Dr.

Justin Kurian
 Introduction
• Genetic variations contribute to interpatient
differences in drug response.

• Pharmacogenetics involves the search for

genetic variations that lead to interindividual
differences in drug response.

• Pharmacogenetics is the study of how genes

affect the way people respond to drug therapy
 Pharmacogenomics and Pharmacogenetics

• Pharmacogenetics : refers to monogenetic

variants that affect drug response.

• For example, a pharmacogenetic study would

examine the influence of the β1-adrenergic receptor gene
on blood pressure response to carvedilol .
• Pharmacogenomics: refers to the entire
spectrum of genes that interact to determine
drug efficacy and safety.

• Example: The interaction between CYP2D6 and

β1-, β2-, and α1-adrenergic receptor genes on
carvedilol effects.
 Goals of Pharmacogenetics

• Optimize drug therapy and limit drug toxicity

based on an individual’s genetic profile.
 Pharmacogenetics - Aims
• Use genetic information to choose a drug, drug dose, and
treatment duration.

• Results of pharmacogenetic research provide opportunities for

clinicians to use genetic tests to predict individual responses to
drug treatments.

• Develop novel strategies for disease treatment and prevention

based on an understanding of genetic control of cellular functions

• Select medications for patients based on DNA profiles

Time line of Genomic discoveries
 HUMAN GENOME PROJECT
• Goal
To determine the entire sequence of the human
genome by 2005.

• Initial working draft of the human genome

sequence was completed in 2000

• 2003 - completed
• National Human Genome Research Institute.

• Improving human health and well-being

• Develop genome-based approaches to predict

drug response.
 Genetic variants linked to drug response.
 Advanced technology to efficiently determine genotype.
 Appropriate integration of genetic testing into the
therapeutic decision process
 Genetic Concepts
• Gene
 The functional and physical unit of heredity
passed from parent to offspring.

 Genes are pieces of DNA.

 most genes contain the information for making

a specific protein.
• Every person has two copies of each
gene, one inherited from each parent.

• Most genes are the same in all people,

but a small number of genes (less than
1 percent of the total) are slightly
different between people.

• Each chromosome contains many

genes.

• Alleles are forms of the same gene with

small differences in their sequence of
DNA bases.
 Alleles:
• An allele is an alternative form of a gene(one
member of a pair) that is located at a specific
position on a specific chromosome.
• An alleleis defined as the sequence of nucleic
acid bases at a given gene chromosomal locus
• Organisms have two alleles for each trait
• heterozygous or homogenous

• one is dominant and the other is recessive

• A gene is a series of codons that specifies a
particular protein.
• Genes contain several regions

• Exons: that encode for the final protein

• Introns : consist of intervening noncoding

regions

• Regulatory regions :that control gene

transcription
• The phenotype refers to the outward
expression of the genotype.
 TYPES OF GENETIC VARIATIONS
• Genetic variations occur as either rare defects or
polymorphisms.

• Polymorphisms are defined as variations that occur

at a frequency of at least 1% in the human
population.

• Example, the genes encoding the CYP450 enzymes CYP2A6,

CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are polymorphic,
with functional mutations of >1% occurring in different ethnic
groups.
 Single nucleotide polymorphisms
• SNPs: are the most common genetic variations
in human DNA.
• once in every 100 to 300 base pairs
• SNPs occur when one nucleotide base pair
replaces another.
• SNPs are single-base differences that exist
between individuals
• Nucleotide substitution results in two possible
Alleles.
• One allele, typically either the most commonly
occurring allele or the allele originally
sequenced- wild type
• Alternative allele is considered the variant
allele
Nucleotide sequence of the β2-adrenergic receptor gene from codons 13 through 19. A.
Nucleotide sequence of the wild-type allele with adenine (A) at nucleotide position 46 (underlined) located in
codon 16 of the β2-adrenergic receptor gene. The AGA codon designates the amino acid arginine (arg), with an
average frequency of 39% in the human population.

B. Nucleotide sequence of the variant allele with guanine (G) at nucleotide position 46 (underlined), located in
codon 16. The GGA codon designates the amino acid glycine (gly), which occurs at an average frequency of 61%.
Although the arg16 polymorphism occurs less commonly than the gly16 polymorphism, it is referred to as the
wild type because it was identified first
• A SNP may change the codon resulting in amino acid substitution,
which may or may not alter the amount or function of the encoded
protein.

