Pharmacogenetics

1. PHARMACOGENETICS
Treating Disease Using an Understanding of Genetics
Prepared by:
Devang Parikh
Department of Pharmacology
S.B.K.S. M.I.&R.C.

2. • Introduction of Pharmacogenetics
• Human Genome Project
• Pharmacogenomic effects on few drugs
• Potentials of Pharmacogenomics
• Pharmacogenomics and Drug Development
• Personalized Medicine
• Pharmacogenomics Knowledge Base- website
KEY OBJECTIVES

3. Rx +  = 
Rx +  = 
????
Rx +  = 
Why Pharmacogenetics ???

4. Rx +  = 
Rx +  = 
Differences in genetic constitution
Rx +  = 

5. All patients with same diagnosis
1
2
Responders and patients
not predisposed to toxicity
Non-responders
and toxic
responders
Treat with alternative
drug or dose
Treat with
conventional
drug or dose
The Promise of Personalized Medicine

6.  Pharmacogenetics
› Study of how genetic differences in a SINGLE
gene influence variability in drug response (i.e.,
efficacy and toxicity)
 Pharmacogenomics
› Study of how genetic (genome) differences in
MULTIPLE genes influence variability in drug
response (i.e., efficacy and toxicity)

7. Time line of genomic discoveries

8.  Determine the sequence of the 3 billion nucleotides that
make up human DNA (completed by April 2003)
 Characterize variability in the genome
 Identify all the genes in human DNA
International HapMap Project:
Identifying common haplotypes in four populations from
different parts of the world
Identifying ―tag‖ SNPs that uniquely identify these
haplotypes
A small number of SNP patterns (haplotypes) can
account for 80-90% of entire human population

9.  Genotype: pair of alleles a person has at a
region of the chromosome
 Phenotype: outward manifestation of a
genotype.
 Monogenic: due to allelic variation at a
single gene
 Polygenic: due to variations at two or more
genes

10.  Mutation: difference in the DNA code that occurs
in less than 1% of population
› Often associated with rare diseases
 Cystic fibrosis, sickle cell anemia,
Huntington’s disease
 Polymorphism: difference in the DNA code that
occurs in more than 1% of the population
› A single polymorphism is less likely to be the
main cause of a disease
› Polymorphisms often have no visible clinical
impact

11. Types of Polymorphisms
 Single Nucleotide
Polymorphism (SNP): GAATTTAAG
GAATTCAAG
 Simple Sequence Length
Polymorphism (SSLP): NCACACACAN
NCACACACACACACAN
NCACACACACACAN
 Insertion/Deletion: GAAATTCCAAG
GAAA[ ]CCAAG

12. DRUG
TARGETS
DRUG
METABOLIZING
ENZYMES
DRUG
TRANSPORTERS
PHARMACOKINETICSPHARMACODYNAMICS
Variability in
Efficacy/Toxicity
•Transporters
•Plasma protein binding
•Metabolising enzymes
•Receptors
•Ion channels
•Enzymes
•Immune molecules

13. Polymorphisms
Drug metabolism
Adverse Drug
Reaction
Disease
susceptibility
Receptor
sensitivity
Drug transport
Responders/
Non-responders
Consequences of polymorphisms

14. These mutations may have
 no effect on enzyme activity(normal)
 Lead to enzyme activity with
Decreased activity
Absent activity
 Duplications lead to increased enzyme activity
 Wild or normal activity enzymes (75 – 85%) of
population
 Intermediate metabolizers (10 -15%)
 Poor metabolizers (5 – 10%)
 Ultra-rapid metabolizers (2 – 7%) of population –
multiple genes

15. Genetic mechanism influence pharmacotherapy
1 - Genetic Polymorphism of genes  which results in
Altered metabolism of drugs (metabolism of TCAs)
Increased or decreased metabolism of a drug may change its
concentration
Of active, inactive or toxic metabolites

16. DRUG TRANSPORTERS

17.  MDR1 encodes a P-glycoprotein (an energy-
dependent transmembrane efflux pump)
There are 7 different ABC transporters
MDR1 is important among them.
Expressions of P-glycoprotein in different tissues
P-glycoprotein serves
a protective role by
transporting toxic
substances
or metabolites out of
cells.

18. Increased intestinal expression of P-glycoprotein
•limit the absorption of P-glycoprotein substrates,
•thus reducing their bioavailability and preventing
attainment of therapeutic plasma concentrations.
Decreased P-glycoprotein expression result in
•supratherapeutic plasma concentrations of relevant drugs
•Thus produces drug toxicity.
 Polymorphism in Exon 26(C3435T), Exon
21(G2677T/A) significantly affect P-gp expression.

