Pharmacogenomics

1. PHARMACOGENOMICS
Sanju Kaladharan
Department of Pharmacology
KMCH college of pharmacy.
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2. • Pharmacogenomics is the study that examines how
genetic variations affect the ways in which people
respond to drugs.
• Pharmacogenomics examine many genomic loci
including large biological pathways to determine the
variability.
• Pharmacogenetics focuses on large clinical effects of
single gene variant in small number of patients.
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4. Merits and demerits
MERITS
• Improve drug safety, and reduce ADRs;
• Tailor treatments to meet patients' unique
genetic pre-disposition, identifying optimal
dosing;
• Improve drug discovery targeted to human
disease; and
• Improve proof of principle for efficacy trials.
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5. Demerits
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6. • Polymorphism
Natural variations in a gene ,DNA sequence or
chromosome that have no adverse effects on the
individual.
• Allele
• An allele is one of a pair of genes that appear at a
particular location on a particular chromosome
and control the same characteristic.
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8. Single Nucleotide Polymorphisms
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9. Polymorphisms affecting drug
metabolism
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10. • Most drugs are usually metabolised in three main ways
• Drugs such as aspirin (acetylsalicylic acid) are initially
converted to an active metabolite (salicylic acid) by
hydrolysis (route 1).
• drugs such as phenytoin (an anti-convulsant drug) are
converted directly into an inactive metabolite by
reduction (route 2).
• Salicylic acid is converted into several forms of inactive
metabolite such as salicylglucuronide, salicyluricacid,
and gentisic acid by conjugation (route 3).
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11. Scenario 1: Route 1 is the major pathway for drug elimination and it is
affected by a polymorphism that results in less active enzyme.
• In this scenario, the effects of genetic polymorphism would be
dependent on whether the drug or its metabolite is active. If the
drug is active and because it uses route 1 as the main pathway for
elimination, it cannot be converted to a metabolite. Therefore drug
will accumulate in the body resulting in toxic activity.
• A good example for this is metoprolol, a selective β1 adrenergic
receptor blocker used in the treatment of hypertension.
• The major route for metabolising this drug is by CYP2D6 (route 1).
• It has been found that poor metabolisers have a 5-fold higher risk of
developing adverse effects during metoprolol treatment than
patients who are not poor metabolisers
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12. Scenario 2: Routes 2 and 3 are the major pathways for
drug elimination and they are affected by polymorphisms
that result in less active enzymes.
• although route 1 is the minor pathway in this scenario, the enzymes
of route 1 would compensate for the affected pathway (route 2).
• Therefore, the drug would still be converted to an inactive
metabolite.
• As explained before, there is considerable overlap in the structural
specificity of some of these enzymes amongst different routes.
• In other words, a drug may be a substrate for more than one
enzyme in the various pathways.
• In scenario 2, if the metabolite is active then there would be
exaggerated response due to the accumulation of active
metabolite.
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13. Scenario 3: Route 1 is the minor pathway for drug
metabolism and it is affected by a polymorphism that
results in a less active enzyme.
• In this scenario, as route 1 is a minor pathway for drug
elimination, there is less chance of drug accumulation as the
enzymes in route 2 would compensate for the less active
enzyme.
• Therefore, in the case of the drug being active the genetic
polymorphism will not show any effects on drug metabolism
• A good example is the analgesic codeine. Since codeine is a
pro-drug and dependant on route 1 for conversion into the
metabolite morphine, genetic polymorphisms affecting route
1 would result in therapeutic failure of this drug.
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14. Scenario 4: Route 1 is the minor pathway and route 3 is the major
pathway for drug elimination. Route 3 enzymes are affected by
polymorphisms that result in poor metabolism
• In this scenario, drug accumulation would not happen as route 1 is
only a minor pathway for metabolism and is not affected by
polymorphisms.
• A good example is caffeine, which is metabolised into various active
intermediate metabolites such as paraxanthines, dimethyl and
monomethyl uric acids, trimethyl- and dimethylallantoin, and uracil
derivatives.
• Paraxanthines are produced via route 1 and they have marked
pharmacological activity.
• Although further metabolism of paraxanthine is affected by genetic
polymorphisms affecting the route 2 enzyme CYP1A2, this would
not produce any adverse effects in individuals taking the
therapeutic dose of caffeine, as paraxanthines only form a small
fraction of all the metabolites of caffeine.
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16. Therapeutic implications of
polymorphisms affecting drug metabolism.
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19. Drug Enzyme involved Effect
codeine Decreased expression of
CYP2D6
Less metabolism of the
drug causes drug to
remain in circulation for
a longer time causing
respiratory side effects
warfarin Reduced CYP2C9 activity
in *2 and *3 variants
15% variability in dose
requirement
tacrolimus CYP3A5*1 variant Require larger dose to
reach targetted Co
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21. CYP2D6 is the rate limiting enzyme in catalysing the conversion
of the prodrug tamoxifen into active metabolites 4-
hydroxytamoxifen and endoxifen which have significantly higher
affinity for the drug target,estrogen receptor. 21Sanju Kaladharan

