Drug metabolism

©Dr. Rupika Delgoda

Natural Products Institute
Faculty of Pure and Applied Sciences
University of the West Indies

Phone: 9702574
Email:
thejani.delgoda@uwimona.edu.jm
 4 lecture course outline

• 1) Principles of metabolism (ADME,

functions, biological significance and
consequences of metabolism)

• 2) Major drug enzymes (phase I&II)

• 3) Factors that influence drug metabolism

• 4) Linkages with drug interactions and

disease
 Structures of drugs
.

Codiene Fluoxetine Tamoxifen:

(Prodrug) (Prozac) Prodrug.
Metabolites: Metabolite: Metabolites:
morphine and norfluoxetine (N- 4- hydroxytamoxifen
codeine-6- demethylated and N-desmethyl-4-
glucuronide fluoxetine hydroxytamoxifen
Natural Products Institute, UWI
Functions of drug metabolism

Typically biphasic :
• Phase I: Functionalisation (introduces a
functional group, such as –OH, -NH2, -SH via
Oxidation, reduction or hydrolysis)
• Phase II: Conjugation (glucoronidation,
sulphation, methylation and acetylation)
What are the significances of these two phases ?
How are they achieved ?
Drug metabolising enzymes (DMEs)
Xenobiotic
Phase I Cytochrome P450s (CYPs)
enzymes: Flavin monooxygenases (FMOs)

Reactive intermediate
Phase II Glutathione S-transferases (GSTs)
Arylamine N-acetyltransferases (NATs)
enzymes: UDP-Glucuronosyltranferases (UGPGTs)
Sulphotransferases (STs)
Epoxide hydrolase

Inactive product Reactive product

Subcellular distribution of DMEs

• Mitochondria : amino acyl transferases

• Endoplasmic reticulum:
Cytochrome P450s (CYP450)
Epoxide hydrolase
Esterases
UDPGT

• Cytoplasm : GST
: ST
: NAT
: Methyltransferases
Biological consequences of metabolism

• Stable inactive products, easily excreted

• Stable products not very different from
parent may act as receptors for further
activity
• Reactive metabolites may bind covalently
with tissues which would result in
– Drug action (e.g.anticancer drugs)
– Drug toxicity (Damage to liver, other organs)
– DNA damage (mutations, thus cancers etc)
 Drug metabolism
XENOBIOTIC
BIOTRANSFORMATION
METABOLISM

Drug
Metabolizing FURTHER
Enzymes BIOACTIVITY

Inactive metabolite
Reactive metabolite Active metabolite
EXCRETION
Pharmacology

DNA/RNA Protein/lipid
Mutagenesis Cytotxicity/Immunotoxicity
Natural Products Institute, UWI
 Terminology
• Xenobiotic:
An exogenous chemical : To have an impact (therapeutic or toxic)
the xenobiotic should be in sufficient quantities at the site of action.
• Pharmacodynamics (PD) and Pharmacokinetics (PK) :
•Pharmacodynamics is often summarized as the study of what a
drug does to the body, whereas pharmacokinetics is the study of
what the body does to a drug
PD: is the study of the physiological effects of drugs on the body and
the mechanisms of drug action and the relationship between drug
concentration and effect.
PK: Pharmacokinetics includes the study of the mechanisms of
absorption and distribution of an administered drug, the rate at
which a drug action begins and the duration of the effect, the
chemical changes of the substance in the body (e.g. by enzymes) and
the effects and routes of excretion of the metabolites of the drug.[1]
CYTOCHROME P450
ENZYMES
Importance of CYP450 enzymes

Metabolism of Major enzyme

New drug entities in the liver
By CYP450 is
Required by FDA

CYP450 enzymes

Metabolises
Very important Over 90%
In pharma Of known
industry drugs
 Background – Chemistry
• Oxidase reaction: oxygen is reduced, but not incorporated
into another molecule. E.g., cytochrome oxidase [citric cycle]:
4e- + 4H+ + O2 2H2O
reverse reaction observed in photosynthesis

• Oxygenase reaction: one or both oxygen atoms of O2 are

incorporated into a substrate molecule.

