Warfarin Dosing

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Warfarin Dosing

• In August 2007, the FDA updated the warfarin
  prescribing guidelines to include genetic
  testing.
• SNP assessment in CYP2C92 and CYP2C93
• SNP assessment in VKORC1

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Pharmacogenetics Drug Safety
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Financial Disclosure

• I have no actual or potential conflict of
  interest in relation to this program.

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Learning Objectives

• Compare and contrast pharmacogenetics and
  pharmacogenomics
• Demonstrate and understanding of basic DNA
  terminology and genomic variations
• Explain personalized medicine from the
  standpoint of drug metabolism, bioactivation, and
  pharmacologic target screening

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Learning Objectives

• Describe the limitations to implementing
  pharmacogenetic screening in health care
• Apply knowledge of pharmacogenetics to the
  initiation of warfarin therapy
• Apply single nucleotide polymorphisms data to
  patient dosing

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Introduction and Background

• Pharmacogenetics
• Early history
• 1931
• Sir Archibald Garrot
• 1954
• During WWII, American soldiers developed severe
  hemolytic anemia after taking primaquine.
• later shown to result from a deficiency in
  glucose-6-phosphate dehydrogenase (G6PD)

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Introduction and Background

• 1954
• individuals who experienced peripheral neuropathy
  in response to the anti-tubuculosis drug
  isoniazid
• lacked the ability to metabolize the drug
• 1956
• prolonged neuromuscular blockade following normal
  doses of succinylcholine
• resulted from a deficiency in psuedocholinesterase
  activity
• In each case, an inherited genetic trait was
  linked to an abnormal drug response!

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Introduction and Background

• Pharmacogenetics vs. Pharmacogenomics
• generally defined as
• the study of the relationship between genetics
  and drug effect
• the application of genetic analysis to predict
  drug response, efficacy, and toxicity

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Introduction and Background

• Pharmacogenetics vs. Pharmacogenomics
• Key differences
• pharmacogenetics
• focused on variation in individual, specific
  genes that influence the response to a drug
• associated with
• a large clinical effect
• mutation in a single gene
• affects a relatively small number of individuals

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Introduction and Background

• Pharmacogenetics vs. Pharmacogenomics
• Key differences
• pharmacogenomics
• focused on variation in a large collection of
  genes, up to the whole genome, that influence
  response to a drug
• associated with
• smaller clinical effect
• involves many mutations or multiple variants
• affects many individuals within a population

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Introduction and Background

• Pharmacogenetics vs. Pharmacogenomics

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Introduction and Background

• Pharmacogenetics vs. Pharmacogenomics
• The promise is personalized medicine!
• Drug therapy tailored to a patients unique
  genetic makeup
• choice of the drug
• choice of the dosing regimen

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Basis for Pharmacogenetics

• Pharmacogenetics vs. Pharmacogenomics
• Concept of pharmacogenetics
• based on several factors
• Most current medications are associated with a
  significant risk for drug toxicity and drug
  inefficacy
• Variability of drug response
• Genetic variation

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Basis for Pharmacogenetics

• Drug toxicity
• Adverse drug reactions (ADRs)
• Each year in the U.S.
• 137 billion due to drug-related morbidity and
  mortality
• Two million (6) hospitalized patients experience
  a serious ADRs
• 106,000 (0.32) hospitalized patients have fatal
  ADRs
• ADRs rank as the fourth-leading cause of death in
  the U.S.
• 50 of ADRs in hospitals have no readily
  discernible or preventable cause
• 16 (193) of pharmaceuticals carry a black box
  warning

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Basis for Pharmacogenetics

• Drug inefficacy
• Response rates vary markedly across therapeutic
  areas
• Estimated response rates
• 80 - analgesics 25 - cancer chemotherapy
• 30 - Alzheimers disease 60 - depression
  (SSRIs)
• 40 - incontinence 47 - HIV
• 50 - rheumatoid arthritis 60 - schizophrenia
• 50 - migraine (prophylaxis) 52 - migraine
  (acute)
• 57 - diabetes 60 - asthma
• 60 - cardiac arrhythmias
• Overall, 50 of patients do not respond to drugs
  in the major therapeutic classes