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23. Genetic Polymorphisms of Drug
Transporters
• The completion of the human genome project resulted in the
identification of a large number of membrane-spanning
proteins involved in endogenous compound and drug
transport which can be divided into two major groups.
• The first group includes members of the solute carrier (SLC)
transporter superfamily, which facilitate the influx or efflux of
a wide range of compounds without the use of ATP.
• The second major group of transporters are the multi-drug
resistance (MDR) ATP binding cassette (ABC) proteins which
carry out ATP-dependent drug efflux.
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24.  Transporters are those proteins that carry either
endogenous compounds or xenobiotics across
biological membranes.
 They can be classified into either efflux or uptake
proteins, depending on the direction of transport.
 The extent of expression of genes coding for
transport proteins can have a profound effect on
the bioavailability and pharmacokinetics of
various drugs.
 Additionally, genetic variation such as single-
nucleotide polymorphisms (SNPs) of the transport
proteins can cause differences in the uptake or
efflux of drugs. 24Sanju Kaladharan

25.  In terms of cancer chemotherapy, tumor cells
expressing these proteins can have either
enhanced sensitivity or resistance to various
anticancer drugs.
 Transporters that serve as efflux pumps on a cell
membrane can remove drugs from the cell before
they can act.
 Transport proteins that are responsible for the
vital influx of ions and nutrients such as glucose
can promote growth of tumor cells if
overexpressed, or lead to increased susceptibility
for a drug if the transporter carries that drug into
the cell.
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26.  There are two superfamilies of transport proteins
that have important effects on the absorption,
distribution, and excretion of drugs:
1. ATP-binding cassette (ABC) superfamilies and
2. Solute-carrier (SLC) superfamilies
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27. Individual Transporters
ABC Transporters
ATP-binding cassette (ABC) transporters are
present in cellular and intracellular membranes
and can be responsible for either importing or
removing of substances from cells and tissues.
 They often transport substances against a
concentration gradient by using the hydrolysis of
ATP to drive the transport.
 There are at least 49 ABC transporter genes,
which are divided into seven different families (A-
G) based on sequence similarity.
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28.  Three of these• particularlyimportantfor
• multiple drug resistance in tumor cells:
1. the ABCB1 gene, encoding MDR1 (also known as P-
glycoprotein);
2. ABCG2 (breast cancer resistance protein);
3. the ABCC family (ABCC1 through ABCC6) or
• multidrug resistance proteins (MRP).
seven gene
drug
families are
transport and
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29.  ABC transporters are characterized as such by
the homology of their ATP binding regions.
 All families but one (ABCG2) contain two ATP
binding regions and two transmembrane
domains.
 The transmembrane domains contain multiple
alpha helices, and number of alpha helices in a
transmembrane domain differs depending on the
family.
 The ATP binding regions are located on the
cytoplasmic side of the membrane.
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32. ABCB1 Transporters: P-glycoprotein
 The ABCB1 gene codes for a glycosylated
membrane protein originally detected in cells that had
developed resistance to cancer
chemotherapy agents.
 The protein is commonly referred to as P-
glycoprotein (P-gp), PGY1, or multidrug
resistance protein-1 (MDR1).
 It is designated as a multidrug resistance protein due to
the fact that its expression in a cell may confer resistance
to multiple classes of drugs with differing chemical
structures and mechanisms of action.
 Various cancers tend to display low initial levels of
P-gp with levels of expression increasing after
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33.  Besides being expressed in cancer cells, P-
glycoprotein is expressed in multiple normal
tissues with excretory or protective function
including intestine, kidney, liver, blood-brain
barrier, spinal cord, testes and placenta.
 P-gp has an important role in forming a protective
barrier against absorption of xenobiotics in these
tissues.
 The substrates for P-gp are often hydrophobic
drugs with a polyaromatic skeleton and a neutral
or positive charge.
 Substrates include cytotoxic chemotherapeutic
agents, protease inhibitors, immunosuppressants,
calcium channel blockers, beta blockers, statins, 33Sanju Kaladharan

34. ABCC Transporter Family
 The protein product of ABCC genes are commonly
known as MRPs or multidrug resistance proteins.
 MRPs often transport anionic compounds.
 Ten members of the MRP family are known and at
least seven may be involved in conferring resistance
to cancer chemotherapeutics (MRP1 to MRP7).
 MRP1 has the most likely significance in clinical
anticancer drug resistance.
 MRPs are located in various tissues with protective
and excretory function such as the brain, liver, kidney,
and intestines.
 They transport a structurally diverse set of
endogenous substances, xenobiotics, and
metabolites. 34Sanju Kaladharan