• For example, guanine (G) is substituted for adenine (A) at nucleotide

46 in the β-adrenergic receptor gene.

• This results in the substitution of glycine for arginine at aminoacid

position (codon) 16 and alterations in receptor down regulation
upon prolonged exposure to β2-receptor agonists .
• SNPs such as this that result in amino acid substitution are referred
to as nonsynonymous

• SNPs that do not result in amino acid substitution are called

synonymous.
• If a SNP changes the amount or function of a protein that
contributes to drug response.

• It may alter a patient’s sensitivity to a drug or predispose the patient

to adverse reactions to drug therapy.
 Other types of genetic variants
• Insertion–deletion polymorphisms - in which a nucleotide or
nucleotide sequence either is added to or deleted from a DNA
sequence.
• Tandem repeats:- in which a nucleotide sequence repeats in tandem
(e.g., if “AG” is the nucleotide repeat unit, “AGAGAGAGAG” is a
five-tandem repeat).
• Frame shift mutation:- there is an insertion/deletion polymorphism
and the number of nucleotides added or lost is not a multiple of three,
resulting in disruption of the gene’s reading frame.
• Defective splicing:- in which an internal polypeptide segment is
abnormally removed, and the ends of the remaining polypeptide
chain are joined.

• Aberrant splice site:- in which processing of the protein occurs at an

alternate site.

• Premature stop codon polymorphisms:- in which there is premature

termination of the polypeptide chain by a stop codon
 Sites – SNPs

• Exon, Intron, or Regulatory regions of a gene

• In exon regions may alter the function of a protein

• In regulatory regions may alter the amount of protein that is

produced.

• In the intron region often are silent

 Diseases - polygenic in nature

• Numerous proteins involved in the renin–angiotensin system,

sympathetic nervous system, and renal sodium transport have
been associated with the risk for essential hypertension.

• Environmental factors interact with genetic factors to influence

disease susceptibility and progression.
Polymorphisms In Genes For Drug-metabolizing
Enzymes

• Polymorphisms in the drug-metabolizing enzymes- first recognized &

most documented
• Drug response and toxicity

• Major phase I enzymes are the CYP450 superfamily of

isoenzymes
• Phase II - N-acetyltransferase, uridine diphosphate
glucuronosyltransferase(UGT), and glutathione S-transferase
• ThiopurineS-methyltransferase (TPMT) and dihydropyrimidine
dehydrogenase(DPD) - Nucleotide base-metabolizing Enzymes.
 Cytochrome P450 Enzymes
• 57 different CYP450 isoenzymes presents in humans.
• 42 involved in the metabolism of exogenous xenobiotics and
endogenous substances such as steroids and prostaglandins.

• Fifteen of these isoenzymes are known to be involved

in the metabolism of drugs.

• Significant inter individual variabilities in enzyme activity exist as a

result of induction, inhibition,and genetic inheritance
 Functional genetic polymorphism
• CYP2A6, CYP2C9, CYP2C19, CYP2D6, and more
recently, CYP3A4/5.

• A polymorphism in the regulatory region of the gene

encoding for CYP1A2 has been identified.

• Its functional importance remains to be determined

 CYP2D6
• Polymorphisms in the CYP2D6 gene.
• 48 gene variants and 53 alleles

• CYP2D6 extensive-metabolizer (EM) and poor-metabolizer (PM)

phenotypes can be predicted with up to 99% confidence with six
genotypic variants.

• The gene encoding CYP2D6 is localized on chromosome 22.

• CYP2D6*1 wild-type variant and exhibits normal enzyme activity

• CYP2D6*2 has the same activity as CYP2D6*1 but is capable of

duplication or amplification.

• Both variants are present in EMs

• Poor CYP2D6 metabolizers carry two defective alleles,
such as
• CYP2D6*3, CYP2D6*4 (more common), CYP2D6*5,
and CYP2D6*6.