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Basis for Pharmacogenetics

• Variability of drug response
• Response to a drug is influenced by both
  environmental and genetic factors

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Basis for Pharmacogenetics

• Variability of drug response
• Genetics
• contribution of genetic factors to a drug
  response can be estimated using twin studies

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Basis for Pharmacogenetics

• Variability of drug response
• Genetic factors
• 75 - 85 of variability in drug half-lives is
  inherited

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Basis for Pharmacogenetics

• Variability of drug response
• Genetic factors
• current drugs act on 500 molecular targets
• 45 - receptors
• 28 - enzymes
• 11 - hormones and related factors
• 5 - ion channels
• 2 - nuclear receptors

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Basis for Pharmacogenetics

• Genetic variation
• DNA
• DNA is comprised of a string of 4 nucleotide
  bases
• A, G, T and C
• Matched A-T and C-G
• Genes
• A segment of DNA containing all of the
  information needed to encode for one protein is
  called a gene.

5-CATGTACCTGGGCCG-3 3-GTACATGGACCCGGG-5
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Basis for Pharmacogenetics

• Genetic variation
• Chromosomes
• Every human cell with the exception of gametes
  contains 23 chromosomes
• ? are diploid (two copies XX)
• ? are haploid (one copy each XY)
• Carry all the genetic coding for all the proteins
  in every cell
• Consist of DNA tightly wound around special
  protein structures called histones

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Basis for Pharmacogenetics

• Genetic variation
• DNA
• Genes
• Chromosomes

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Zipped Files
Decompression
Executable Files
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Transcription Translation
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Basis for Pharmacogenetics

• Genetic variation
• Sequencing of the human genome (2003)
• Some things we learned
• 3.2 billion base pairs
• 30,000 genes (estimated)
• 1 contained in exons
• 24 in introns
• 75 is intergenic
• All human beings share 99.9 DNA sequence
• diversity at the genetic level is encoded by 0.1
  variation in our DNA

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Basis for Pharmacogenetics

• Genetic Variation
• Determining genetic differences between
  individuals
• Polymorphisms
• Common variation in DNA
• often defined as greater than 1 in a given
  population
• Occur on average every 1331 bp
• frequency can be much greater in a given gene.
• Estimated to be 11 million polymorphisms in the
  genome

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Basis for Pharmacogenetics

• Polymorphisms
• Two main types
• SNPs polymorphisms that occur at a single
  nucleotide
• Can be located in either coding regions (DNA that
  is transcribed occur less frequently) or
  non-coding regions
• Coding polymorphisms are further classified as
• Non-synonymous (missense) results in
  translation of a different amino acid
• Synonymous (sense) results in translation of
  the same amino acid
• Nonsense results in the insertion of a stop
  codon

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Basis for Pharmacogenetics

• Polymorphisms
• SNPs
• Non-coding polymorphisms
• when located in promoters, introns, or other
  regulatory regions may alter transcription factor
  binding, mRNA transcript stability or RNA
  splicing

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Basis for Pharmacogenetics

• Polymorphisms
• Two main types
• Coding Non-coding SNPs

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Basis for Pharmacogenetics

• Polymorphisms
• Indels
• insertion or deletion of multiple nucleotides
• commonly result in gene insertions, duplications
  or deletions

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Basis for Pharmacogenetics

• Polymorphisms (mainly SNPs) are used to
  characterize genetic differences between
  individuals
• However,
• a pharmacogenetic trait cannot be linked to just
  one SNP
• In this case, haplotypes can be used to associate
  a genotype with a phenotype

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Basis for Pharmacogenetics

• Haplotypes
• Defined as
• Group of SNPs located closely together on a
  chromosome
• are inherited together
• Most genes contain between 2 and 53 haplotypes
• avg. 14
• Haplotypes themselves may not have a direct
  effect on drug response
• their proximity to a causative SNP allows them to
  act as a marker for a particular drug response

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Basis for Pharmacogenetics

• Haplotypes
• Haplotypes themselves may not have a direct
  effect on drug response
• their proximity to a causative SNP allows them to
  act as a marker for a particular drug response

Haplotype
chromosome
SNP
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Basis for Pharmacogenetics

• Haplotypes
• ß2 adrenergic receptor haplotypes
• 12 haplotypes identified in the 5 UTR and open
  reading frame
• Several haplotypes are associated with gt 2-fold
  increase in response to albuterol
• Individual SNPs within the haplotypes were not!