35. ABCC1 Transporters
 It confers resistance to anthracyclines and vinca
alkaloids.
 MRP1 transports primarily neutral and anionic
hydrophobic compounds and their glutathione,
sulfate, and glucuronide conjugates.
 A few cationic substances can also be
transported.
 It is located in lung, blood-cerebrospinal fluid
barrier, and testes.
 Substrates
alkaloids,
leukotriene
include anthracyclines, vinca
methotrexate,glutathione conjugates,
C4, bilirubin,glutathione, and35Sanju Kaladharan

36. ABCG2 Transporters
 ABCG2 is alternatively known as Breast Cancer
Resistance Protein (BCRP), placenta-specific
ABC transporter (ABCP), and mitoxantrone
resistance protein (MXR).
 It is very important in limiting bioavailability of
certain drugs, concentrating drugs in breast milk,
and protecting the fetus from drugs in maternal
circulation.
 It is highly expressed in the gastrointestinal tract,
liver, and placenta, and influences the absorption
and distribution of a wide variety of drugs and
organic anions.
 Substrates are Doxorubicin, daunorubicin, 36Sanju Kaladharan

37. Solute Carrier Proteins
 Solute carrier proteins (SLCs) are important in
transport of ions and organic substances across
biological membranes in the maintenance of
homeostasis.
 Examples of some of the endogenous solutes
that are transported include steroid hormones,
thyroid hormones, leukotrienes, and
prostaglandins.
 The solute carrier protein class includes the
transporters
transporters),
known
the
as OATs (organic
OATPs (organic
anion
anion
transporting polypeptides, which are structurally
different from OATs), OCTs (organic
transporters),
cation
transportand PepTs (peptide 37Sanju Kaladharan

38.  SLCs are expressed in a variety of tissues such
as liver, kidney, brain, and intestine.
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39. TRANSPORTER GENE
NAME
TISSUE LOCALISATION SUBSTRATES
Serotonin transporter SLC6A4 Neurons, heart
valve, intestine
Serotonin
Reduced folate
Carrier (RFC-1)
SLC19A1 kidney, leukemic cells,
wide distribution
Methotrexate,
leucovorin,
OATP1B1 SLCO1B1 liver, brain Pravastatin,
digoxin,
mycophenolate
Methotrexate,
mycophenolate
Cephalexin, other
β-lactam
antibiotics,
ACE inhibitors
Methotrexate
OATP1B3 SLCO1B3 liver
PEPT1 and PEPT2 SLC15A1,
SLC15A2
PEPT1: small intestine,
duodenum
PEPT2: broad distribution
RFC-1 SLC19A1 Broad distribution
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40. CNT1, CNT2,
CNT3
SLC28A1,
SLC28A2,
SLC28A3
Intestinal/renal
epithelia, liver,
macrophages,
leukemic cells
Didanosine,
idoxuridine,
zidovudine,
ENT1, ENT2,
ENT3, ENT4
SLC29A1,
SLC29A2,
SLC29A3,
SLC28A4
Intestine, liver,
kidney,
placenta
Pyrimidine and/or
purine nucleosides,
adenosine,
gemcitabine,
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43. Drug Transporter Effect
digoxin C3435T
polymorphism of
ABCB1 gene
encoding p-gp
Reduced serum
digoxin
concentration
diflometacan ABCG2
heterozygous
genotype C421A
300% higher plasma
levels
Estrone sulfate,
estradiol 17β-D-
Glucoronide
OATP-c*9 and *5
(gene SLC21A6)
Reduced uptake
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44. ABCC*2 haplotypes causes less exposure to intestinal cell by
reducing hepatibiliary secretion and thus reduce incidence of
diarrhoea 44Sanju Kaladharan

