION
In vitro methods Enzyme sources are human derived systems.
Consists of: liver microsomes hepatocytes and cell lines liver slices
individually cDNA-expressed enzymes

In vivo methods Requires the collection and analysis of

appropriate biological samples. Samples include urine, faeces,
expired air, blood/plasma, bile, milk, saliva, synovial fluid and
tissues.

 Samples divided into 2 groups: 1).those requiring complete

collection eg: urine and faeces 2).those collected at regular
intervals eg: blood Two approaches to identification of
metabolites in the collected samples: 1).identification of
metabolites in the biological samples when reference compounds
are available 2).isolation and identification of all major
metabolites