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Basis for Pharmacogenetics

• Haplotypes
• Both SNPs and haplotypes can be used to map
  genetic changes associated with a drug response

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Pharmacogenetics in Drug Therapy

• Drug metabolism
• Thiopurine S-methyltransferase (TPMT)
• The TPMT gene is located on chromosome 6
• The main function of TPMT is to catalyze the
  S-methylation of the immunosuppressants
  azathioprine and 6-mercaptopurine
• Used in
• prevention of acute rejection in transplant
  recipients
• Inflammatory Bowel Disease (IBD) particularly
  Europe
• acute lymphoblastic anemia (6-mercaptopurine)

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Pharmacogenetics in Drug Therapy

• Drug metabolism
• Thiopurine S-methyltransferase (TPMT)
• Four major SNPs correlate to a loss of enzyme
  activity
• TPMT3A, TPMT3B, TPMT3C
• TPMT3A accounts for 85 of mutated alleles
• Encompasses two SNPs
• G460A (A154T) and A719G (Y240L)

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Pharmacogenetics in Drug Therapy

• Drug metabolism
• Thiopurine S-methyltransferase (TPMT)
• 0.3 of individuals are homozygous for TMPT
  deficiency
• 10 are heterozygous individuals and show an
  intermediate phenotype
• Patients with TPMT deficiency
• Increased risk for severe hematopoeitic toxicity
  if treated with conventional doses of thiopurines!

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Pharmacogenetics in Drug Therapy

• Drug metabolism
• The CYP Families
• proportion of drugs metabolized by the major P450
  enzymes

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Pharmacogenetics in Drug Therapy

• CYP2D6
• The gene is located on chromosome 22
• Nearly 100 drugs are substrates for this enzyme
• ß-adrenergic blockers Antidepressants Neuroleptic
  s
• metoprolol amitriptyline haloperidol
• propanolol clomipramine resperidone
• desipramine thoridazine
• Antiarrhythmics fluoxetine
• encainide fluvoxamine Others
• sparteine imipramine codeine
• flecainide nortryptaline dextramethophan
• propafenone paroxetine tramadol

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Pharmacogenetics in Drug Therapy

• CYP2D6 polymorphisms
• poor metabolizer (PM) phenotypes
• CYP2D63 A2637 del (frameshift)
• CYP2D64 G1934A (splicing defect)
• most common in Caucasian populations
• CYP2D65 Gene deletion (no enzyme)
• CYP2D610 C188T
• most common in Asian populations
• CYP2D64 is almost completely absent in this
  group
• CYP2D617 C1111T
• most common in African populations

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Pharmacogenetics in Drug Therapy

• CYP2D6 duplication
• extensive metabolizer (EM) phenotype
• repetition of a 42 kb XbaI fragment containing
  the CYP2D62 gene that results in 2-13 copies of
  the enzyme
• the frequency of individuals possessing CYP2D6
  duplication suggests a geographical gradient,
  possibly resulting from dietary pressures
• 1 - Sweden
• 4 - Germany
• 7-10 - Spain
• 10 - Italy
• 30 - Ethopians

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Pharmacogenetics in Drug Therapy

• Examples
• Nortriptyline

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Pharmacogenetics in Drug Therapy

• CYP2C19
• Only two SNPs have been identified thus far
  CYP2C192 and CYP2C193
• Each results in a non-functional protein product
• individuals homozygous for either CYP2C192 or
  CYP2C193 have no functional enzyme
• results in increased drug levels and improved
  therapeutic outcome

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Pharmacogenetics in Drug Therapy

• CYP2C19
• CYP2C192 and CYP2C193

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Pharmacogenetics in Drug Therapy

