Pharmacogenetics presentation...............

1. PHARMACOGENETICS
DR.SOURAV CHAKRABARTY
PGT(MD)
DEPT OF PHARMACOLOGY
B.S. MEDICAL COLLEGE.

2. INDEX
1. INTRODUCTION.
2. PHARMACOGENETICS,PHARMACOGENOMICS
&’PERSONALISED MEDICINE.’
3. REVIEW OF ELEMENTARY GENETICS.
4. HISTORY OF PHARMACOGENETICS.
5. EFFECTS ON DRUG RESPONSE BY GENES.
6. DRUG DEVELOPMENT & PHARMACOGENETICS.
7. USES OF GENETIC METHODS TO IDENTIFY VARIED
DRUG RESPONSE.
8. PHARMACOGENETICS IN CLINICAL PRACTICE
9. CONCLUSION

3. INTRODUCTION
• Drug Response :Environmental factors and genetic
factors.
• Pharmacogenetic disorders(plasma cholinesterase
deficiency, acute intermittent porphyria, drug
acetylation deficiency and aminoglycoside ototoxicity.
• Pharmacogenomic tests:Tests for variations in human
leukocyte antigen (HLA) genes.
• Genes influencing drug metabolism.
• Drug targets such as the epidermal growth factor
receptor HER2, tyrosine kinase inhibitors and the main
target for warfarin, vitamin K epoxide reductase
(VKOR).

4. Exogenous & Endogenous factors contribute to variation in drug response

5. CONTD…………..
• every year about 2 million people are hospitalized for drug
adverse reactions. And every year 100,000 people die
because of these reactions.
• This makes it the 6th leading cause of death worldwide
• 49% of adverse drug reactions associated with drugs that
are substrates for polymorphic drug metabolizing enzymes.
• Interindividual variation :can be
pharmacokinetic/pharmacodynamic/ idiosyncratic.
• If not taken into account, can result in lack of efficacy or
unexpected side effects
• Twin studies:very useful to explore genetic basis of drug
response variation.

6. • Pharmacogenetic contribution to pharmacokinetic
parameters. t1/2 of antipyrine is more concordant in identical
in comparison to fraternal twin pairs. Bars show the t1/2 of
antipyrine in identical (monozygotic) and fraternal (dizygotic)
twin pairs. (Redrawn from data in Vesell and Page, 1968.)

8. • PHARMACOGENETICS = Pharma and genetics
• Pharma the Greek word i.e. PHARMACON, related to
Drugs.
• Genetics related to genes / genome
• The study of the genetic basis for variation in drug
response.
• PHARMACOGENOMICS: Surveying the entire
genome to assess multigenic determinants of drug
response.
• PERSONALISED MEDICINE: Individualising drug
therapy in light of genomic information.

9. • To use genetic information specific to an
individual patient to preselect a drug that will
be effective and not cause toxicity.
• Better than relying on trial and error
supported by physical clues.
• USFDA :Addition of pharmacogenomics
labelling information to the package inserts of
over 50 drugs.

10. PERSONALIZED MEDICINE
Understanding human
genome
Simpler methods
identify genetic
information
Genetic information
specific to individual
Preselect
effective drug
No
toxicity
No trial
& error

12. REVIEW OF ELEMENTARY GENETICS
Definitions:
a gene is the basic instruction—a sequence of nucleic
acids (DNA or, in the case of certain viruses RNA), while
an allele is one variant of that gene. Referring to having
a gene for a disease for example, sickle-cell
disease is caused by a mutant allele of a haemoglobin
gene.
• 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

13. CONTD………..
• Mutations :Heritable changes in the base sequence of DNA.
• Occur during crossing over of DNA during Meiosis.
• Polymorphism :Variation in the DNA sequence that is present at an
allele frequency of 1% or greater in a population.
• Arise initially because of a mutation.
• If nonfunctional stable.
• If disadvantageous die out during subsequent generations .
• Two major types: single nucleotide polymorphisms (SNPs) and
insertions/deletions (indels)
• cosmopolitan or population (or race and ethnic) specific.
• 95% of the genome is intergenic, most polymorphisms are unlikely
to directly affect the encoded transcript or protein.
• Most pharmacogenetic traits are multigenic rather than monogenic.