OH

CO18OH
+ 18
O2
CO18OH

OH

E.g ., pyrocatechase is a dioxygenase [catechol transformed into

cis,cis muconate]
 Cytochrome P450s are
monooxygenases

C
H
+ O2 + NADH + H+ C OH + H2O + NAD+

• One oxygen atom of O2 is incorporated into the substrate:

• Substrates include endogenous compounds like steroids,
but also drugs, pesticides, carcinogens, etc.
Primary purposes of CYPs
CYP isoforms have evolved to :

– Make a molecule less lipophilic as

rapidly as possible

– Make some molecules more vulnerable

to Phase II conjugation.
 Reactions catalyzed by P450s
Aromatic hydroxylation
OH
Aliphatic hydroxylation
O O
[O]
R [O] OH
CH3
R
H3C HN H3C HN

Deamination N-Oxidation
HO O O
R R CH3 R CH3
CH3 [O] CH3 [O] CH3
R R N [O] N

NH2 NH2 [NH3] CH3 [H+] CH3

N-dealkylation Sulfoxidation
R R
R
R' [O] R'
CH3 [O] S S
R N
N [O] R NH2
OH [H+]
H [CH2O] O
H

O-dealkylation
CH3

R O [O] R O [O]
R OH
OH
[CH2O]
Cytochrome P450s are heme-thiolate proteins that show maximal
light absorption at 450 nm when reduced & complexed in vitro with
exogenous carbon monoxide.

P450s contain a single

Heme group, that plays H
O
H

a major role in activating

molecular oxygen.
In the native state, water HS

coordinates the Fe atom Cys

P450 protein
Characteristic UV-
Vis signature; Can
be used to observe
spin state changes
as a result of Metoprolol and 2D6

ligand binding
0.1 Conc (uM)
0.6 200 uM 2.7
0.08
500 uM 5.3
0.5 0.06
800 uM 9.1
1500 uM
0.04

Absorbance (a.u.)
12.8
0.4 1200 uM 0.02
25.5
Absorbanc

2000 uM 0
-0.02340 390 440 490 50.3
0.3
e

0 uM
-0.04 86.4
0.2 -0.06 132.6
-0.08 369.5
0.1 -0.1 1048
-0.12 2276.6
0 4761.6
300 400 500 600 700 wavelength (nm)
wavelength nm
 Nomenclature
• Grouped together into families and subfamilies that share
sequence identity (amino acid homology)
• Those sharing >---% identity is classed into a family
(denoted by a numerical) and those with >---% identity are
further classified into a subfamily (denoted by a roman
numerical) and each member given a number.
• E.g: CYPs 1A1, 1A2, 1B1
• CYP 2B1
• CYPs 2C9, 2C19
• CYP 2D6
• CYP 2E1
• CYPs 3A4, 3A5
• There are many other P450s but the above are the most
significant for metabolism of xenobiotics.
 P450s in Drug Metabolism
•P450s are responsible for inactivating most drugs

•Main isozymes responsible for drug metabolism in humans:

CYPs 3A4 >> 2D6 > 2C9, 2C19 >> 1A2, 2E1

•Typical substrates for the CYPs are:

•1A2: methyl-xanthines(caffeine, theophylline)
•2C9: non-steroidal anti-inflamatoryagents (ibuprofen,
diclofenac)
•2C19: S-mephenytoin(as marker)
•2D6: beta-blockers, anti-arrhythmics,
tricyclicantidepressants
•2E1: volatile anaesthetics
•3A4: wide range of substrates
Human Liver P450 Isoforms Expression

• Over 17 cytochromeP450 isoforms have been identified to date, and

known to be expressed in the liver.
• The major isoforms responsible for drug metabolism are presented

1A2, 12.7

2A6, 4 Other , 28
2B6, 0.2

2C, 18.2

2D6, 1.5
2E1, 6.6
3A, 28.8
 The participation of hepatic P450
isoforms in xenobiotic metabolism