45. G-Protein Coupled Receptor
 7 trans membrane helices connected by alternating cytosolic and
extra cellular loop
 C terminal: inside the cell
 N terminal : extra cellular region
 Extra cellular portion has unique messenger binding site
 Cytosolic loop allow receptor to interact with G protein.
 The eventual effect of agonist -induced activation is a change in the
relative orientations of the TM helices (likened to a twisting motion)
leading to a wider intracellular surface and "revelation" of residues
of the intracellular helices and TM domains crucial to signal
transduction function (i.e., G-protein coupling).
 Inverse agonists and antagonists may also bind to a number of
different sites, but the eventual effect must be prevention of this TM
helix reorientation
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46. Genetic variation in G protein
coupled receptors
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47. Genetic variation in G protein
coupled receptors
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53. PHARMACOGENOMICS IN DRUG
DISCOVERY AND DEVELOPMENT
• Through examination of individual response profiles and
elucidation of different effect of different compounds on
gene expression will lead to target identification,drug
discovery and compound selection.
 Identification of novel proteins involved in disease
processes
 Targetting of proteins with variant structure resulting
from genetic polymorphism.
 Refinement of existing targets to improve specificity of
drug action.
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54. Approaches to drug discovery and
development
• Development of new drugs to overcome drug
resistance or target new drug targets.
• Optimisation of drug metabolism and
pharmacokinetics(DMPK) to minimise
variations in drug levels
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55. Overcoming drug resistance
• Imatinib >>nilotinib>>nasatinib
Drug Target Mutation sites Effect
Imatinib BCR-ABL tyrosine
kinase,mast/stem
cell growth factor
receptor(SCFR,CD1
17),PDGFR
T315I,F359V(contac
t regions of drug
with ABL domain),
P-loop of ATP
binding pocket of
kinase
domain(suitable
conformation for
binding)
25% of patients
with
gastrointestinal
stromal tumors
suffered relapse.
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56. Optimisation of DMPK
• DMPK optimisatisation is a practical and effective
approach in developing especially orally active drugs
that have predcatable pharmacokinetic profiles and
can be administered with reduced need for
monitoring and dose adjustment in drug therapy.
Eg:oral anticoagulants
• Warfarin>> clopidogrel>>prasugrel>>apixaban
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57. Drug Target Variation causing
factor
Drug
intermediates
Effect
WARFARIN VKORC1 CYP2C9 hypersensitation or
true resistance
CLOPIDOGREL Antiplatelet
and factor Xa
CYP influenced
Hepatic carboxyl
esterases
deactivates active
thiol intermediate
(CYP2C19(2ox
o),CYP3A4(
active thiol))
Lesser degrees of
platelet inhibition and
increased risk of
cardiovascular events
in esterase over
expressed population
PRASUGREL Antiplatelet
and factor Xa
Esterase(less
variant)
CYP3A4 Greater platelet
aggregation with
lesser variability
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59. Pharmacogenetics in practice
• In a large population a medication that is proven
efficacious in many patients often fails to work in
some other patients.
• Major genetic factors affecting individual drug
response include
Therapeutic targets
Drug metabolising enzyme
Drug transporters
Targets of ADR
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60. Therapeutic target
Eg1: warfarin
C1173T polymorphism in
intron 1 of VKORC1 result in
dose change from 15mg/day
to 16mg/day
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61. Eg2.
Anti HIV drugs:vicriviroc,maraviroc
Target associated Variants Effects
CCR2,a chemokine
receptor for
monocyte chemo
attractant protein1.
Polymorphism at
codon 64 (V64I)
with Ile allele
HIV progress to
AIDS2 four years
later than those
carrying wild type
allele
CCR5,a chemokine
receptor used by
HIV as a coreceptor
to enter into the
target cell
White persons have
32 base pair
deletion but it is not
find in Africans
Deletions make
receptor
nonfunctional and
less HIV
transmission
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62. Eg3 β agonist and ADRB2
Drug Gene mutation Effects
Albuterol 2 SNPs of
ADRB2 results
in mutations
R16G Q27E
Evokes a larger
and more rapid
broncho
dilation
response in
arg16/arg16
than in carriers
of gly16 allele
(arg16/gly16,gly
16/gly16)
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63. Drug metabolisms
• Cytochrome P450 catalyses the mono
oxygenation of lipophilic drugs to give rise to
metabolites with altered activity and
increased water solubility
• Variable expression of genes encoding these
enzyme make effect on drug response
depending upon the affinity of the receptors
of the metabolite and orginal drug molecule.
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64. TARGETS OF ADR
• Idiosyncratic drug reactions characterised by their
rare occurance and requirement of multiple
exposure are most extreme of individual
variability in drug safety.
1. On target drug toxicity:inhibition or activation
of a therapeutic target eg:excessive bleeding
from high doses of warfarin
2. Off target drug toxicity: interaction between a
drug and a target protein differbt from the
therapeutic target.eg:statin induced myopathy.
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65. drug gene effect
flucloxacillin HLA-B*1 attributed
to SNP in MHC
Cholestatic
hepatitis(drug
induced liver injury)
simvastatin Various(about
3lakh) at various loci
SNP associated with
SLCO1B1
myopathy
Various cardiac and
non cardiac drugs
KCNE2 encoding a
subunit of cardiac
potassium channel
Long QT syndrome
(arrhythmia –
torsades de pointes)
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66. DRUG HYPERSENSITIVITY
• Eg:abacavir hypersensitivity associated with
HLA-B*5701(effective antigen presenting
molecule) polymorphism.
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67. Thank you
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