14. MARKERS OF GENETIC
VARIATION
Types of Polymorphisms
• Single Nucleotide
Polymorphism (SNP): GAATTTAAG
GAATTCAAG
• Insertion/Deletion: GAAATTCCAAG
GAAA[ ]CCAAG

15. CONTD……………
• SNPs occur every 100–300 bases along
the 3 billion base human genome.
• The greatest number of DNA variations
associated with diseases or traits are
missense and nonsense mutations,
followed by deletions.

16. HISTORY
Time line of genomic discoveries

17. CONTD………
• First pharmacogenetic examples to be discovered was
glucose-6-phosphate dehydrogenase (G6PD) deficiency.
• Albinism and ‘Inborn errors of metabolism’ in the early part
of the 20th century by Archibald Garrod, a British physician
who initiated the study of biochemical genetics.
• In the 1950s Walter Kalow discovered atypical
cholinesterase while studying suxamethonium sensitivity.
• Detected by a blood test that measures the effect of the
inhibitor dibucaine .
• Malignant Hyperpyrexia:Mutation of the Ryanodine receptor,
located on sarcoplasmic reticulum mediate the release
of calcium ions resulting in a drastic increase in intracellular
calcium thus, muscle contraction .
• Triggered by exposure to certain drugs used for general
anesthesia (Halothane etc)

18. CONTD………….
• Acute intermittent prophyria .
• use of sedative, anticonvulsant or other drugs in patients
with undiagnosed porphyria can be lethal. CYP inducer i.e.
barbiturates, griseofulvin, carbamazepine, estrogen can
precipitate acute attacks in susceptible individuals.
• fast acetylators’ and ‘slow acetylators’ of Isoniazid.
• The N – acetyl transferase (NAT) enzyme is controlled by
two genes, (NAT 1) and (NAT 2) of which NAT2 A and B are
responsible for clinically significant metabolic
polymorphism.
• Fast:peripheral neuropathy
• Slow:hepatotoxicity,
• Aminoglycoside ototoxicity:Mitochondrially inherited.
• 1970s and 1980s:debrisoquine &(CYP2D6) deficiency was
isolated.

19. EFFECTS OF GENES ON DRUG
RESPONSE
• PHARMACOKINETIC:
• Too much/not enough drug @site of action.
I. Metabolism
II. Transporters
III. Plasma protein binding
1. Thiopurine drugs (Tioguanine, Mercaptopurine and its prodrug
Azathioprine) and TPMT(Thiopurine-S-methyltransferase) activity:Bone
marrow and liver toxicity.
• About 1 in 300 Caucasians and African-Americans are TPMT- deficient
2. 5-Fluorouracil (5-FU) and DPYD(dihydropyrimidine dehydrogenase )
activity:Decreased metabolism
leukocytopenia, stomatitis, diarrhea, nausea and vomiting.
3. Tamoxifen AND CYP2D6:
4. Irinotecan AND UGT1A1*28: In Gilbert’s syndrome,50 fold reduction in
irinotecan metabolism and such patients can be at risk of toxicity.

20. DRUG METABOLIZING ENZYMES
Phase I: biotransformation reactions: oxidation, hydroxylation, reduction, hydrolysis
Phase II: conjugation reactions—to increase their water solubility and elimination from the
body. The reactions are glucuronidation, sulation,acetylation, glutathione conjugation

21. CYP450 CONTENT IN HUMAN LIVER
Low levels of P4502D6 & P4502C19
P4502D6
Other
P4501A2
P4502C19 P4502E1
P4502B6 P4502A6
P4502C9
P4502C8
P4503A4

22. MUTANT ALLELES OF PHASE I ENZYMES
CYP 450
gene
Mutant Alleles Substrates
CYP2C9*1 *2, *3, *4, *5, *6
Warfarin, losartan
phenytoin, tolbutamide
CYP2C19*1
*2, *3, *4, *5,
*6, *7, *8
Proguanil, Imipramine,
Ritonavir, nelfinavir,
cyclophosphamide
CYP2D6*1
*1XN, *2XN,
*3,*4,*5, *6
*9,*10,*17
Clonidine, codeine,
promethazine,
propranolol, clozapine,
fluoxetine, haloperidol,
amitriptyline
Red: Absent; Blue: Reduced; Green: Increased activity