19. 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
Substrates of P-glycoprotein
Dipeptide transporter, organic anion
and cation transporters, and
L-amino acid transporter.
Other Polymorphic
Drug Transporters

20. Drug Transport

21. 2 – Genetic variants may produce  unexpected drug
effect (toxicity or anaphylactic reaction)
Hemolysis in glucose -6 –phosphate dehydrogenase
deficiency
3 – Genetic variation in drug targets
May alter the clinical response & frequency of side
effects
Variants of β –adrenergic receptor alter response to β –
agonists in asthma patients

22. DRUG METABOLISM

23.  Evidence of an inherited basis for drug response
dates back in the literature to the 1950s
› Succinylcholine: 1 in 3000 patients developed
prolonged muscle relaxation.
•usual paralysis lasted 2 to 6 min in patients.
•occasional pt exhibited paralysis lasting hrs
•cause identified as an ―atypical‖ plasma cholinesterase
(1/100 affinity than normal enzyme)
Hydrolysis by
pseudocholinesterase
choline succinylmonocholine
O C CH2CH2
O
(H3C)3NH2CH2C C
O
O CH2CH2N(CH3)3
+ +
SUCCINYLCHOLINE

24. 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

25. 1A2
19%
2D6
3%
2E1
10%
3A4/5
42%
2C9
2C19
26%
1A2
5%
2D6
24%
2E1
1%
3A4/5
51%
2C9
2C19
19%
Primary CYP Enzymes in Drug Metabolism
% of total enzyme % of drugs
metabolised

26. CYP2C9: Phenytoin, warfarin, NSAIDs etc
CYP2C19: Omeprazole, proguanil, diazepam
CYP2D6: More than 60 drugs
CYP2E1: Ethanol
CYP1A6: Nicotine
Phase - I enzymes known to have
polymorphism

27. 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
Mutant alleles of Phase I enzymes
Red: Absent; Blue: Reduced; Green: Increased activity

28.  NAT2: Isoniazid, hydralazine,
 GST: D-Penicillamine
 TPMT: Azathioprine, 6-MP
 Pseudocholinesterase: Succinyl choline
 UGT1A1: Irinotecan

29. 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;
Mutant alleles of Phase II enzymes

32. Normal CYP2D6 : 150 mg/day
Mutant CYP2D6 : 10-20 mg/day

33. RECEPTOR SENSITIVITY

34. Receptor Sensitivity/Effect
1 receptor gene
Arg389Gly
Ser49Gly
Subjects with Gly 389 have reduced
sensitivity to beta-blockers
Subjects with Gly 49 have increased
sensitivity to beta-blockers
2 receptor gene
Arg16Gly
Gln27Glu
Response to salbutamol is 5.3 fold
lower in Gly16 asthmatics.
Subjects with Glu27 have strong
resistance to beta 2 agonists

35. 10 fold difference in concentration required between genotypes(adenylyl
cyclase activity)

36. RESPONDERS &
NON-RESPONDERS

37. Disease
Gene and
Polymorphism
Allele/
Genotype
Effect
Asthma
ALOX5
Promoter region
mut
Respond poorly to
antileukotriene
treatment with ABT-
761
Atherosclerosis
CETP
TaqIB B2/B2
Poor response to
treatment with
pravastatin
Smoking
cessation
CYP2B6
C1459T TT
Greater craving for
cigarettes and
higher relapse rates
Heart failure
2 AR gene
Gln27Glu Glu27 Better response to
carvedilol treatment

38. ADVERSE DRUG REACTIONS

39.  Inter –individual difference in genetic
constitution
 inter ethnic group variability
49% of ADRs are associated with Drugs that are
substrates for Polymorphic Drug metabolising enzyme.

40. Subjects who are carriers of at least one
mutant allele (*2 or *3) are 4 times more
susceptible to bleeding complications
in spite of low dose administration

41. • 1º and 2º prevention of venous blood clots
• patients with prosthetic heart valves or atrial fibrillation
• 1º prevention of acute myocardial infarction in high-risk men
• prevention of stroke, recurrent infarction, or death in patients
with acute myocardial infarction
• has a narrow therapeutic window
• considerable variability in dose response among subjects
• subject to interactions with drugs and diet
• laboratory control that can be difficult to standardize
• problems in dosing as a result of patient nonadherence
Warfarin- anti-coagulant therapy

42. • prothrombin time and the international normalized ratio (INR)
are monitored
• doses are adjusted to maintain each patient's INR within a
narrow therapeutic range(2.5-3.5)
• INR of < 2 is associated with an increased risk of
thromboembolism
• INR of > 4 is associated with an increased risk of bleeding
Clinical management