• Total absence of active enzyme and an impaired ability

to metabolize CYP2D6-dependent substrates.
CYP2D6 Activity in three different
ethnic populations
 Examples of CYP2D6 substrates

• Neuroleptic medications
• Antidepressants:- Tricyclic antidepressants and mianserin
• antiarrhythmic drugs
• β-adrenergic antagonists s- metoprolol

• Overall drug metabolism and the drug’s therapeutic index, clinically

significant side effects may occur in PMs as a result of elevated parent
drug concentrations.
 Examples
• Neuropathy after treatment with the antianginal agent perhexiline .
more in PMs than EMs
• More adverse effects with propafenone and neuroleptic agents
such as perphenazine

• Poor CYP2D6 metabolizers experience little or no analgesic relief

after taking codeine and tramadol

• Codeine and tramadol are converted by CYP2D6 to morphine and

O-desmethyltramadol

• CYP2D6-catalyzed conversion of tamoxifen to the more potent

antiestrogen metabolite 4-hydroxytamoxifen
• Drug abuse - “protected”

 EMs -metabolically convert- PMs

 fluoxetine 20 mg- CYP2D6 inhibitor

Patients who are EMs have a wide range of CYP2D6
Activity
• Ultrarapid metabolizers (UMs) on one end of the
spectrum.

• Subjects with diminished activity on the other end.

• UMs carry a duplicated or amplified mutant allele.

• Two or multiple copies of the functional CYP2D6*1 or

CYP2D6*2 allele - high CYP2D6 activity.
• Nontherapeutic plasma concentrations of nortriptyline- Ums

CYP2D6

• Nortriptyline 10-hydroxynortriptylin
• High prevalence of CYP2D6*10 in Asian population

• Associated with lower enzyme activity

• Higher drug concentrations and/or lower dosage

requirements of neuroleptic medications and mianserin

• Presence of the CYP2D6*17 in African heritage

• Metabolize CYP2D6 substrates at a slower rate

• Cost of treatment and genotyping.

• AmpliChip CYP450 Test
 CYP2C19
Two principal defective alleles
• CYP2C19*2 - aberrant splice site
• CYP2C19*3 - premature stop codon
• which result in inactive CYP2C19 enzymes and the PM phenotype
• PMs – more than 12-fold increase in the AUC of omeprazole compared with EMs.

• Defective CYP2C19 allele improved helicobacter pylori cure rates

after dual or triple therapy with omeprazole as well as with
lansoprazole

• Cure rate 100% in PMs and 60% & 29% in hetro and
homogeneous EMs
 CYP2C9
Warfarin, phenytoin, tolbutamide

• CYP2C9*2 and CYP2C9*3

• Exhibit single amino acid substitutions at positions
critical for enzyme activity
 90% reduction in S-warfarin clearance in CYP2C9*3

• CYP2C9 variant alleles was observed in 81% of patients requiring low-dose

warfarin therapy (<1.5 mg/day).
• CYP2C9*3 allele - 0.5mg/kg
 CYP2A6

• CYP2A6*1 (wild type)

• CYP2A6*2 (single amino acid substitution)
• CYP2A6*3 (gene conversion)—and
• Three gene-deletion alleles—CYP2A6*4A,

CYP2A6*4B ,and CYP2A6*4D.

• Deletion of the CYP2A6 gene is very common in

Asian patients
• Nicotine is metabolized by CYP2A6.
• Clinical relevance of the CYP2A6 polymorphism- nicotine abuse

• Nonsmokers were more likely to carry the defective CYP2A6 allele

than were smokers
• Smokers who had the defective CYP2A6 allele smoked fewer
cigarettes and were more likely to quit.
• defective CYP2A6 allele- inability to metabolize nicotine
• Enhanced nicotine tolerance and increased adverse effects from
nicotine
 CYP3A4/5

• CYP3A subfamily- CYP3A4, CYP3A5, and CYP3A7

• CYP3A4 variants with amino acid substitutions in exons 7 and 12-

altered catalytic activity- nifedipine
• CYP3A5- polymorphic in 60% of African Americans and 33% of white people

• CYP3A5*1 allele, CYP3A5*3(aberrant splice site) in intron 3 have

no functional CYP3A5
 PHASE II AND NUCLEOTIDE-BASE METABOLIZING
ENZYMES
• Genetic polymorphisms in TPMT, DPD,and UGT
enzymes- demonstrated in cancer treatment
• TPMT gene has three mutant alleles

• TPMT*3A(the most common), TPMT*2, and TPMT*3C

• TPMT mutant alleles- serious anemias during mercaptopurine treatment.