• 1/3 of all drugs are

metabolized by CYP3A4
• 1/5 of all drugs are
metabolized by CYP2D6.
• This increases the odds of
drug-drug interactions: When
metabolized by the same
isoform, only one of two drugs
administered at the same time
can occupy the binding site (can
cause toxicity, side and
prolonged effects)
 CYP1 family
• CYP1A1: metabolism of a number of polycyclic aromatic hydrocarbons (PAH)
such as benzo(a)pyrene, as well as DES,2 and 4-OH estradiols (catechol-
estrogens) which are oxidized to their quinoneform. Mostly extrahepatic(small
intestine, placenta, lung, skin). Induced in the liver by cigarette smoke or food
(egBrusselsprouts’ indole-3-carbinol). •Quinones, if not destroyed, produce DNA
damage .
• Inter-individualvariations in the induction of CYP1A1, further related to genetic
differences in the aromatic hormone receptor (Ah) levels, explain the differences
in the individual susceptibility to cigarette-induced cancer –since the induction is
initiated after binding of aromatic hydrocarbons to Ah.
• CYP1A2 : metabolises 4 aminobiphenyl. Induced by smoking, grilled meat,
brocolli. metabolizes arylamines, nitrosamines, aromatic hydrocarbons. It is
expressed mostly in liver, stomach and intestine; also induced by smoking.
• Activates aflatoxinB1 to carcinogenicity.
• Also related to gastric carcinogenesis
• CYP1B1: Overexpressed in tumour tissues such as those in the prostate.
 CYP2C family
• CYP2C: large and important family
– 2C8 metabolises retinoic acid
– 2C9 metabolises tolbutamide, warfarin
– 2C19: metabolises phenytoin
• Found primarily in the liver and the intestine
• Genetic differences in CYP2C mark “poor metabolizers”
• This can lead to variations in drug metabolism in Asians
(<30%), African-Americans (<20%), e.g., decreased
clearance for S-warfarin can cause bleeding
 CYP2D and 2E familites
• CYP2D6: metabolises debrisoquine, spartein,
codiene, polymorphic (more later)
– Risk of lung cancer, Parkinson’s disease
• CYP2E: ethanol inducibile.
– Activates carcinogens
 CYP3A

• Major form in the liver

• Metabolises the large amount of known
drugs; many structural variants.
• Eg.erythromycin, testosterone, nifedipine,
dextromethorphan
 CYP450 table of substrates,
inhibitors and inducers
• You will be handed a list of drugs that are metabolized by a specific
cytochrome P450 isoform and also those known to be inhibitors and
inducers
•
Flockhart DA. Drug Interactions: Cytochrome P450 Drug Interaction Table.
Indiana University School of Medicine (2007).
http://medicine.iupui.edu/flockhart/table.htm. Accessed [date].
• Cytochrome P450 Drug Interaction Table
• www.drug-interactions.com
Adverse Drug Reactions (ADRs)

ADR: any noxious, unintended and

undesired effect of a drug at doses
used in humans for prophylaxis,
diagnosis and therapy

• > 2 million injuries in hospitals

• 100, 000 deaths per year
• Fourth leading cause of death in the U.S.
• Increases exponentially for >4 drugs
• Elderly at most risk
References: Lazarou et al, 1998 , JAMA, 1200-5
Delgoda and Westlake, 2004, Toxicol. Rev, 23(4), 239-249.
Phase II enzymes
Arylamine N-
acetyltransferase, NAT

C CH 3
NH 2
N
ACoA CoA H
 Substrates of human NAT1 and NAT2

COOH
Substrates of human NAT1
COOH
OH
NAT
NH 2 NH 2 substrates
p-aminobenzoic acid p-aminosalicylic acid

Substrates of human NAT2

O NH
NH 2 NH-NH 2

N
N N

Isoniazid Hydralazine NH2

NH2

O NH CH2CH2N(CH2CH3)2

2- napthylamine 2-aminofluorene
N Procainamide
Me
O N
H2 N S NH
O N NH2
Me H 2N NH2

Sulphamethazine
Benzidine 4-aminobiphenyl
O
H2 N S NH 2
O
Figure 1.13. Some carcinogens subject to NATmetabolism
Dapsone
 Association with diseases
• Exposure to certain arylamines have had increased
likelihood of developing cancers. The most clear
link established has been that of NAT2 acetylator
status and occupational bladder cancer. NAT2
slow acetylator phenotype, who are exposed to
environmental carcinogens (work or smoking) are
at a significantly higher risk of developing bladder
cancer.
• The carcinogenesis process often involves CYP
enzymes too (1A2, 1A1, 2E1).
Glutathione S-transferases (GST)
• These catalyse the conjugation of glutathione with a range
of electrophilic compounds including alkyl, arylhalides,
lactones, epoxides, esters and activated alkenes. The
parent compound is generally detoxified.
 UDPGT
Nucleophile
COOH
O
HO
HO O
OH
HN
O O-
- O N
O P O P O CH2
O
O O

