Transcript Pathways of drug metabolism

Introduction to
pharmacogenomics and
personalised medicine
Jerzy Jankowski, MD
Department of Clinical Pharmacology
www.zfk.ump.edu.pl
The Relationship Between Dose and
Effect
Pharmacotherapy – clinical problems
Adverse Drug Reactions (ADRs)





56% of drugs that cause ADRs are metabolized
by polymorphic phase I enzymes, of which
86% are CYP P450;
only 20% of drugs associated with ADRs are
substrates for non-polymorphic enzymes
ADRs cause > 100 000 deaths/y in the USA
Up to 7% of all hospital admissions in the UK
and Sweden are due to ADRs
ADRs cost the US society ~ US$ 100 billion
Pharmacotherapy – clinical problems
Efficacy

30-60% of subjects treated with drugs do not
respond to drug therapy
Pharmacogenomics
Is the study of how an individual’s genetic
makeup affects the body’s response to drugs.
The key to creating personalised drugs with greater
efficacy and safety
Combines traditional pharmaceutical sciences with
an understanding of common DNA variations in
the human genome
The most common DNA variations- SNPs
Genetic polymorphisms
Exist in a human population when allelic variants
occure with a frequency of
1% or greater
TYPES OF GENETIC VARIANTS

single nucleotide polymorphisms ( SNPs )
1 SNP every 300 – 1000 base pairs

Insertions/deletions ( INDELS )
in comparison with SNPs, indels are much less frequent,
especially in coding regions of genes

Copy number variations ( CNVs ) – large segments of
DNA ( gene duplications, gene deletions, gene inversions )
SINGLE NUCLEOTIDE
POLYMORPHISM


SNPs in the coding region cSNPs:
- non-synonymous or missense
(protein strucutre, stability, substrate affinty
or introduce a stop codon)
- synonymous or sense (transcript stability,
splicing)
Noncoding SNPs may occur in 3´ and 5´ UTR, in
promoter or enhancer regions, in introns or in
intergenic regions
MOLECULAR MECHANISMS OF
GENETIC POLYMORPHISMS
ETHNIC DIVERSITY



Polymorphisms differ in their frequencies within
human populations
Polymorphisms are classified as:
- cosmopolitan ( present in all ethnic group )
- population specific ( or ethnic and race )
The presence of ethnic and race - specific
polymorphisms are consistent with geographical
isolation of human populations
Pharmacogenomics
SNPs are often linked to an
individual’s response to a drug.
Anticipated benefits of
pharmacogenomics

More powerful medicine
Drugs more targeted to specific diseases,
maximising therapeutic effects while decreasing
damage to nerby healthy cells

Better, safer drugs the First Time
The best available drug therapy from the
beginning; shorter recovery time; the
likelikehood of adverse reactions is eliminated
Anticipated benefits of
pharmacogenomics

More accurate methods of determinig
appropriate drug dosages
Current methods of basing dosages on weight
and age will be replaced with dosages based on
person’s genetics

Advanced screening for disease
Treatments can be introduced at the most
appropriate stage to maximize their therapy
Anticipated benefits of
pharmacogenomics

Better vaccines
Made of either DNA or RNA, promise all the
benefits of existing vaccines without all the risks

Improvements in the drug discovery and
approval process
The cost and risk of clinical trials will be
reduced by targeting only those persons capable
of responding to a drug

Decrease in the overall cost of health care
The fate of a drug in the body
Is affected by:
Liberation
Absorption
Distribution
Metabolism
Excretion
LADME
PHASE I AND PHASE II REACTIONS
IN DRUG DYSPOSITION
Pathways of drug metabolism

Phase I reactions: oxidation
reduction
hydrolysis

Phase II reactions: glucuronidiation
sulfation
acetylation
methylation
PHASE I REACTIONS
convert the parent drug to a more
polar (water-soluble) and/ or more
reactive product by unmasking or
inserting a polar group such as
-OH, -SH, -NH2
PHASE II REACTIONS
increase water solubility by
conjugation of the drug molecule with
a polar moiety such as glucuronate,
sulfate, acete, glutathione, glycine and
methyl groups
Both types of reaction convert relatively
lipid- soluble original drug molecules
into more water-soluble metabolites
that are more easily excreted.
Determinants of drug
biotransformation

biological: gender
age
renal and liver fun.
disease- related fac.

