PHARMACOGENETICSTreating Disease Using an Understanding of Genetics
Prepared by: Devang Parikh Department of Pharmacology S.B.K.S. M.I.&R.C.
• 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
Pharmacogenetics
Rx + =
Rx + = ????
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Why Pharmacogenetics ???
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Differences in genetic constitution
Rx + =
Why Pharmacogenetics ???
All patients with same diagnosis
1
2Responders and patients not predisposed to toxicity
Non-respondersand toxic
responders
Treat with alternativedrug or dose
Treat with conventionaldrug or dose
The Promise of Personalized Medicine
Genetics or Genomics?
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)
History
Time line of genomic discoveries
Human Genome Project
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 worldIdentifying “tag” SNPs that uniquely identify these
haplotypesA small number of SNP patterns (haplotypes) can account
for 80-90% of entire human population
Some definitions
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
Differences in the Genetic Code
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
Types of Polymorphisms
Single Nucleotide Polymorphism (SNP): GAATTTAAG
GAATTCAAG Simple Sequence Length
Polymorphism (SSLP): NCACACACAN
NCACACACACACACANNCACACACACACAN
Insertion/Deletion: GAAATTCCAAGGAAA[ ]CCAAG
Markers of Genetic Variation
Pharmacogenomics
DRUGTARGETS
DRUGMETABOLIZING
ENZYMES
DRUGTRANSPORTERS
PHARMACOKINETICSPHARMACODYNAMICS
Variability in Efficacy/Toxicity
•Transporters•Plasma protein binding•Metabolising enzymes
•Receptors•Ion channels•Enzymes•Immune molecules
Polymorphisms
Drug metabolism
Adverse Drug Reaction
Disease susceptibility
Receptor sensitivity
Drug transport
Responders/Non-responders
Consequences of polymorphisms
These mutations may have no effect on enzyme activity(normal) Lead to enzyme activity with
Decreased activityAbsent 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
Pharmacogenetic Trait Clinically Relevant
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
DRUG TRANSPORTERS
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
substancesor metabolites out of
cells.
Molecular genetics of MDR1/ P-gp
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.
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, andL-amino acid transporter.
Other Polymorphic Drug Transporters
Drug Transport
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
Pharmacogenetic Trait Clinically Relevant
DRUG METABOLISM
Drug Metabolism Pharmacogenomics 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 bypseudocholinesterase
choline succinylmonocholine
O C CH2CH2
O
(H3C)3NH2CH2C C
O
O CH2CH2N(CH3)3+ +
SUCCINYLCHOLINE
Drug Metabolizing Enzymes
Phase I: biotransformation reactions: oxidation, hydroxylation, reduction, hydrolysisPhase II: conjugation reactions—to increase their water solubility and elimination from the body. The reactions are glucuronidation, sulation,acetylation, glutathione conjugation
1A219%
2D63%
2E110%
3A4/542%
2C92C19 26%
1A25%
2D624%
2E11%
3A4/551%
2C92C1919%
Primary CYP Enzymes in Drug Metabolism
% of total enzyme % of drugs metabolised
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
CYP 450 gene Mutant Alleles Substrates
CYP2C9*1 *2, *3, *4, *5, *6Warfarin, 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
Phase II enzymes known to have polymorphism
NAT2: Isoniazid, hydralazine, GST: D-Penicillamine TPMT: Azathioprine, 6-MP Pseudocholinesterase: Succinyl choline UGT1A1: Irinotecan
Gene Mutant Alleles Substrates
NAT2 *2, *3, *5, *6,*7, *10,*14 Isoniazid, hydralazine,
GSTM1A/B, P1
M1 null, T1 nullD-penicillamine
TPMT *1,*2,*3A,C, *4-*8 Azathioprine, 6-MP
UGT1A1 *28 Irinotecan
Red: Absent; Blue: Reduced;
Mutant alleles of Phase II enzymes
Starting dose of nortriptylineNormal CYP2D6 : 150 mg/dayMutant CYP2D6 : 10-20 mg/day
RECEPTOR SENSITIVITY
Receptor Sensitivity/Effect
1 receptor gene
Arg389Gly
Ser49Gly
Subjects with Gly 389 have reduced sensitivity to beta-blockersSubjects 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
10 fold difference in concentration required between genotypes(adenylyl cyclase activity)
RESPONDERS & NON-RESPONDERS
Disease Gene and
PolymorphismAllele/
GenotypeEffect
AsthmaALOX5
Promoter region mut
Respond poorly to antileukotriene treatment with ABT-761
AtherosclerosisCETPTaqIB B2/B2
Poor response to treatment with pravastatin
Smoking cessation
CYP2B6C1459T TT
Greater craving for cigarettes and higher relapse rates
Heart failure2 AR geneGln27Glu Glu27 Better response to
carvedilol treatment
ADVERSE DRUG REACTIONS
What is the reason for high rate of ADRs of Type A ?