OH OH

UDP-glucuronosyl transferase

HN
- -
O O
COOH
HO
O
+
Nucleophile
-
O P O P O CH2
O
O
N
HO O O
OH

OH OH
Natural Products Institute
• Catalyses the glucuronidation of a variety of
endogenous substrates, including hormones,
bilirubin, fat-soluble vitamins and xenobiotics
(paracetamol, chloramphenicol, morphine).
Factors affecting drug metabolism

-Genetic polymorphisms
- Other drugs
- Age
- Diet
- Smoking
- Gender
- Ethanol
- Disease
 Genetic variations

• Polymorphisms: a region of the genome that varies between

individual members of a population with significant
frequency (usually at least 1% prevalence).

Pharmacogenetics :
– The influence of heredity on the response to drugs and
their fate in the body; aims to understand inter-individual
variations to drug response
– The use of genotyping to individualise drug therapy
 CYP polymorphisms

• In 1977 adverse response to debrisoquine and

spartein lead clinicians to identify CYP2D6 and
the presence of polymorphisms in CYP enzymes.
A simple test using a probe drug can identify the
polymorphism in a person.
• SNPs, 2 nucleotide or even complete deletions
• Poor metabolisers can have up to 10 times plasma
levels of those of the extensive metabolisers.
• These variations are found to have different
distributions in different ethnicities.
 Summary of ethnic variability
CYP BLACK WHITE ASIAN Ethiopian/
Saudi Arabian

PM UM PM UM PM UM PM UM
Prevalence(%)

2D6 0-20 2 5-10 1-10 1 0-2 1.8-2 10-29

2C9 ND ND 0.2-1 NI 2-3 ND ND ND

2C19 1-5 ND 2-4 NI 10-25 ND 2 ND

Consequently: S-mephenytoin and PM-Poor Metabolizer
Diazepam given in lower doses to Oriental UM-Ultra-Rapid Extensive
and Black communities than Caucasians Metabolizer
ND- Not determined
(CYP2C19 mediated) NI –Not identified
 Clinical implications
• Failure to clear (by PMs) betablockers such as timolol,
propanolol, pindolol and metoprolol can lead to
complications suchas as bronchosplasm, heart problems,
headaches, nightmares, depression etc.
• Schizophremic patients with CYP2D6 polymorphisms are
prone to side effects to the point that the drugs for the
disease cannot be tolerated.
• Codeine, a prodrug is less effective in CYP2D6 poor
metabolisers, whose release into morphine (O-
demethylation) is less effective.
 Polymorphisms in Phase II
enzymes
• First found case of pharmacogenetics in 1950s,
where the rate of clearance of the drug isoniazid
(treatment for TB) in a healthy population could
be divided into 2 nodes of frequency distribution
instead of a bell shaped normal distribution.
Multiple forms of NAT2 gene was found. Fast
and slow aceylators were identified.
• Five forms of GSTs have been classified as
α(GSTA),µ (GSTM), π (GSTP), σ (GSTS), θ
(GSTT), with each family sharing atleast 50%
sequence identity: Polymorpshisms linked with
susceptibility to cancer.
Drug interactions: Impact of a
second drug

• Pharmacodynamics
• Pharmacokinetics
ADME
Impact of a second xenobiotic

Can influences metabolism in two ways:

1. Inhibition of drug metabolising enzymes

2. Induction of drug metabolising enzymes

 Metabolic interference
Enzyme Induction

Drug 1

Metabolism Drug 2 or
Natural Product

Soluble product
 Metabolic interference
Enzyme Inhibition

Drug 1

Metabolism Drug 2 or
Natural Product

Soluble product
Examples of drug impact on DMEs
Drug /xenobiotic Enzyme Action Result
Grapefruit Juice CYP3A4 Inhibition Elevated levels of co-administered drugs such
as felodipine, terfenadine, saquinavir, etc

St.John’s Wort CYP3A4 Induction Depleted levels of co-administered drugs such

as cyclosporin, ethinylestradiol etc
Ketoconzole CYP3A4 Inhibition Elevated levels of drugs such as terfanadine,
tolbutamide, cyclosporin