23. MUTANT ALLELES OF PHASE II ENZYMES
Gene Mutant Alleles Substrates
NAT2
*2, *3, *5, *6,*7,
*10,*14
Isoniazid, hydralazine,
GST
M1A/B, P1
M1 null, T1 null
D-penicillamine
TPMT *1,*2,*3A,C, *4-*8 Azathioprine, 6-MP
UGT1A1 *28 Irinotecan
Red: Absent; Blue: Reduced;

24. GENETIC POLYMORPHISM BASED ON DRUG METABOLIZING ABILITY
PHENOTYPE GENOTYPE EFFECTS
A. extensive or normal
drug metabolizers (EM)
(75 – 85%)
homozygous or
heterozygous for wild type
allele.
Normal metabolism.No
dose modification needed.
B.intermediate metabolizer
phenotype (IM) (10 -
15%)
heterozygous for the wild
type allele
may require lower than
average drug dose for
optimal therapeutic
response.
C. poor metabolizers (PM)
(5 – 10%)
mutation or deletion of
both alleles
accumulation of drug
substrates in their systems
with attendant effects.
D. ultrarapid metabolizers
(UM) (2 – 7%)
gene amplification . drug failure

25. GENETIC VARIATION IN DRUG RECEPTOR:
(i)ATP binding cassette (ABC) family :
I. multi drug resistance gene also classified as ABCB 1 i.e.
(ABCB1/MDR1), :MDR1 encodes a P-glycoprotein (an
energy-dependent transmembrane efflux pump)
II. ABCC1, ABCC2, uric acid transporter (ABCG2),
III. breast cancer resistance protein BCRP also classified ABCG2
i.e. (BCRP/ABCG2).
(ii) The solute transporter superfamily (SLC):
I. organic anion transport polypeptide (SLC 21/OATP),
II. organic cation transporter SLC 22 OCT),
III. zwitterion/cation transporter (OCTNs),
IV. folate transporter(SLC19A1),
V. neurotransmitter transporter (SLC6,SLC17,&SLC18)
VI. serotonin transporter (5HTT).
• Important roles in the GI absorption,biliary and renal
elimination and distribution to target sites of their substrates.

26. SUBSTRATES OF P-GLYCOPROTEIN
Category Substrates of P-gp
Anti-cancer agents Actinomycin D, Vincristine,etc
Cardiac drugs Digoxin, Quinidine etc
HIV protease inhibitors Ritonavir, Indinavir etc
Immunosuppressants Cyclosporine A, tacrolimus etc
Antibiotics Erythromycin,levofloxacin etc
Lipid lowering agents Lovastatin, Atorvastatin etc
Dipeptide transporter, organic anion
and cation transporters, and
L-amino acid transporter.
Other Polymorphic
Drug Transporters

27. PHARMOCODYNAMIC
•
• Receptors
• Ion channels
• Enzymes
• Immune molecules
• Drug target-related genes.
1. TRASTUZUMAB AND HER2 receptor:EGF antagonist that binds
Human epidermal growth factor receptor 2—HER2.
2. DASATINIB, IMATINIB AND BCR-ABL1 receptor:A mutation (T315I) in
BCR/ABL confers resistance to the inhibitory effect of dasatinib and
patients with this variant do not benefit from this drug.
. Combined (metabolism and target)gene tests:
Warfarin and CYP2C9 & VKORC1(vitamin K epoxide reductase )
genotyping:

28. G-protein Coupled Receptors (GPCR):Over 50% of all drug targets
have G-protein coupled receptors (GPCR). Genes of GPR has more
coding regions than non – GPCR genes making them more
important for pharmacological investigations.
GABAA Receptor Mutation in GABAA receptor ion
channel:diminished protection of anti epileptic drugs.
Insulin Receptor(INSR):Mutation of the gene encoding the receptor
will result in poor response particularly in type 2 diabetes.Also
contribute to genetic susceptibility to the polycystic ovarian
syndrome.
B2 Receptor:Patients with B2 receptor arginine genotype
experience poor asthma control with frequent symptoms and a
decreasing scores of poor exploratory volume compared with those
with glycine genotype.17% of whites and 20% of blacks carry the
arginine genotype