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.
CYP2C9 and ADR of Warfarin
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
•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
•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
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
CYP2C9 POLYMORPHISM
VKORC1POLYMORPHISM
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/dVKORC1 A/B: 4.9 ± 0.2 mg/dVKORC1 B/B: 6.2 ± 0.3 mg/dMean 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 activeVit K hydroquinone
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
TPMT polymorphism induced ADR
Drugs Demonstrated to Precipitate Hemolytic Anemiain Subjects with G6PD Deficiency
Nitrofurantoin Primaquine DapsoneMethylene Blue Sulfacetamide Nalidixic AcidNaphthalene Sulfanilamide SulfapyridineSulfamethoxazole
INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT ETHNIC POPULATIONS
Ethnic Group Incidence(%)Asiatics Chinese 2 Filipinos 13 Indians-Parsees 16 Japanese 13
Pharmacogenomic Biomarkers as Predictors of Adverse Drug Reactions
Gene Relevant DrugTMPT 6-mercaptopurinesUCT1A1*28 IrinotecanCYP2C0 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 OmperazoleHLA-B5701 AbacavirHLA-B1502 CarbamazepineG6PD Deficiency Rasburicase; Dapsone; Primaquine;
Chloroquine
MDR1 Protease inhibitorsADD1 DiureticsIon channel genes QT prolonging antiarrhythmicsCRHR1 Inhaled steroids
DISEASE SUSCEPTIBILITY
Disease Gene PolymorphismAllele/
GenotypeEffect
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
Disease GeneAllele/
GenotypeEffect
Acute MI CYP2C9eNOS
*3T786C
susceptibility to AMI.
Alzheimer’s disease ApoE
ε 2
ε 4/ ε4
Reduced risk
Poor prognosis
CancerGST M1 Null
T1 Null susceptibility to lung
and bladder cancer
NAT NAT2 *10 susceptibility to colorectal cancer
Drugs Demonstrated to Precipitate Hemolytic Anemiain Subjects with G6PD Deficiency
Nitrofurantoin PrimaquineMethylene Blue Sulfacetamide Nalidixic AcidNaphthalene Sulfanilamide SulfapyridineSulfamethoxazole
INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT ETHNIC POPULATIONS
Ethnic Group Incidence(%)Asiatics Chinese 2 Filipinos 13 Indians-Parsees 16 Japanese 13
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.
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
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
• 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
Potential of Pharmacogenomics
Clinical Relevance
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
The Future of Pharmacogenomics
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
Role of Pharmacogenomics in the Drug Development Process
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
Drug development…is a long one!
IdeaMarketed
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
…and an expensive one!
It costs >$800 million to get a drug to market
Applying PharmacogenomicsApplying Pharmacogenomics
.
DISEASE GENETICS
TARGETVARIABILITY
SELECTINGRESPONDERS
PHARMACO-GENETICS
Discovery Development
Choosing the Best Targets
Better Understanding of
Our Targets
Improving Early Decision Making Predicting
Efficacy and Safety
Pharmacogenomic Paradigm in the Drug Development Process
Current Options Options with Pharmacogenomics
Pro
por
tion
of
pat
ien
ts s
how
ing
poo
r or
no
resp
onse
Low
High
Continue clinical trialsto market
Abandon drugbefore market
Optimize clinical trials,making them
smaller and shorter
Continue trials safelyby excluding at-risk pts
Personalized Medicine and the Pharmaceutical Industry
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
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 response1. Store and organize primary genotyping data2. Correlate phenotypic measures of drug response
with genotypic data3. Curate major findings of the published literature4. Provide information about complex drug pathways5. Highlight genes that are critical for understanding
pharmacogenomics
Patient requires Treatment
Examination by the Physician
Genomic testingTraditional investigations
EXPERT SYSTEM
Decision making by Physician, assisted by an Expert System (interactive interpretation)
Prescribes individualized drug treatment
..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
“Oh! She is a poor metabolizer”
Personalizedmedicine
S M A R T C A R D
Person’s name
GENOME
(Confidential)
“Here is my sequence”
elusive dream or
imminent reality?
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