lifestyle: smoking
alkohol consumtion
diet
drug -drug interaction

inherited
GENETIC CONTRIBUTION TO
PK PARAMITERS
75 – 85%
Determinants of drug
biotransformation

The potential risk factors of drug inefficacy
and toxicity

Differences in drug metabolism can lead to
severe toxicity or therapeutic failure
Determinants of drug
biotransformation
Of greater importance are inherited
determinants that affect
the kinetics and dynamics
of numerous drugs.
Pharmacokinetic variability
Refers to variability in delivery of drug to, or
removal from, key molecular sits of action
that mediate efficacy and/or toxicity.
Drug- metabolising enzymes (DMEs) and
drug transportes (P-gp) are involved in this
processes.
Pharmacodynamic variability
refers to variable drug effects despite
equivalent drug delivery to molecular sits of
action.
This may reflect variability in the function
of the drug targets (receptors or enzymes)
Genetic variation in genes for DMEs,
drug receptors (DR) and drug
transportes (DT) is associated with
variability in efficacy and toxicity of
drugs
The fate of drug in the body

The majority of pharmacogenomic differences
represent variability in drug metabolism

Most of the remaining represent alternations in:
Receptors
Transporters
Protein binding

Pharmacogenetic differences in absorption or
excrition of drugs are relatively uncommon
Polygenic Determinants of Drug
Effects
The phenotypes of drug metabolism

The extensive metabolizer (EM) (dominant trait;
inherited as either homozygous or heterozygous for the
wild- type allele (wt)

The poor metabolizer (PM) recessive autosomal
trait due to mutation and/or deletion of both allels;
inherited as either homozygous or heterozygous for
mutant allele

The ultra- extensive metabolizer (UEM)
recessive autosomal trait due to gene amplification
Metabolism of Debrisoquin
Cytochrome P450 (CYP450) enzymes

The most important enzymatic system

Biosynthesis and degradation of endogenous
compounds (steroids, lipids, vitamines)

Degradation of exogenous compounds (diet,
environment, medications)

Highly polymorphic
Clasiffication of CYP450
enzymes

Amino acid similarities

Designated by a family number, a subfamily
letter, a number of an individual enzyme within
the subfamily and an asterisk followed by a
number and a letter for each genetic (allelic)
variant

www.imm.ki.se/CYPalleles/
CYP 450 ENZYMES
Cytochrome P450 (CYP450) enzymes

57 CYP450 genes

CYP1, CYP2, CYP3 families appear to
contribute to the metabolism of drugs

These CYP enzymes are involved in
approximately 80% of oxidative drug
metabolism and account for 50% of the overall
elimination of commonly used drugs
Cytochrom P450 3A (CYP 3A)

3A4, 3A5, 3A7 and 3A43 isoenzymes in adults

Chromosom 7q22.1

Probably the most important of all DMEs

Aboundant in intestinal epithelium and in the liver

50% of the CYP450 activity in the liver

Involved in the metabolism of more then half drugs that
undergo oxidation
Drug
interactions
involving
inhibition of
CYP 3A
Drug interactions involving
inhibition of CYP 3A

The interaction between grapefruit juice nad CYP 3A
substrates

250ml of this juice inhibits intestinal CYP 3A for 2448h.