29. Neurotransmitter Transporters: SLC6, SLC17 and SLC18 families.
•sites of action of various drugs of abuse e.g cocaine, amphetamine and other
clinically approved drugs like desipramine, reserpine, benztropine and
tiagabine.
•Genetic variation may affect the efficacy of such drugs.
Ion Channels:KCNJ10, KCNJ3, CLCN2, GABRA1, SCN1B and SCN1A.
•Some polymorphism of this channel has been linked to idiopathic generalized
epilepsy.
•The 5-HT3 receptor is a ligand-gated ion channel composed of five subunits.
• five different human subunits are known; 5-HT3A-E, which are encoded by
the serotonin receptor genes HTR3A, HTR3B, HTR3C, HTR3D and HTR3E,
respectively.
•Functional receptors are pentameric complexes of diverse composition.
•Different receptor subtypes seem to be involved in chemotherapy-induced
nausea and vomiting (CINV), irritable bowel syndrome and psychiatric
disorders.
• 5-HTR3A and HTR3B polymorphisms may also contribute to the etiology of
psychiatric disorders and serve as predictors in CINV and in the medical
treatment of psychiatric patients.

30. IDIOSYNCRATIC
1. ABACAVIR AND HLAB*5701:severe rashes.
2. ANTICONVULSANTS AND HLAB*1502:severe
life-threatening rashes including Stevens
Johnson syndrome and toxic epidermal
necrolysis .
3. CLOZAPINE AND HLA-DQB1*0201:
agranulocytosis

31. PHARMACOGENOMIC BIOMARKERS AS
PREDICTORS OF ADVERSE DRUG REACTIONS
Gene Relevant Drug
TMPT 6-mercaptopurines
UCT1A1*28 Irinotecan
CYP2C0 and VKORC1 Warfarin
CYP2D6 Atomoxetine; Venlafaxine; Risperidone; Tiotropium
bromide inhalation; Tamoxifen; Timolol Maleate;
Fluoxetine HCL; Olanzapine; Cevimeline hydrochloride;
Tolterodine; Terbinafine; Tramadol; Acetamophen;
Clozapine; Aripiprazole; Metoprolol; Propranolol;
Carvedilol; Propafenone; Thioridazine; Protriptyline HCl;
Tetrabenazine; Codeine sulfate; Fiorinal with Codeine;
Fioricet with Codeine
CYP2C19 Omperazole
HLA-B5701 Abacavir
HLA-B1502 Carbamazepine
G6PD Deficiency Rasburicase; Dapsone; Primaquine; Chloroquine
MDR1 Protease inhibitors
ADD1 Diuretics
Ion channel genes QT prolonging antiarrhythmics
CRHR1 Inhaled steroids

32. Polymorphism-Modifying Diseases and
Drug Responses:
• MTHFR polymorphism, for example, is linked to
homocysteinemia, which in turn affects thrombosis
risk.
• . polymorphisms in ion channels (e.g., HERG, KvLQT1,
Mink, and MiRP1) affect risk of cardiac dysrhythmias,
accentuated in the presence of a drug prolonging QT
interval(macrolide antibiotics, antihistamines.
• Polymorphisms in HMG-CoA reductase degree of
lipid lowering following statins and degree of positive
effects on high-density lipoproteins among women on
HRT.

33. PHARMACOGENETICS AND DRUG
DEVELOPMENT
• Pharmacogenomics may contribute to a “smarter” drug
development process
– Allow for the prediction of efficacy/toxicity during clinical
development
– Make the process more efficient by decreasing the number of
patients required to show efficacy in clinical trials
– Decrease costs and time to bring drug to market.
• Genome-wide approaches hold promise for identification of new
drug targets and therefore new drugs.
• To identify which genetic subset of patients is at highest risk for a
serious ADR, and to avoid testing the drug in that subset of
patients.
• Usually dosing alteration done,NOT drug preclusion.

