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

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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?