Grapefruit juice is contraindicated when drugs
extensively metabolised by CYP 3A are used

CYP 3A inhibition is reversible 2-3 days
Drug interactions involving
inhibition of CYP 3A

The problem of drug interactions can be serious

For exapmle: interaction of erythromycin and
inhibitory drugs (nitroimidazole, diltiazem, verapamil,
troleandomycin)

When an orally administered drug undergoes extensive
first-pass metabolism, its bioavailability in the face of
CYP3A inhibition may increase severalfold, thus
prolonging the presence of the drug in the body
Cytochrom P450 3A (CYP 3A)

Activity vary markedly among individuals of a given
population

Multiple genes are involved in its regulation

Activity modulated by several factors including drugs

Drug interactions may increase or reduce CYP 3A
activity (expanding the range of variablility to about 400fold)
Cytochrom P450 3A (CYP 3A)

Variability in drug levels of this magnitude,
potentially presents a major therapeutic problem
in dosage optimization.

For example: dosage of cyclosporine in patiens
receiving also ketoconazole

For example: dosage of cyclosporine in patients
receiving also rifampin
Drug interactions involving
induction of CYP 3A

The induction of CYP 3A significantlly
decreases (up to 95%) the plasma levels of
certain drugs administered concurrently

CYP 3A activity is especially sesnitive to
modulation

Previously effective drug dosages become
ineffective
Drug interactions involving
induction of CYP 3A

The consequences of CYP 3A induction are not
immediate

Steady- state levels are reached in 2-3 weeks

„Washing out” the induction effect also takes
several weeks

Effectivnes of drug therapy is reduced
CYP 3A4

42 alleles ( to date )

Functional characterisations of most CYP 3A4
variants – a limited impact on protein expresion and
activity

CYP 3A4*1B and CYP 3A4*20 alter CYP function

CYP 3A4*1B – 4% in Caucasians, 67% in Black sub.
CYP 3A5

25 alleles ( to date )

CYP 3A5*3A – decreased enzymatic activity
in vivo and in vitro

Alleles *3B - *3L only in vitro
CYP 3A7 AND CYP 3A43

7 alleles ( to date )

CYP 3A7*1A, *1B, *1C, *1D, *1E, *2, *3

5 alleles ( to date )

CYP 3A43*1A, *1B, *2A, *2B, *3

No significant role in drug metabolism
CYP 3A

CYP 3A genes are under the transcriptional control
of nuclear receptors such as:
- the pregnane X receptor ( PXR )
- the constitutive androstane receptor ( CAR )
- the hepatocyte nuclear factor-4α ( HNF4α )

CAR and HNF4α regulates the constitutive CYP3A

PXR mediates the induction by exogenous comp.
CYP 3A GENES REGULATION

Genetic polymorphisms of nuclear receptors may
influence CYP 3A gene expression and CYP activity

Becouse of the simultaneous effect of nuclear
receptors on CYP 3A genes, marked increases of
CYP 3A activity are observed following the exposure
to CYP 3A inducers
Cytochrom P450 2D6



Plays a role in the metabolism of 100 most
commonly used drugs
78 variants
Mechanisms of gentic polymorphism:
 SNPs
(insertions/ deletions)
 Complete gene deletion
 Gene duplication or multiplication
CYP2D6 genetic polymorphism
Designation
Nature of mutation
CYP2D6 *2
G1749C, silent mutation, C2938T, amino acid
change, multiple copies result in increased
activity
3.5
CYP2D6 *3
A2637 deletion in exon 5
2.7
CYP2D6 *4
G1934A, splice side defect, no activity
28.6
CYP2D6 *5
CYP2D6 gene deletion
11.6
CYP2D6 *6
T1795, deletion, premature stop codon
1.8
CYP2D6 *7
A3023C, amino acid change
1.5
CYP2D6 *8
G1846T
CYP2D6 *9
3 bp deletion, loss of K281
<1.5
CYP2D6 *10
C188T, G4268T; amino acid substitutiondecreased catalytic activity
<0.5
CYP2D6 *11
G971C, splice defect, no activity
Allelic frequency
(%)
1
<1
Cytochrom P450 2D6


The CYP 2D6 polymorphisms are associated with:
The PM phenotype (complete lack of active proteine, a
recesive trait):
CYP 2D6*4 allele 70% in PMs
CYP 2D6*5 allele 25% in PMs
CYP 2D6*3 allele 3% in PMs
Heterozygous individuals
normal metabolic ratio (SNP)
increased metabolic ratio (multipoint mutant allele)
35% of individuals with a heterozygous genotype
Cytochrom P450 2D6