Quinidine CYP2D6 Inhibition Toxicities arising from elevated levls of drug

such as fluoxetine
Nicotine CYP1A2 Induction Ineffective therapy of drugs such as clozapine

Rifampicin CYP3A4 Induction Ineffective therapy of drugs such as

BC34B
ethinylestradiol 48
 Induction of Phase I enzymes
Clinical examples
A 49 year old male epileptic was prescribed phenytoin at 600mg/day and
carbamazepine at 2000mg/day. The patient’s condition was controlled with
minimal side effects for 3 months. The phenytoin was then abruptly
discontinued. Within 4 days, the patient became gradually more lethargic and
confused, until one week later hospitalization was necessary. The
carbamazepine dosage was reduced to 1200 mg./day and confusion and
sedation disappeared.

A 64 year old obese male was prescribed simvastatin 10mg daily. Over the next 3
months, lack of clinical response led to a 5 fold increase in dosage. He was then
admitted to hospital with rhabdomyolysis (is the rapid breakdown (lysis) of
skeletal muscle tissue (rhabdomyo) due to injury to muscle tissue.) Unknown to
his GP he self administered St.John’s Wort which he discontinued when his
mood was sufficiently elevated, around 10 days prior to the toxicity
manifesting itself.
.
 Enzyme Inducers
• Anticonvulsants: phenytoin, carbamazepine, phenobarbitone induce
many CYPs 1A2, 2C9, 2C19 and 3A4
• Steroids: dexamethasone, prednisolone and glucocorticoids induce
CYP3A4
• Polycylic aromatic hydrocarbons: present in cigarette smoke, industrial
solvents, barbequed meat and include dioxins, PCBs and induce
CYP1A1, 1A2.
• Antibiotics: rifampin induce most CYPs, 1A2,2C9,2C19 and 3A4
• Recreational agents: nicotine inducers 1A2, alcohol inducers 2E1,
marijuana inducers 3A4
 Mechanisms of Induction

• Inducers are able to increase the expression and

thereby activity of a certain enzyme by activating
key transcription factors
• CYP3A4 is induced by activating the transciption
factor called Pregnane X Receptor (PXR)
• CYP1A1 is induced by activating
arylhydrocarbon receptor (AhR)
• CYP2B6 is induced by activating constitutive
androstane receptor (CAR)
 Clinical aspects
• The process is relatively slow; ie usually days or
even weeks
• The potential changes in drug concentrations can
be great enough to cause treatment failure
• The induction process is usually but not always
reversible over a similar time frame to its
appearance, although reversal can be slower.
• Where the patient is stabilized on a high induced
drug dosage, if there is a treatment break of up to
several days, drug accumulation and toxicity will
occur.
 Enzyme Inhibition