43. Warfarin, which is metabolized by CYP2C9, inhibits the vitamin K cycle via actions on
thiol-dependent enzymes, such as VKORC1, that are required for regeneration of active
vitamin K
Pereira, N. L. and Weinshilboum, R. M. (2009) Cardiovascular pharmacogenomics
and individualized drug therapy Nat. Rev. Cardiol. doi:10.1038/nrcardio.2009.154

44. Clearance of S-warfarin and
time to achieve steady-state (5x
T1/2)
*1/*1: ~ 3 days
*1/*2: ~ 6 days
*1/*3: ~ 12 days
Haplotype A (-1639GA, 1173CT):
lower maintenance dose
Haplotype B (9041GA): higher
maintenance dose
VKORC1 A/A: 2.7 ± 0.2 mg/d
VKORC1 A/B: 4.9 ± 0.2 mg/d
VKORC1 B/B: 6.2 ± 0.3 mg/d
Mean maintenance dose: 5.1 ± 0.2
mg/d
principal enzyme that catalyzes the
conversion of S-warfarin to inactive 6-
hydroxy and 7-hydroxy metabolites
Converts inactive Vit K in to active
Vit K hydroquinone

45.  Patients having TPMT*2, *3A and *3C
alleles have low enzyme activity
 They are at risk for excessive toxicity,
especially fatal myelosuppression,
even at standard dose of azathioprine,
mercaptopurine and thioguanine

46. Drugs Demonstrated to Precipitate Hemolytic Anemia
in Subjects with G6PD Deficiency
Nitrofurantoin Primaquine Dapsone
Methylene Blue Sulfacetamide Nalidixic Acid
Naphthalene Sulfanilamide Sulfapyridine
Sulfamethoxazole
INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT
ETHNIC POPULATIONS
Ethnic Group Incidence(%)
Asiatics
Chinese 2
Filipinos 13
Indians-Parsees 16
Japanese 13

47. 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

48. DISEASE SUSCEPTIBILITY

49. Disease Gene Polymorphism
Allele/
Genotype
Effect
Hypertension
AGT M235T T allele  BP
ACE ACEI/D DD  risk
AT1R A1166C C  risk
β1 AR Arg389Gly Arg389
 risk
Atherosclerosis CETP TaqIB B2/B2  risk
Genetic polymorphism & disease susceptibility

50. Disease Gene
Allele/
Genotype
Effect
Acute MI
CYP2C9
eNOS
*3
T786C
susceptibility to AMI.
Alzheimer’s
disease
ApoE
ε 2
ε 4/ ε4
Reduced risk
Poor prognosis
Cancer
GST
M1 Null
T1 Null
 susceptibility to lung
and bladder cancer
NAT NAT2 *10
 susceptibility to
colorectal cancer

51. Drugs Demonstrated to Precipitate Hemolytic Anemia
in Subjects with G6PD Deficiency
Nitrofurantoin Primaquine
Methylene Blue Sulfacetamide Nalidixic Acid
Naphthalene Sulfanilamide Sulfapyridine
Sulfamethoxazole
INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT ETHNIC
POPULATIONS
Ethnic Group Incidence(%)
Asiatics
Chinese 2
Filipinos 13
Indians-Parsees 16
Japanese 13

52. Pharmacogenomic Biomarkers as Predictors of
Adverse Drug Reactions
Gene Relevant Drug
TMPT 6-mercaptopurines
UCT1A1*28 Irinotecan
CYP2C0 and VKORC1 Warfarin
CYP2D6 Tricyclic antidepressants
Beta blockers
Tamoxifin
CYP2C19 Omperazole
HLA-B5701 Abacavir
HLA-B1502 Carbamazepine
HLADRB1*07 and DQA1*02 Ximelagatran
MDR1 Protease inhibitors
ADRB1 Beta blockers
ADRB2 B agonists
ADD1 Diuretics
Ion channel genes QT prolonging antiarrhythmics
RYR1 General anesthetics
CRHR1 Inhaled steroids
HMGCR Statins
Adapted from: Ingelman-Sundberg M. N Engl J Med 358:637-639, 2008.
Roden DM et al. Ann Intern Med 145:749-57, 2006.