The UEM phenotype duplication/amplification of
CYP2D6*2 (indentified in the heterozygous form)
The
amplified gene product has functionally
the same but catalytically increased activity
Cytochrom P450 2D6
Cytochrom P450 2D6

Clinical importance of CYP 2D6
polymorphism:
 The
greater risk of adverese reactions in PMs
(high plasma level of the affected drug)
 Lack
of efficacy in UEMs (low plasma level of
the affected drug)
Cytochrom P450 2D6


CYP 2D6 inhibitors (qinidine, fluoxetine,
paroxetine) are able to convert EMs into PMs.
Phenomenon termed
PHENOCOPYING
Cytochrom P450 2D6

Clinical relevance of phenocopying

Combination of SSRI and TAD
(2-4 - fold increase in plasma level of tricyclic
antidepressant; inhibition lasts for several weeks
because of persistant inhibitory metabolites)

Codeine – loss of analgesic efficacy
Cytochrom P450 2D6
Inhibitory interactions don’t occur in
persons with PM, who lack active
enzyme.
Cytochrom P450 2C19





Proton- pump inhibitors (omeprazole, lansoprazole )
diazepan, propranolol, clopidogrel)
Chromosome 10q24.1
36 alleles ( to date )
CYP 2C19 polymorphism associated with either
PMs or UEMs phenotype (CYP 2C19*17)
CYP 2C19*2 CYP 2C19*3 account for more than
95% of cases of PM
CYP 2C19 POLYMORPHISM




CYP 2C19*2 – 681 G→A in exon 5 leading to a
splice-defective site
CYP 2C19*3 – 636 G→A in exon 4 leading to a
premature stop codon
Loss of function variants
CYP 2C19*17 – 806 C→T in 5´ flanking region of
the gene leading to an increased transcription and
increased CYP 2C19 activity
CYP 2C19 POLYMORPHISM
CLINICAL SIGNIFICANCE

Eradication of Helicobacter pylori with PPIs is more
effective in CYP 2C19*2 and CYP 2C19*3
homozygotes and CYP 2C19 *2/*3 heterozygotes
then in patients who are CYP 2C19*1 homozygotes

Clopidogrel, an inactive prodrug, requires
metabolisation and activation by hepatic CYPs
including CYP 2C19. Loss-of-function alleles are
associated with reduced effectiveness of the drug
CYP 2C19 POLYMORPHISM
CLINICAL SIGNIFICANCE

CYP 2C19*17 allele is significantly associated with
enhanced response to clopidogrel and increased risk
of bleeding
Cytochrom P450 2C9

Warfarin, tolbutamid, phenytoin, glipizide, losartan,
NSAIDs
Cytochrom P450 2E1

Metabolism and bioactivation of procarcinogens and some drugs

Alternative system of metabolising ethanol

Gene located on chromosom 10 with seven
different loci

Indentified two variants of the gene (C, c2)
Clinical significance hasn’t yet been established
PHASE II DRUG METABOLISM
UDP-GLUCURONOSYLTRANSFERASE





Prevent the accumulation of toxic lipophilic
compounds and initiate their elimination through
more hydrophilic vehicules
Hydrophilic sugar moiety ( glucuronide ) from
uridine diphosphate glucuronic acid
Two families: UGT1A and UGT2 ( 2A, 2B )
UGT1A members are all encoded by a singel-gene
locus on chromosome 2q37.1
UGT2B and UGT2A3 encoded by several likely
duplicated genes located on 4q13.2
UDP-GLUCURONOSYLTRANSFERASE

UGT1A family has the ability to conjugate several
xenobiotics such as drugs and carcinogens
UDP-GLUCURONOSYLTRANSFERASE