• CYP2C9
• Encodes the p450 enzyme that metabolizes the
  anticoagulant warfarin
• Warfarin
• most widely prescribed oral agent for the
  treatment of thromboembolic diseases
• antagonist at vitamin K epoxide reductase
• required to maintain levels of reduced vitamin K,
  which allows carboxylation of glutamate receptors
  on coagulation factors
• wide inter-individual variability in therapeutic
  efficacy
• 0.5 mg/day 50 mg/day
• underdosing leaves patients at risk for clotting
  while overdosing increases the risk for
  hemorrhaging

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Pharmacogenetics in Drug Therapy

• CYP2C9
• 200 polymorphisms have been identified in CYP2C9
• 30 coding region variants
• CYP2C92
• most common in Caucasian populations
• CYP2C93
• most common across all ethnicities
• individuals with either CYP2C92 or CYP2C93
  exhibit increasing sensitivity to warfarin
• greatest sensitivity if homozygous for CYP2C93
• 23 haplotypes have been identified within CYP2C9
• only the 2 or 3 SNPs are associated with
  increased warfarin sensitivity

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Pharmacogenetics in Drug Therapy

• Vitamin K oxidoreductase (VKORC1)
• molecular target of warfarin
• 27 SNPs identified in VKORC1 that are contained
  within 5 different haplotypes
• Based on haplotypes, 2 phenotypically distinct
  groups were identified (A or B)
• AA require 3 mg/day warfarin
• BB require 7 mg/day warfarin
• polymorphisms in VKORC1 account for 20 30 of
  warfarin sensitivity

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Pharmacogenetics in Drug Therapy

• Warfarin Dosing
• In August 2007, the FDA updated the warfarin
  prescribing guidelines to include genetic
  testing
• VKROC1 and CYP2C9

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Pharmacogenetics in Drug Therapy

• Warfarin Dosing
• Pharmacokinetics
• racemic mixture of R and S isomers
• S 5X more potent than R
• S-warfarin is transformed by CYP2C9 R-warfarin
  is mainly transformed by CYP1A2
• rapidly absorbed by GI tract with high
  bioavailability
• plasma concentrations peak approximately 90
  minutes after administration
• half-life 36-42 hours, binds to plasma proteins
  (mainly albumin)

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Example dosing table for warfarin based on SNP
type
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Pharmacogenetic Influence in Therapeutics

• Pharmacogenetics has been slow to be implemented
  clinically
• As of 2003, 51 drugs contain pharmacogenetic-relat
  ed information on their product label

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Pharmacogenetic Influence in Therapeutics

• Limitations
• Cost
• Existing DNA sequencing technology makes genetic
  screening inherently expensive
• Who is responsible for the cost burden associated
  with genotyping
• patient, government, or insurance company?
• influence on drug development
• CYP2D6-metabolized drugs
• Potential emotional and financial liability
  associated with genetic information
• Availability and timeliness of genetic testing

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Pharmacogenetic Influence in Therapeutics

• However
• Recent study suggests a tremendous interest among
  patients and clinicians for pharmacogenetics to
  be more involved in therapeutic decision making
• In U.S.
• 80 viewed genetically-guided personalized
  medicine favorably
• 50 are willing to undergo genetic testing to
  determine medications used in therapy
• March 2005, FDA officially encouraged
  pharmacogenomic data to be submitted with drug
  approval application materials

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Dedicated Bench-top DNA Detection System
Rx Consumer Kiosk
Patient Information Results Presentation Prescript
ion Support Consumer Education
1
3 and 4
2
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Warfarin Dosing

• In August 2007, the FDA updated the warfarin
  prescribing guidelines to include genetic
  testing.
• SNP assessment in CYP2C92 and CYP2C93
• SNP assessment in VKORC1

57
1.) Pharmacogenomics is the study of the
relationship between genetic variation and drug
responses in many individuals within a
population.

• True
• False

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2.) Polymorphisms in which metabolic enzyme
activity is associated with both poor metabolizer
(PM) and extensive metabolizer (EM) phenotypes?

• NAT2
• CYP2D6
• TPMT
• VKORC1

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3.) The promise of personalized medicine is to
tailor drug therapy, including drug choice and
dosing regimen, based on a patients unique
genetic makeup.

• True
• False

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4.) Each of the following are limitations to
implementing pharmacogenetic screening in health
care EXCEPT

• Cost
• Privacy
• Speed/availability of genetic testing
• Patient support