34. THERAPEUTIC DRUGS AND CLINICALLY
AVAILABLE PHARMACOGENOMIC TESTS:
• Tests for
• (a) variants of different human leukocyte antigens
(HLAs),strongly linked to susceptibilities to several severe
idiosyncratic reactions;
• (b) genes controlling aspects of drug metabolism;
• (c) genes encoding drug targets
• Mostly use germline DNA, that is, DNA extracted from any
somatic, diploid cells, usually white blood cells or buccal
cells (due to their ready accessibility).
• Usually made on venous blood samples which contain
chromosomal and mitochondrial DNA in white blood cells
• The genomic tests are performed on DNA from samples of
the tumour obtained surgically.

35. CONTD………
• amplification of the relevant sequence(s) and
molecular biological methods, often utilising chip
technology, to identify the various polymorphisms
• BUT, relatively few are used routinely in patient care.
• Because genomic variability is so common (with
polymorphic sites every few 100 nucleotides), "cryptic"
or unrecognized polymorphisms may interfere with
oligonucleotide annealing, thereby resulting in false
positive or false negative genotype assignments.
• It is important to select polymorphisms that are likely
to be associated with the drug-response phenotype.

36. LIMITATIONS OF PHARMACOGENETICS
• Complex targeting due to multiple gene
involvement
• Difficult and time consuming to identify small
variations in genes
• Interaction with other drugs and environment
to be determined

37. PHARMACOGENETICS IN CLINICAL PRACTICE
• . The development has been slowed by various scientific,
commercial, political and educational barriers.
• 3 major types of evidence that should accumulate in order
to implicate a polymorphism in clinical care.
A. Screens of tissues from multiple humans linking the
polymorphism to a trait;
B. Complementary preclinical functional studies indicating
that the polymorphism is plausibly linked with the
phenotype;
C. Multiple supportive clinical phenotype/genotype studies
• Ideal example:Impact of the polymorphism in TPMT on
mercaptopurine dosing in childhood leukemia.

38. CONTD………
• Most drug dosing takes place using a population
"average" dose of drug.
• Much more hesitation from clinicians to adjust
doses based on genetic testing.
• Broad public initiatives,i.e.NIH-funded
Pharmacogenetics and Pharmacogenomics
Knowledge Base provide useful resources to
permit clinicians to access information on
pharmacogenetics.
• Complexity of dosing will be likely to increase
substantially in the postgenomic era.

39. Pharmacogenetics and Pharmacogenomics
Knowledge Base (PharmGKB)
• Publicly accessible knowledge base
– www.pharmgkb.org
• Goal: establish the definitive source of information
about the interaction of genetic variability and drug
response
1. Store and organize primary genotyping data
2. Correlate phenotypic measures of drug response with
genotypic data
3. Curate major findings of the published literature
4. Provide information about complex drug pathways
5. Highlight genes that are critical for understanding
pharmacogenomics

40. ..what many thought would not happen has
already happened
Roche Diagnostics Launches the
AmpliChip CYP450 in the US,
- the World’s First Pharmacogenomic
Microarray for Clinical Applications

41. CONCLUSION
• Nonetheless, the potential utility of pharmacogenetics
to optimize drug therapy is great.
• Advantage They need only be conducted once
during an individual's lifetime.
• With continued incorporation of pharmacogenetics
into clinical trials, the important genes and
polymorphisms will be identified.
• Refinement of dosing in the context of drug
interactions and disease influences.
• More precise ‘personalised’ therapeutics for several
drugs and disorders.

42. S M A R T C A R D
Person’s name
GENOME
Personalized
medicine
(Confidential)
“Here is my
sequence”

44. BIBLIOGRAPHY
1. THE PHARMACOLOGICAL BASIS OF
THERAPEUTICS ,GOODMAN & GILMAN,12TH
EDITION,2011,PAGE 145-165.
2. RANG & DALE’S PHARMACOLOGY,7TH
EDITION,2012,PAGE 132-137.
3. METHODS IN MOLECULAR BIOLOGY,VOL
448,PHARMACOGENOMICS IN DRUG
DISCOVERY & DEVELOPMENT,GARY
HARDIMAN,PAGE 21-29.

45. Thank You for
your Attention!