53. Biomarker Drugs Associated with this Biomarker
C-KIT expression Imatinib mesylate
CCR5 -Chemokine C-C motif
receptor
Maraviroc
CYP2C19 Variants Clopidogrel; Voriconazole; Omeprazole; Pantoprazole;
Esomeprazole; diazepam; Nelfinavir; Rabeprazole
CYP2C9 Variants Celecoxib; Warfarin
CYP2D6 Variants 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
Deletion of Chromosome 5q(del(5q) Lenalidomide
DPD Deficiency Capecitabine; Fluorouracil Cream; Fluorouracil Topical
Solution & Cream
EGFR expression Erlotinib; Cetuximab; Gefitinib; Panitumab
Familial Hypercholesterolemia Atorvastatin
G6PD Deficiency Rasburicase; Dapsone; Primaquine; Chloroquine
Her2/neu Over-expression Trastuzumab; Lapatinib
HLA-B*1502 allele presence Carbamazepine
HLA-B*5701 allele presence Abacavir
KRAS mutation Panitumumab; Cetuximab
NAT Variants Rifampin, isoniazid, and pyrazinamide; Isosorbide dinitrate
and Hydralazine hydrochloride
Philadelphia Chromosome-positive
responders
Busulfan; Dasatinib; Nilotinib
PML/RAR alpha gene expression Tretinoin; Arsenic Oxide
Protein C deficiencies Warfarin
TPMT Variants Azathioprine; Thioguanine; Mercaptopurine
UGT1A1 Variants Irinotecan; Nilotinib
Urea Cycle Disorder (UCD)
Deficiency
Valproic acid; Sodium Phenylacetate and Sodium Benzoate;
sodium phenyl butyrate
Vitamin K epoxide reductase
(VKORC1) Variants
Warfarin

54. Routine Use of Genetics is Coming Soon!
• Good prognosis vs. poor prognosis
• Which patients need more intensive or longer therapy
• Which patients should receive specific types of therapy
• Which patients should not receive specific types of therapy

55. • How Using Genetics Can Improve Medical Safety
and Efficacy
• Rapidly expanding field that will have a major
impact on how we treat diseases
• Help identify who will respond to a specific therapy
• Help identify who is at risk for side effects of
treatment
• Help identify the appropriate dosing for individual
patients
• Assist in determining which patients are or are not
good candidates for a specific type of therapy

56.  Creating opportunities to increase the value
of the drugs we develop using genetics
› Distinguish subgroups of patients who
respond differently to drug treatment
› Aid interpretation of clinical study results
› Obtain greater understanding of disease
 Predict disease severity, onset, progression
 Identify genetic subtypes of disease
 Aid in discovery of new drug targets

57.  Genome wide approach versus candidate gene
approach
 Thousands of SNPs
 Thousands of patients
 Replication studies
 Sophisticated databases housing pharmacogenomic
information
 Drug selection and dosing algorithms incorporating non-
genetic and genetic information
 Integrating genetics with other technologies
 Transcriptomics, Proteomics, Metabonomics, Imaging, PK/PD
modelling
 A combined approach to diagnosis & prescription

58.  80% of products that enter the development
pipeline FAIL to make it to market
 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

59. Idea
Marketed
Drug
Years
11-15 Years
Discovery Exploratory Development Full Development
Phase I Phase II Phase III
0 155 10
Patent life 20 years
Phase IV
It costs >$800 million to get a drug to market

60. Applying Pharmacogenomics
.
DISEASE
GENETICS
TARGET
VARIABILITY
SELECTING
RESPONDERS
PHARMACO-
GENETICS
Discovery Development
Choosing the
Best Targets
Better
Understanding of
Our Targets
Improving Early
Decision Making
Predicting
Efficacy and
Safety

61. Current Options Options with Pharmacogenomics
Proportionofpatientsshowing
poorornoresponse
Low
High
Continue clinical trials
to market
Abandon drug
before market
Optimize clinical trials,
making them
smaller and shorter
Continue trials safely
by excluding at-risk pts

62.  Targeted Therapies:
› Herceptin: treatment of HER2 positive metastatic
breast cancer
› Gleevec: treatment for patients with Philadelphia
chromosome-positive chronic myeloid leukemia
› Erlotinib: treatment for non-small cell lung cancer
 Most effective in epidermal growth factor receptor positive
tumors
› Maraviroc (not approved): treatment for HIV
 Studies have incorporated a screening process for different
receptors that HIV uses to gain access to cells
› Iloperidone (not approved): schizophrenia treatment
 Company identified a genetic marker that predicts a good
response to the drug

63.  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

64. Patient requires Treatment
Examination by the Physician
Genomic testing Traditional
investigations
EXPERT SYSTEM
Decision making by Physician, assisted by an
Expert System (interactive interpretation)
Prescribes individualized drug treatment

65. Roche Diagnostics Launches the
AmpliChip CYP450 in the US,
- the World’s First Pharmacogenomic
Microarray for Clinical Applications

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

67. elusive dream
or
imminent
reality?