• patients with a defective allele of the DPD gene cannot

metabolize 5-fluorouracil- neurotoxicity.
 Examples of Genetic Polymorphisms in Drug-Metabolizing Enzymes
and Response to Drug Therapy
Genetic Variants/Genes Drug Drug Effect Associated with
Polymorphism

CYP2D6*4, CYP2D6*5 Perhexiline Neuropathy

Codeine
Tramadol Significant reduction in analgesic
effect

CYP2D6*2 (n > l) Tricyclic antidepressants Inadequate antidepressant response

(e.g., desipramine,
nortriptyline)

CYP2D6*10 Antipsychotics (e.g., Elevated plasma concentrations and

haloperidol) exaggerated responses

CYP2CP*2, CYP2C9*3 Warfarin Hemorrhage

CYP2C19 Omeprazole Improved cure rates for Helicobacter
pylori

`Glutathione-S-transferase Primaquine Hemolytic reactions

 Examples of Genetic Polymorphisms in Drug-Metabolizing
Enzymes and Response to Drug Therapy
Thiopurine methyltransferase Mercaptopurine Bone marrow depression

N-Acetyltransferase slow Isoniazid More prone to peripheral

acetylator neuropathy
Procainamide More prone to development of
Hydralazine systemic lupus erthematosus-like
syndrome
Sulfonamides Increased hematologic and
gastrointestinal adverse reactions

Uridine diphosphate Irinotecan Increased severity of diarrhea and

glucuronosyltransferase neutropenia in carrier of (TA)
TAA allele
 POLYMORPHISMS IN DRUG TRANSPORTER
GENES
• Drug transport proteins
• Across the gastrointestinal tract,
• Drug excretion into the bile and urine
• Drug distribution across the blood–brain barrier

• Genetic variations in drug transport proteins

• P-glycoprotein - exhibit genetic polymorphism.

• P-glycoprotein is an energy-dependent
transmembrane efflux pump encoded by the ABCB1
gene (MDR 1 gene) – ABC
• P-GP is one of the product of MDR-1 gene , which
is efflux transporter in oral drug absorbtion,
distribution and urinary and biliary excretion of the
drugs.

• It has the functions of –

 Exporting the anti cancer agents from cancer
cells.
 Promoting multi drug resistance to chemotherapy
 Protective role by transporting toxic substrates
out of cells by its location in intestinal enterocytes,
hepatocytes etc.
EFFECT OF P-GP

It affects the distribution of drugs as

Digoxin
Immunosuppressants
cyclosporine
Tacrolimus
antiretroviral protease Inhibitors.
• A number of polymorphisms are seen in MDR1 gene

• Common SNP’s which causes polymorphism occur

in exons 12, 21 and 26 of MDR1 gene.

• The MDR1 exon 21 and 26 polymorphs affect

plasma digoxin concentrations in patients with
cardiac arrythmias, heart failure and digoxin dosing.

• The MDR1 exon 26 polymorphism is associated with

plasma concentrations of protease Inhibitors in
patients with HIV.
• So after therapy for 6 months with Efavirenz/
Nelfinavir more CD4 cell counts observed in
individuals with exon 26 TT genotype [wild]
COMPARED WITH those without.

• Other examples of drug transporting proteins

are
 Dipeptide transporters
 Org anion/ cation transporter
 L-amino acid transporter.
B] GENETIC POLYMORPHISM IN DRUG TARGETS
 Genetic polymorphism occurs for drug target proteins
 receptors
 enzymes
 ion channels
changes in drug effect occurs.

 Drug target genes may work along with genes that affect
PK properties to contribute to overall drug response.