• A 29 year old male used terfenadine twice daily for one year to treat
allergic rhinitis. The patient drank grapefruit juice two or three times
weekly. On the day of his death, he consumed 2 glasses of juice, took
his terfenadine dose and then mowed his lawn; within one hour he
became ill, collapsed and died. Post mortem terfenadine and its
metabolite plasma levels were reported to be at arrhythmogenic levels.
• A 64 year old female with a history of depression was stabilized on
amitriptyline but without improvement in mood. Her GP added
fluoxetine and within 3 weeks the patient’s symptoms subsided,
although 1 week later, she collapsed at home was found in a coma.
• A 44 year old female epileptic was stabilized on carbamazepine but on
the advice of a friend started taking a liquorice preparation for stomach
problems. Over a period of 2 days she became gradually more sedated
and confused, until she had difficulty standing up. She was admitted to
hospital and recovered within 3 days.
 Mechanisms of inhibition
• Competitive Inhibition
– Substrate and inhibitor compete for the same active site
– E.g. Azoles: Ketoconazole is a potent inhibitor of CYP3A4
• Noncompetitive inhibition
– Involves two sites where the substrate and inhibitor bind. Once the
inhibitor binds this allosteric site, the confirmation of the active is changed
and is less likely to bind the substrate.
– E.g. Omeprazole and lansoprazole are non competitive inhibitors of
CYP3A4
• Uncompetitive inhibition
– Inhibitor binds only to the enzyme/substrate complex ; relatively rare
– E.g. flavanoids found in citrus fruits inhibit CYP3A4 uncompetively
• Mechanism based inhibitors
– Binds similar to a competitive inhibitor, uses the reducing power and
occupies the active site for a longer period most often leading a destruction
of the active site of enzyme.
– E.g; Among most potent are grapefruit juice, norfluoxetine which destroy
the enzyme and takes several days to resolve.
 Effects of diet on drug metabolism
• Polyphenols: naringenin, bergamottin from grapefruit juice
and flavanoids such as quercetin, fisetin found in soy are
potent inhibitors of CYP family thus impacting the
metabolism of numerous drugs
• Barbecued meat: Such cooking exposes the consumer to
polycyclic aromatics, nitrosoamines and hetercyclic
aromatic amines, which all induce CYP1A family. If
consumption is large, clearance of drugs such as
amitryptalline, clozapine, paracetamol will be increased
significantly.
• Cruciferous vegetables: Glucosinolates, isothiocyanates
can induce GST activity in the gut and liver.
 Effects of age on drug metabolism
The elderly: decreased in mass and blood flow to liver, the low supply of
oxygen, decrease in renal function, increased fat deposits, and reduction
in gut blood flow (which affects absorption), all contribute to a lowered
enzyme function in-vivo in the elderly. Drugs’ bioavailability may
increase due to decreased first pass effect. A person’s life long smoking
and drinking habits can also complicate the situation. Among drugs
known to be cleared more slowly in older persons are antipyschotics,
paracetamol, antidepressants.

• Neonates: CYP activities are less than half and perhaps as little as 1/3 of
adult activity. However, the difference between neonates and adults
disappear by 2-6 months of CYP activity. Glucoronidation in babies are
low. This is clear in the relatively high incidence of jaundice in newborns,
which is due to the immaturity of UGT-mediated bilirubin clearance.
Overall, drug clearance in premature neonates can be 10 times less than
adults and they possess little ability to glucornidate drugs. Term neonates
also have immature Phase I and II systems but by 2-3 years old the system
is virtually adult.
 Gender effects

• In general experimental probe drugs are

metabolized more quickly by women than men.
There are suggestions that some CYPs (2C9/19)
would be more sensitive to female sex hormone
cycles than others (CYP3A4), but little is known
of the effects of menopause.
• Females are also more susceptible to drug adverse
reactions than males, which may be a reflection of
their increased formation of toxic metabolites.
 Disease
• There are a number of conditions and their
treatments that influence CYP activities and
expression, which include interferon treatments
for melanomas. Renal disease has a marked effect
on drug clearance.
• Hepatitis B infection appears to suppress some
hepatic detoxification processes, leaving such
patients susceptible to liver toxicity and cancer.
 Other factors

• Smoking: Nicotine induces CYP1A2 and

CYP2E1, which alters the clearance of
paracetamol, isoniazid and tamoxifen
– Marijuanna induces CYP3A4 and shown to
clear antipyrene faster.
• Red wine: inhibitors of CYPs1A1, 2B6,
2E1 and 3A4
 DMEs and Link with diseases

• Exposure to certain arylamines have had increased

likelihood of developing cancers. The most clear
link established has been that of NAT2 acetylator
status and occupational bladder cancer. NAT2
slow acetylator phenotype, who are exposed to
environmental carcinogens (work or smoking) are
at a significantly higher risk of developing bladder
cancer.
 DMEs and link with diseases
The carcinogenesis process often involves CYP
enzymes (CYPs1A2, 1A1, 1B1, 2E1).
BPD

CYP1A1 & Anti-BPDE:

BaP CYP1B1 Ultimate
carcinogen
HO

OH

Anti-BPDE
CYP1A1 & OH

CYP1B1 HO

DNA
Trans DDBP Adducts

CYP enzymes
H

OH
H
OH

A schematic representation of CYP involvement in and the different metabolites formed from the metabolism of BaP. Other than CYP enzymes,
such as expoxide hydrolase has been identified as critical in BaP’s metabolism. Papers by Kessler and Rita, 1997 and Rojas et al., 2004 assisted in the
derivation of the above scheme.3-hydroxybenzo [a]pyrene (3HBaP), (-)-benzo[a]pyrene-trans-7,8-dihydrodiol (BPD),