UGT 1A1 has over 30 genetic variants
One of the genetic variants of UGT 1A1 occurs in
the TATA promoter region and has variable
repeats of thymine-adenine ( TA )
The wild-type promoter ( UGT1A1*1 ) has 6 TA
repeats
UGT 1A1*28 variant has ( TA )
An increase in TA repeats within TATA region
results in reduced UGT 1A1 activity
UGT1A1*28 POLYMORPHISM AND
TOXICITY OF IRINOTECAN



An inverse relationship between SN-38
glucuronidation rates and frequency of diarrhea
An association between UGT1A1*28 and
pharmacokinetic values and toxicity endpoints
with irinotecan therapy ( grade 4 neutropenia,
grade 3 diarrhea )
9-fold higher risk of developing grade 4
neutropenia in patients who were homozygous
for the UGT1A1*28
THIOPURINE SMETHYLTRANSFERASE ( TPMT )




Thiopurine drugs mercaptopurine, azathioprine,
thioguanine are all prodrugs converted to
thioguanine nucleotide (TGN) metabolites
Purine antimetabolites used as immunosuppresants
and to treat neoplasias ( ALL of Chiledhood )
White populations can be separated into 3 groups
based on the level of TPMT activity in their red cells
TPMT activity is inherited as a monogenic
autosomal co-dominant trait
THIOPURINE SMETHYLTRANSFERASE ( TPMT )




The phenotypic test for the level of TPMT activity
in red cells
The genotypic tests based on DNA
TPMT*3A is the most common variant allele
(~4%) associated with very low levels of TPMT in
whites
TPMT*3A no observed in China, Korea, Japan
P- glycoprotein (P- gp) pump
The multidrug resistance pump
 A transmembrane protein
 A member of the ATP- binding cassette family
of transport proteins
 Encoded by the multidrug resistance gene
(MDR1/ABCB1) on chromosome 7q21.1
 MDR1 (ABCB1) appears to be involved in drug
transport and the development of drug
resistance

P- glycoprotein (P- gp) pump

The apical surface of epithelial cell of the:
small
and large intestines
biliary ductules
ductules of the pancreas
proximal tubules of the kidneys

Choroid plexus
The role of P-gp

P-gp acts as a transmembrane efflux pump
(removes drugs from the cell)

Sites of expression of P-gp suggest a role in
decreasing absorption from the gut
secretion of endogenous and
exogenous hydrophobic toxins
List of P-glycoprotein substrates

Steroid compounds:

Anticancer agents:
Aldosterone Progesterone Hydrocortisone Cortisol
amethasone
Corticosterone Dex
Doxorubicin Daunorubicin Vinca alkaloids Actinomycin D

Immunosuppressive agents:
Cyclosporin

FK 506
Methotrexate
Protease inhibitors:
Indinavir Nelfinavir Ritonavir

Antibiotics:
Erythromycin

Cardiac drugs:
Digoxin

Rifampicin
Quinidine Lovastatin
Antihistamines:
Terfenadine

Others:
Domperidone
Loperamide
Epotoside
P- glycoprotein (P- gp) pump

The same site of expression for both P-gp
and CYP 3A4 in the small intestinal
epithelial cells is the reason that P-gp:
Can
influence the intracellular levels of
many CYP 3A substrates
It may also affect the availability of
those substrates to CYP 3A and thereby
the extent of their CYP3A- mediated
metabolism
P- glycoprotein (P- gp) pump

P- gp expression and function shows wide
interindividual differences influcenced by
both:
Environmental
Genetic factors (SNP in exon 26
3435CT, exon 21 2677GT)
Assessment of Individual’s Polymorphism

Phenotyping – administration of the
respective drug, followed by determination of
the metabolic ratio

Genotyping – identification of specific DNA
variations that result in a specific phenotype:
over expresion (gene duplication)
absence of active enzyme (null allele)
formation of a mutant protein with ↓
activity (inactivating allele)
Indications for genotyping

signs of toxicity or therapeutic failure upon
therapy administration

Drugs selection that require genotyping before
therapy introduction

Drug of choice is a substrate for a polymorphic
enzyme
www.zfk.ump.edu.pl