 Exs are that of polymorphisms in -adrenergic receptors

and their sensitivity to -agonists in asthmatics, angiotensin
converting enzyme (ACE) and its sensitivity to ACE
inhibitors, sulphonyl urea receptor and response to OHA’s.
 DRUG EFFECT ASSOCIATED WITH
GENE [exon] DRUG/DRUG CLASS
POLYMORPHISM
Blood pressure reduction and clinical
α-Adducin Hydrochlorothiazide outcomes
ACE Inh Blood pressure reduction, regression of left
ACE ventricular Hypertrophy
Angiotensinogen ACE Inh Blood pressure reduction

B1 receptor B- Blockers Blood pressure lowering

B2 receptor B- Blocker Bronchodialtion

D2 receptor Levodopa Cough

D3 receptor Levodopa dyskinesias

Estrogen receptor Estrogen Bone mineral density

 ADVANTAGES

 Sensitivity to specific medications analysed as gene

sequence varies among individuals. Eg ACE Inh

 Genetic polymorphisms that underlie disease

pathogenesis can also be major determinants of drug
efficacy

Eg- mutations in the apolipoprotein E gene and

responsiveness of patients with Alzheimer's disease to
tacrine therapy.
• Risk of adverse drug effects has been linked to
genetic polymorphisms that predispose to toxicity

Eg- dopamine D3 receptor polymorphism and the

risk of drug-induced tardive dyskinesia , potassium
channel mutations and drug-induced dysrhythmias
etc
B1] RECEPTOR GENOTYPES AND DRUG RESPONSE
 The β1 and β2 receptor genes has been the focus to β
agonist and antagonists.

 B1 receptors- located on heart, kidney- lowers BP.

 There is variation in response to BP lowering agents at
alleles 49 and 389.

The HTN patients having ‘Ser 49 and Arg 389 alleles’’ were
found to have greater reduction in BP than ‘Gly 49 and Gly
389’’.

 This data suggests that β1 genotypes is important

determinant of BP lowering.
 A significant percentage fo HTN patients fail to achieve
adequate BP control with β blockers alone, so finding
genotype is important.

 β-Blockers continued in patients who respond well to β

blockers while other class of drugs used if no response.

 β2 receptors are located on the bloodvessel, smooth

muscles- brochoconstriction and vasodilation.

 Inh β2 agonists most effective for reversal of

bronchospasm, but effects varies among people
 More than 11 SNP’s are identified in B2 receptor genes of which 3
occur commonly.

 Two occur at alleles 16 and 27 and third occur in genes promotor

region.

 Three investigators studied influence of B2 receptor at 16 on

vasodilation ---

• 2 of 3 groups showed that Arg genotypes increase vasodilation.

• 1 showed that Gly receptors increase vasodilation.

• Another group showed that combination of SNP’s in promoter

region - increase vasodilation
 So it shows multiple receptor gene variations
correlates with B2 receptor receptor.

Ex 2- Clozapine is example of drug with evidence of

multiple receptor genes to show effect.

 Clozapine is for Schizophrenia in histamine and

serotonin 2A and 2C receptor genes gives 97%
response- showing that combination of polymorphs
rather than single shows response.

B2] ENZYME GENES AND DRUG RESPONSE

 The ACE is ex of enzyme with genetic contribution to
drug response.
 Pharmacokinetics/Pharmacodynamics (PK/PD)
Considerations and
Pharmacogenetics/Pharmacogenomics (PGt/PGx)

• Unexpected pharmacodynamic responses and pharmacokinetic variations

among individuals during drug therapy involve genetic factors

• Genetic factors contribute variation at many levels, including

drug transport, metabolism, and interaction at the receptor site.
• Applying genetics to study interindividual variations in drug
response may greatly improve drug therapy.

• Understanding and monitoring the underlying pharmacogenetic factors will allow

physicians to optimize efficacy and minimize side effects.

• PGt/PGx is influencing the study of pharmacokinetics and

pharmacodynamics

• Models that will predict dosing and an individual's drug clearance more
accurately will require more details concerning the individual patient's genetic
profile, demographic and pathologic information, as well as the drug's PK
profile
• successful application of genetic screening tests to identify patients with
specific risks in drug response or drug toxicity .

• Genetic diagnostic tests becoming more common and affordable

• It is expected that individual drug dosing will become more accurate and
ultimately result in vast improvements in therapeutic response and better
drug tolerance.

• Use of diagnostic DNA microarrays or gene chips simplify and expand

testing and have clinical applications in
• Disease prevention, drug selection, and dose calculation